key: cord- -f eb nf authors: van rensburg, v; pillay-fuentes lorente, v; decloedt, e title: current evidence for directed and supportive investigational therapies against covid- date: - - journal: nan doi: . /ajtccm. .v i . sha: doc_id: cord_uid: f eb nf coronavirus disease (covid- ) is a global health crisis. there is currently a great need for effective and safe therapies directed at the disease, but no drugs are presently registered for use in covid- . several directed therapies have been proposed, and most are still in clinical trials. currently available published, peer-reviewed results mostly involve small sample sizes with study limitations restricting the interpretation of the findings. many trials currently published also do not have a control group, limiting the interpretation of the effect of the intervention. investigational directed therapies as well as investigational supportive therapies against covid- are reviewed here. chloroquine and hydroxychloroquine show promise as directed therapies, but current trial results are conflicting. lopinavir/ritonavir also shows potential, but was started late in the disease course in most trials. no randomised controlled evidence is currently available for remdesivir and favipiravir. corticosteroid use is not recommended for directed therapy against covid- , and the role of tocilizumab is currently unclear, based on limited evidence. early initiation of investigational directed therapies may provide benefit in selected patients. the results from larger randomised controlled trials will clarify the place of these therapies in covid- treatment. global clinical need for effective therapy has led to the investigational repurposing of registered drugs, as well as the early-phase testing of new antiviral drugs. many of these drugs have already been investigated for sars-cov- , middle east respiratory syndrome (mers) or ebola virus, with inconsistent efficacy results. [ , ] there are currently no clear data from randomised controlled clinical trials on the effect of these drugs on covid- outcomes, but several hundred clinical trials are presently registered to investigate their efficacy and safety in humans. [ , ] below we briefly and narratively summarise the current evidence published in peer-reviewed scientific journals for the listed investigational therapies. chloroquine and hydroxychloroquine chloroquine (cq) has been the backbone of malaria treatment and prevention for many decades. it is also used as an immunomodulator for rheumatological conditions such as systemic lupus erythematosus and rheumatoid arthritis (ra). [ ] cq has good in vitro activity against sars-cov- . [ ] the postulated mechanisms of action in covid- infection are threefold: [ ] increasing endosomal ph that inhibits the sars-cov- spike protein cleavage required for viral/endosomal fusion after entry; interference with the glycosylation of cellular receptors (possibly through modification of angiotensin-converting enzyme (ace ), purported to be involved with viral entry); [ , ] and immunomodulation. multiple trials across the globe are currently underway to assess the efficacy of cq for the treatment and prevention of covid- , but no published, peer-reviewed results are available at the time of writing. [ , , , ] similarly to cq, hydroxychloroquine (hcq) has in vitro activity against sars-cov- . [ ] hcq was found to be more potent than cq at inhibiting sars-cov- in vitro, [ ] and is expected to have a similar mechanism of antiviral action to cq. [ ] hcq is generally regarded as having fewer long-term adverse effects than cq at equivalent doses, most notably retinopathy. [ , ] the mechanisms underlying this difference are currently unknown, but may be related to differences in drug accumulation, or the reduced propensity of hcq to bind to eye tissues, compared with cq. [ ] cardiotoxicity, especially qtprolongation, is, however, a significant risk with both drugs, even early in the treatment course. [ ] the mechanism of drug-induced qt prolongation is through inhibition of the herg/kv . potassium channels in the heart. [ ] inhibiting these channels increases the cardiac action potential duration, prolonging the qt interval and increasing the risk for torsades de pointes. [ ] the risk of qt-prolongation is further compounded in patients with underlying cardiac disease or cardiac risk factors, and with the administration of concomitant qtprolonging medications or enzyme inhibitors. [ , ] the pervasive use of certain tuberculosis drugs and hiv antiretroviral therapy in the southern african setting may further increase the qt-prolongation risk. [ ] several risk-stratifying tools are currently available for drugs with qt-prolonging potential. a newly launched platform, medsafety scan (https://medsafetyscan.org), has been made available online to guide clinicians on risk and clinical decision-making when they input patients' qt-prolonging drugs and selected clinical factors. the risk of cq and hcq to cause qt prolongation is based on the well-known crediblemeds (https://crediblemeds.org) database. the currently available evidence on the use of hcq in covid- comes from four published, peer-reviewed trials. gautret et al. [ ] suggested in a small, open-label, non-randomised trial that hcq together with azithromycin (azm) may be more effective in reducing viral shedding by day post-treatment than hcq alone, or controls. of note is that the effect of azm was not prospectively assessed with hcq, as the azm was only given to some patients as part of standard of care if the clinical presentation warranted it. the association of azm and hcq in the reduction of viral shedding was therefore made after the fact. some limitations of this trial were the small sample size and lack of power (only six participants received hcq and azm), no reporting of clinical outcomes and exclusion of participants who died or were sent to the intensive care unit (icu). the authors commented that the addition of azm to hcq may increase the risk of qt-prolongation, but that the potential antiviral activity of azm and the synergistic effect with hcq may justify the combination on a case-by-case basis. gautret et al. [ ] published a follow-on uncontrolled study with a larger sample size (n= ). primary endpoints were the need for oxygen therapy or transfer to the icu after at least days of treatment, contagiousness as assessed by viral real-time reversetranscriptase polymerase chain reaction (rt-pcr) and culture, and length of stay in the study ward. dosages used were oral hcq sulphate mg three times a day for days, combined with azm mg on day , followed by mg per day for the next days. hcq with azm was not started in patients in whom the cardiac risk was deemed too high. the primary endpoint was reached in . % of participants, who required oxygen therapy or transfer to the icu. viral rt-pcr was negative in % at day , and % at day post treatment. viral cultures were negative in . % of participants at day . sixty-five of the participants were discharged from the study ward during the trial period, with a mean time from initiation to discharge of . days. the findings of this trial are difficult to interpret, as participants were not randomised, nor was there a control group. study limitations further include that most participants ( %) were considered low risk for clinical deterioration at enrolment, and this low risk continued until discharge in most ( . %). four asymptomatic patients were also included in the trial, and more than three-quarters of participants were younger than the high-risk age group of ≥ years old. [ ] the majority of participants had a low risk for clinical deterioration, and the observation of negative rt-pcr tests in % of participants by day after treatment initiation -therefore at a median of day after symptom onset -could well be in keeping with the natural course of the disease. [ , ] in response to the first publication by gautret et al., [ ] molina et al. [ ] replicated the design and dosages of hcq and azm in their own trial to assess whether similar findings were observed. they prospectively enrolled consecutively hospitalised patients with severe covid- . paired rt-pcr results were available in participants, with still testing positive at days - after treatment initiation. the authors conclude that their findings stand in contrast to those reported by gautret et al., [ ] and cast doubt on the strong antiviral efficacy of the combination of hcq and azm. [ ] limitations of this trial include a small sample size, no control group and limited information on clinical outcomes. a chinese study evaluated participants randomised to receive hcq sulphate mg once a day for days with standard of care (n= ), or standard of care alone (n= ). [ ] the study endpoints were virological clearance by day , or death. the authors found no difference between the two groups for virological clearance ( . % in the interventional group v. . % in the standard of care group; p> . ). none of the participants died during the -week follow-up period. [ ] in summary, trials assessing hcq and cq in covid- to date have mostly been uncontrolled, and have shown conflicting results. owing to methodological limitations, the results are difficult to interpret, and there are currently insufficient clinical data to recommend either for or against the use of cq and hcq in covid- outside of the clinical trial setting. [ ] lopinavir/ritonavir (with or without interferon) lopinavir and ritonavir are both protease inhibitors. ritonavir is used in combination with lopinavir to increase lopinavir plasma concentrations by inhibiting the enzymatic metabolism of lopinavir. [ ] the rationale for the use of lopinavir/ritonavir (lpv/r) in covid- stems from its in vitro activity against sars-cov- , [ ] as well as from a retrospective, multicentre cohort study evaluating lpv/r as early treatment in sars-cov- , which demonstrated decreased mortality and intubation rates. [ ] numerous case reports and case series have shown successful management of covid- patients with lpv/r. [ ] [ ] [ ] [ ] [ ] [ ] nevertheless, some patients on lpv/r still progressed to severe disease, although in most such cases lpv/r was only started late in the disease course. it is therefore unclear whether the observed effect was due to the intervention or to the natural course of the disease. in a retrospective cohort study of patients receiving lpv/r / mg twice a day, all patients tested negative review with rt-pcr by day . [ ] wan et al. [ ] reported the clinical features and treatment of covid- in a case series of patients in china. they reported that lpv/r and interferon administered to all patients in early-stage disease resulted in the discharge of patients and the death of patient at the end of the -day study period. a total of patients were still hospitalised at the end of the study period. the doses of lpv/r, and the doses and type of interferon used, were not stated. in vitro studies of interferon have demonstrated inhibition of sars-cov- infection, but only in combination with other antiviral agents. [ ] several clinical trials are currently evaluating various treatment combinations with interferon. [ , ] the most robust evidence for lpv/r in sars-cov- currently comes from a chinese randomised controlled trial that evaluated its efficacy and safety. [ ] the primary outcome was the time to clinical improvement. one hundred and ninety-nine participants were individually randomised to lpv/r (n= ) at a dose of / mg twice a day with standard of care, or standard of care alone (n= ), for days. the results showed that the time to clinical improvement between the two groups was similar, with a median of days for both (hazard ratio for clinical improvement . ; % confidence interval . - . ; p= . ). [ ] recorded adverse events included nausea, vomiting and diarrhoea, all of which were more common in the lpv/r group than the standard of care group. four gastrointestinal-related serious adverse events reported in the lpv/r group were deemed to be related to lpv/r. limitations included the open-label design of the trial, the initiation of investigational treatment late in the disease course (a median of days after symptom onset) and the fact that both groups were heterogeneous and received various additional treatments. in summary, lpv/r does not currently have robust evidence for use in covid- , but the mostly conflicting results from currently available data should be interpreted in light of the study designs and timing of lpv/r in the disease course. at the time of writing, lpv/r is not recommended for use, other than as part of clinical trials. [ ] remdesivir remdesivir is in early-phase drug development, and currently being investigated for covid- therapy. remdesivir was originally developed for use against the ebola virus in , [ ] and has shown promising in vitro activity against sars-cov- . [ ] it acts as a nucleotide analogue, causing premature termination of viral rna replication by inhibiting rna polymerase. [ ] although remdesivir is not yet registered by any medicines regulatory authority for human use, its safety profile was established in a randomised controlled trial during the ebola virus outbreak in the democratic republic of congo. [ ] several clinical trials are currently registered for the investigation of remdesivir, [ , ] but no robust results are yet available. remdesivir has, however, been approved for compassionate use in several countries, and a recent preliminary report described the outcomes in a cohort of patients hospitalised for severe covid- who were treated with remdesivir on a compassionate-use basis. [ ] the authors described the outcomes of participants, and reported that after a median follow-up of days, % of the participants had an improvement in terms of their required oxygen support. of participants who initially required mechanical ventilation, remained ventilated, were switched to non-invasive ventilation, were weaned off oxygen and were discharged. six participants who were initially ventilated died, and the overall mortality rate was %. a significant limitation of this report is the protracted duration of symptoms before remdesivir was started (median (interquartile range) ( - ) days). additionally, the absence of a control group limits the interpretation of the effect of remdesivir. concomitant therapies and viral loads were also not reported. in summary, there are currently insufficient clinical data available to recommend either for or against the use of remdesivir in covid- , outside of clinical trials. [ ] favipiravir favipiravir is an antiviral drug approved for the management of influenza in japan. [ ] it is currently being evaluated in clinical trials for the management of covid- . [ ] it is a nucleic acid analogue that is incorporated into viral rna to affect viral replication and possibly rna polymerase. [ ] at the time of writing, there were no published peer-reviewed trials or case studies evaluating favipiravir in covid- , and its use is not currently recommended outside of clinical trials. tocilizumab tocilizumab is a monoclonal antibody used for the treatment of several rheumatic conditions, including ra and juvenile idiopathic arthritis. [ , ] the us food and drug administration additionally approved tocilizumab in for the treatment of cytokine release syndrome (crs, also known as cytokine storm) caused by chimeric antigen receptor t-cell therapy, an immunomodulatory approach used in oncology. [ ] tocilizumab is a recombinant humanised monoclonal antibody against the interleukin (il- ) receptor. it binds both soluble and membrane-bound il- receptors, inhibiting il- signal transduction. [ ] patients with severe disease during the previous outbreaks of sars-cov- [ ] and mers [ ] were found to have elevated concentrations of il- . patients who died due to covid- were also found to have increased markers for crs, including raised il- . [ ] it is therefore postulated that the current severe organ dysfunction observed in covid- may be due to crs, and that il- inhibitors such as tocilizumab may attenuate this immune reaction. [ , , ] a retrospective observational study from a single centre in china reported on the use of tocilizumab in covid- patients. [ ] the study population was older patients, aged between and years. most patients were severely or critically ill, and over half received a course of methylprednisolone. one-third of the patients ( / ) received two or more doses of tocilizumab. the authors report increasing il- concentrations after tocilizumab initiation followed by a decrease in most patients who improved clinically. five patients died or had disease aggravation, with il- concentrations increasing in all five. limitations include the small sample size and the non-reporting of the duration of symptoms before treatment was started, or whether other investigational therapy was given as well. several case reports and a case series have also been published describing the effect of tocilizumab in patients with various underlying conditions. [ ] [ ] [ ] [ ] all showed improvement, but these reports included patients who were started on tocilizumab after inadequate response to standard of care. it is unclear what the effect of tocilizumab was in these cases, as it was started late in the disease course and the clinical improvement may review have been in keeping with the natural course of the disease. numerous randomised trials are currently underway to evaluate tocilizumab in covid- . [ , ] the who advises against the use of corticosteroids in the management of targeted covid- owing to potential harmful effects. [ ] this is largely based on previous experiences of corticosteroid therapy in influenza and mers-cov. a systematic review of treatment options in sars-cov- infection included corticosteroids' effects on mortality, in vitro inhibition of sars viral replication and acute respiratory distress syndrome. [ ] the study concluded that the evidence for the effects of corticosteroids in the management of sars-cov- was either inconclusive or showed that they conferred possible harm. [ ] however, the studies included were of poor-quality evidence. a cochrane review [ ] of observational studies of corticosteroids as adjunctive therapy in the treatment of influenza found an increased risk of mortality. the included studies demonstrated significant heterogeneity across the group as a whole, and the quality of evidence was graded as low. [ ] a subsequent observational cohort study investigating influenza adjusted for baseline and time-dependent characteristics, and matching treatment groups according to propensity scores, found no association between the use of corticosteroids and mortality in influenza. [ ] in mers-cov, there was an association between delayed viral clearance and the use of corticosteroids, but no association with -day mortality. [ ] the major limitation of this study was the retrospective observational design, with possible confounding by indication. nevertheless, owing to the possibility of potential harm by corticosteroids in viral infections, the who recommends against the routine use of corticosteroids in covid- patients, unless used with caution for other indications where there are supportive data, such as sepsis and acute exacerbations of asthma and chronic obstructive pulmonary disease, or as part of clinical trials. [ ] patients infected with covid- subsequently require a risk-benefit assessment prior to the use of corticosteroids. at the time of writing there were no published, peer-reviewed studies on the use of corticosteroids in covid- , but several clinical trials are underway. [ , ] several of the above trials have shown that results from investigational therapies that were started late in the disease course are difficult to interpret, as the outcomes observed may have been masked by irreversible systemic injury (no difference compared with standard of care), or natural course of the disease (improvement regardless of the therapy). clinical trials will subsequently require large sample sizes to determine if the investigational directed therapy is in fact effective for smaller treatment effects later in the disease course. in addition, investigational supportive therapies such as tocilizumab may be more effective when given later in the disease course with more severe immunopathology, acting to decrease organ damage in the presence of crs. conversely, some therapies may be more beneficial when given early. oseltamivir, an antiviral drug used for influenza virus infection, has been shown to be more effective when given - hours after symptom onset. [ ] the rationale is that influenza viral replication peaks at - hours after symptom onset, [ ] and viral replication inhibitors such as oseltamivir should be given no later than at the peak of viral replication. drugs that purportedly inhibit sars-cov- replication (such as the investigational antivirals) or viral entry and replication (cq and hcq) may therefore be more effective when given earlier in the covid- disease course. patients who may benefit from earlier treatment initiation include those with confirmed covid- who do not require hospitalisation, but have significant risk factors. there is a pressing need globally to find effective treatment options for covid- . current strategies to curtail the pandemic are aimed at infection prevention and control, and supportive management of those with infections of varying severity. the hope of effective and safe large-scale preventive strategies such as vaccines seems to still be several months away. in the interim, numerous clinical 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chloroquine: a potential drug in the covid- scenario date: - - journal: trans indian natl doi: . /s - - -w sha: doc_id: cord_uid: nf zvc today, the whole world is fighting a public health emergency called ‘covid- ’ caused by a new infectious virus called sars-cov . any person can catch covid- from an infected person via aerosol droplets when the person coughs, sneezes, or speaks. to limit such a transmission, world health organization (who) has recommended people to wear masks and physically distance themselves by staying at least m ( feet) away from others. as aerosol droplets (by cough or sneeze) land on objects and surfaces around the person such as tables, doorknobs and handrails, and remain active on these surfaces for hours to days, people are advised to use soaps for at least s. and alcohol-based sanitizers as well. as the public made efforts, clinicians and researchers investigated and found that drugs which were initially used to treat other diseases may work as a treatment option for covid- . one of those drugs was chloroquine and its related derivative called hydroxychloroquine. in this review article, we have systematically searched for details of covid- pandemic till may and assembled few data pertaining to (i) corona viruses; (ii) sars-cov , the virus that causes covid- ’ and (iii) how chloroquine and hydroxychloroquine mediates anti-viral effect in both prophylactic and therapeutic setting. these data have been acquired mostly from pubmed and websites of who and indian council for medical research (icmr). we did a systematic search and found that the properties of chloroquine are very much essential for the covid- scenario. we also bring to you some evidence that the anti-lysosomal activity of chloroquine may be increased by botanicals like betulinic acid. viruses are submicroscopic infectious particles with the capacity to infect bacteria, algae, plants, animals and humans. these can be found in every ecosystem on earth. there are numerous different varieties of viruses and many are yet to be discovered. viruses are different from other living organisms as these require a host cell to reproduce. when virus infects a host cell (or the virus enters a living organism), the host cell gets 'enslaved' to the virus and synthesizes thousands of identical copies of the original virus. thereafter, the host cell dies in the process of releasing newly synthesized viral particles. a complete virus basically exists as 'virions' comprising of either dna or rna as genetic material encased in a protein or sometimes lipid coat. the genetic material may be single stranded or double stranded, linear or circular. the amount and arrangement of the genetic material and proteins determines the shape and sizes of the viruses. viruses thus have complicated shapes ranging from helical to icosahedral forms. how a virus originated on earth is still a mystery and it has been thought that these were formed from dna fragments that once escaped from a cell. with time, many viruses learned to evade host immune systems via efficient strategies producing diseases for both humans and animals. however, the last several years has witnessed a surge in many new viruses especially those from animals by a process called zoonosis. this basically occurred due to intrusion of human activity into wildlife habitats. an example of ranjana singh and viji vijayan have contributed equally to this work. this is deforestation which led to changes in living habitat of bats, and infections from bats to other organisms like palm civets, pangolins, camels, horses which acted as intermediary hosts to infect humans. the increased world-wide travel is a reason that has accelerated the spread of viruses. for example, the avian flu virus h n was transported from asia to europe by migratory birds. the aids pandemic that was induced by human immunodeficiency viruses (hiv) was originally a zoonotic transmission from monkeys to humans, followed by efficient further distribution within the human population (modrow et al. ) . the transmission of such virus can occur by different means like vectors, respiratory droplets, feces, blisters, mucus, sexual contact, etc. coronaviruses (covs) are a group of single-stranded rna viruses belonging to a sub-family called coronaviridae that together with torovirinae form the coronaviridae family of the nidovirales order. these are the largest known rna viruses with the potential to infect birds, humans and some other vertebrates. although the first member of the coronavirus family was discovered in the s, coronaviruses gained notoriety with the severe acute respiratory syndrome (sars) outbreak in (belouzard et al. ). the international committee on taxonomy of viruses (ictv) have segregated covs into four genera, historically based on serological analysis and now on genetic studies: α, β, γ, and δ. these viruses are enveloped, spherical and distinct in appearance, as they possess club-shaped projections on their surface. the rna genome of these viruses ranges from , to , bases (longest among rna viruses). the genome is packed into a helical nucleocapsid surrounded by a host-derived lipid bilayer. rna has a cap structure at the ′ end and multiple poly (a) tail at the ′ end. rna is positive and, hence, can serve as messenger rna (mrna), allowing the translation of replicase/transcriptase and structural proteins. the replicase/transcriptase is composed of two overlapping open reading frames (orfs): orf a and orf b which encode non-structural proteins and classical structural proteins, viz. (i) spike protein (s), (ii) envelope protein (e), (iii) membrane protein (m), and (iv) nucleocapsid protein (n) (lodish et al. ) . m and e proteins are involved in virus assembly. s protein is leading mediator of viral entry and critical player in determining host range. modification in s protein alters cell and tissue tropism, association with other viral and host factors and changes in host pathogenicity (belouzard et al. ) . covs mainly cause respiratory and intestinal infections with an array of clinical manifestations. so far, covs have been identified that produce infections in humans. these are the α-type hcov- e and hcov-nl ; the β-type hcov-hku , sars-cov, mers-cov, and hcov-oc ; and -ncov (that caused the present pandemic). hcov- e, hcov-oc , hcov-nl , and hcov-hku were mildly pathogenic; while sars-cov or severe acute respiratory syndrome cov, and mers-cov or middle east respiratory syndrome coronavirus were highly pathogenic. the highly pathogenic covs infect the lower respiratory tract causing fatal acute lung injury (ali) and acute respiratory disease syndrome (ards). we are yet to determine the severity of sars-cov (abd el-aziz and stockand ). what is covid- ? covid- is a peculiar form of respiratory disorder that emerged in in a business hub called wuhan, which is located in the hubei province of china. initially, china described the disease as 'atypical' pneumonia and then declared the causative agent to be a corona virus (cov). the corona study group (csg) of the international committee on taxonomy of viruses (ictv) after assessing the etiological agent named it sars-cov (severe acute respiratory syndrome corona virus ) and the disease outbreak as covid- (corona virus disease-year of identification). the disease rapidly spread to more than countries mostly via people with a travel history. as on th april approximately , , deaths were reported worldwide owing to covid- (who b) . characterization of sars-cov is under progress. how did covid- originate? the source of covid- outbreak is yet to be determined. although some preliminary investigations in china identified samples in the huanan seafood wholesale market of wuhan city to be positive for sars-cov , the zoonotic source of the outbreak is still ambiguous. all throughout the pandemic, it was argued by many researchers that bats which harbor a lot of viruses (but remain unaffected by these) are a reason for the covid- outbreak. this was because some bats called 'intermediate horseshoe bats' (rhinolophus affinis) of rhinolophidae family (widespread in central china, southeast asia and indian subcontinent) carry bat corona viruses called batcov ratg (popular in yunnan province) with % whole genome similarity with sars-cov (calisher et al. ; zhang et al. ) . however, direct transmission of sars-cov from these bats was not considered as evidences from recent epidemics demonstrate that efficient human-to-human transmissibility while retaining high human pathogenicity is possible only with involvement of an intermediate host. according to the current evidence, virus isolated from malayan pangolins (manis javanica) celled pangolin-cov has . % and . % genetic similarity to sars-cov and batcov ratg , respectively, and hence contemplated to be one of the natural reservoirs of sars-cov . what are the symptoms of covid- ? for few people, covid- is mild with symptoms like fever, cough, fatigue, aches and nasal congestion and in others, covid- is asymptomatic. the severe stage of covid- occurs when the infection advances to ards and multiple organ failure mainly because of the inability to control the 'cytokine storm' or the wild cytokine production taking place in the body. increased age-related problems and medical complications like diabetes, blood pressure or heart diseases are observed to worsen covid- (tisoncik et al. ; abd el-aziz and stockand ; wang and mao ) . similarities of sars-cov and sars-cov : sars cov shows some similarities with sars cov. sars cov originated in the guangdong province of china in november . palm civets were identified as intermediary hosts for sars cov which contracted the virus from bats (falsey and walsh ) . sars-cov, however, was more `severe than covid- and produced influenza-like symptoms in humans like fever, myalgia, headache, diarrhea, shivering, cough and shortness of breath. who described the epidemic as a serial killer that spanned from th november to th september affecting individuals in different nations and causing death of people (mortality rate of . %) (who ) . presently, there are reports that sars-cov virus is undergoing mutations similar to sars-cov virus. in both cases, mutations have occured in the spike protein. this glycoprotein is important for the association of virus with angiotensin-converting enzyme- receptors (ace ) on cells of lung, intestine, liver, heart, vascular endothelium, testis, and kidney (hamming et al. ) . ace is part of the 'renin-angiotensin' hormonal system of the body. structural and biochemical studies conducted so far have shown that s protein of a sars virus comprises of functional subunits. (i) the s subunit or receptor binding domain is responsible for binding of virus to host cell receptor and (ii) s is responsible for fusion of virus with cellular membranes. both sub-units are separated but remain noncovalently bonded in a 'pre-fusion conformation' stabilized by s (andersen et al. ; walls et al. ) . although both sars-cov and sars-cov viruses have been observed to infect host cells via same ace receptor, there have been some differences noted like an increased affinity of the virus towards the receptor in sars cov . this has been presumed to be due to a mutation, i.e., n t in spike protein of sars-cov that has somehow increased the binding affinity of virus for ace (andersen et al. ) . very recent reports demonstrate that the s protein of pangolin-cov is similar to sars cov than ratg . there is evidence that key amino acid residues in spike protein that are in interaction with human ace are consistent between pangolin and sars-cov ; while, only amino acid mutations are present in ratg . nonetheless, both pangolin-cov and ratg have lost putative furin recognition at s /s cleavage site that is seen in sars cov . these data emerge from findings of liu et al. ( ) who detected corona viruses in lung-infected samples of dead malayan pangolins along with sendai virus from the guangdong wildlife rescue center of china which showed low identities ranging from . to . % with known sars-cov. what are the possible ways sars-cov infect a host cell? from the available data we have concise the information pertaining to sars-cov infection into host cell that is depicted as fig. . step entry of virus: viral entry is a fine interplay between the virion and the host cell and is initiated by association of the viral particle with specific proteins on the host cell surface. thereafter, the enveloped viruses fuse their envelope with the host cell membrane to deliver their nucleocapsid to the host cell. the s protein plays a dual role in this viral entry process by mediating receptor binding and membrane fusion, which involves large conformational changes of the s protein. during covid- , sars-cov s-protein binds to host cell's receptor ace (belouzard et al. ; andersen et al. ) step the activation of the virus: endosomal ph acidification is a fusion trigger for corona viruses and those like sars-cov rely on endosomal proteases for productive entry. as virus enters the host cell, it moves through the early and late endosomes that have low ph. in 'early' endosome, host proteases like cathepsin l facilitates cleavage of s protein in the s -s boundary region leading to fusion of the viral envelope. enzyme digestion experiments have shown that first cleavage event at the s -s boundary probably enables the second cleavage event at s ' region, that is responsible for fusion activation. sars-cov spike protein also exhibits a certain degree of plasticity in the position of the cleavage site for priming of fusion sars-cov. in 'late' endosome, the viral genome with its nucleocapsid is released into the host cytoplasm (cassell et al. ; yang et al. ; wang et al. ; belouzard et al. ) step viral replication: replication occurs in the cell cytoplasm and the positive-strand viral genomic rna is transcribed into a negative rna strand that is used as a template for the synthesis of viral mrna (devaux et al. ) step viral protein synthesis: orf a and orf b genes of viral genome produce two polyproteins (pps) that are pp a and pp b which takeover host ribosomes for their own translation and replication. as ribosome machinery of the infected cells moves in favor of the virus, it synthesizes non-structural proteins (nsps) for virus. these assemble into the replicase-transcriptase complex to support viral subgenomic mrna synthesis. nsp and nsp have specific functions such as suppression of host gene expression (schoeman and fielding ; devaux et al. ; prajapat et al. ) step transport across er and tgn (trans-golgi network): after replication, the envelope proteins such as m, e, and s are translated and gets transport towards the endoplasmic reticulum (er)-golgi intermediate compartment (ergic) complex to form the structure of viral envelope. replicated genome binds to n protein and forms the ribonucleoprotein (rnp) complex (prajapat et al. ) step assembly: viral genomic rna is then packed into the nucleocapsid and then envelope proteins are included during the budding step to form mature virions. the m protein, which localizes to the trans-golgi network, plays an essential role during viral assembly by interacting with the other proteins of the virus mrna (devaux et al. ) step budding and exocytosis: further after assembly, the virus particle comes out of the ergic via a budlike structure. the budding is determined by the localization of its membrane protein m and finally, newly formed viral particles are transported to the cell surface in vesicles and are released by exocytosis mrna (devaux et al. ) plasma membrane step step step step step step step the mechanism of viral infection is still not complete and under study. so far, the data show that the activation of the virus occurs as it enters the host cell. during this time, a cleavage process occurs at polybasic (furin) cleavage site at s -s boundary (formed by insertion of nucleotides), which is followed by an irreversible conformational change. through bioinformatics tool, researchers have identified the presence of a proline residue in the cleavage site and so the inserted sequence is "prra". presence of proline is predicted to cause addition of o-linked glycans to s , t and s that margin the cleavage site and produce a mucin-like domain that can shield epitope or key residues of sars-cov spike protein (andersen et al. ) . these observations reinforce the fact that sars-cov has been optimized for ace, but the increased virulence in some humans is not clear. previously it has been shown that in avian influenza, the speedy replication and transmission of virus in high population of chicken 'selects' for attainment of polybasic cleavage sites in a protein called hemagglutinin or ha (immunodominant viral antigen of h n virus). such acquirement of polybasic cleavage sites in hemagglutinin (insertion or recombination) has been observed to convert low pathogenic viruses to very high pathogenic ones (andersen et al. ) . ha has a similar function with the spike protein of sars-cov but whether a similar mechanism is operating in covid- needs to be validated. we have also seen that for sars-cov, the evolutionary starting point (as evidenced by palm civets) was a prototype virus group with low-pathogenicity with singlenucleotide variations (snv) which caused amino acid changes in the s protein. this group caused the early phase of the - epidemic. later, further changes, i.e., snvs caused amino acid residue changes that produced a "highly virulent group" which caused the middle phase of the epidemic of . thereafter, snvs caused four amino acid changes and brought about a group responsible for the late phase and the global epidemic. the neutral mutation rate of this virus during was almost constant, at around × − nt − day − , which was the same with the most recognized rna viruses. afterwards, a second interspecies jump recorded in late to early , caused resurgence sars in china. this was, however, thought to be an independent interspecies transmission event, instead of residual cases of the major epidemic because the virus had lower affinity of virus for human ace (cheng et al. ; kan et al. ; zhang et al. ) . such mutations may be operational in the case of sars-cov virus as well and, hence, the differences in severity we seen in italy, china, united kingdom, japan, usa and india. what governs the affinity of a virus towards the human host cell? as for the case of sars-cov, it was shown that the binding specificity of virus to host cell was due to prime amino acid residues in s protein at positions , , and . interestingly, the patient samples in the epidemic had n and t in their s protein, while most civet samples had k/r and s , which is a combination that reduces the affinity of virus for receptor. at the later stages of the epidemic ( ) ( ) , both the samples from humans and civet showed the presence of n and s , signifying an intermediate stage of mutation of the s protein. additional change to the n and t combination is believed have allowed competent human-to-human transmission. a different set of studies conducted using samples isolated from game animals (animals hunted for sports) from guangdong also showed high nucleotide similarity with sars-cov. these exhibited an insertion of -bp between orf a and orf b in animals, which was deleted either before or soon after crossing the species barrier to humans (cheng et al. ). we are yet to figure out what are the differences in amino acid residues of sars-cov that made it effectively adhere to the ace receptors on endothelial cells and lungs and other cells of the human body. today, governments face a very urgent need for measures to tame of the rapid spread of sars-cov . hence, clinicians and researchers across the globe are actively engaged in identifying effective therapeutic strategies to curb this deadly disease. to rush through the pandemic, one option researchers proposed was to 'repurpose' drug that has worked for similar diseases, till herd immunity is achieved (zumla et al. ; li and de clercq ) . when covid- was detected in wuhan, china resorted to test the effectiveness of anti-viral drugs and other infectious diseases against covid- . how long has it been since we know chloroquine (cq, -aminoquinoline)? it is believed that a scientist named hans andersag and his coworkers discovered a derivative of cq at bayers laboratories in (coatney ) . after a long line of controversy as a toxic agent, cq reinstated its importance in by proving its effectiveness against extra-intestinal amebiasis (conan ) . after the world war ii, who deployed cq as one of its principal arms for combating malaria and now cq is on the organization's list of essential medicines for the same. today, cq is known to be effective against malarial parasites of plasmodium genus such as plasmodium malariae, plasmodium ovale and plasmodium vivax but not against plasmodium falciparum owing to the resistance developed (mejia et al. ) . how does cq help eliminate malarial parasite? during malaria, the malarial pathogen attacks red blood cell and degrades hemoglobin in vacuoles of the parasitic cell and acquires those amino acids for constructing its proteins and energy metabolism. cq is basically a lysomotrophic agent and crosses plasma membrane and organelle membranes by simple diffusion process. once inside the acidic organelles, cq gets protonated and cannot leave the site by diffusion. cq, therefore, gets concentrated in the acidic organelles herein the parasitic vacuoles, changing the ph and facilitating the formation of a complex of 'heme and chloroquine' that prevents parasites of plasmodium genus from drawing nutrients from the red blood cell. cq can be transported out of cells via a protein called multi-drug resistance protein or mrp- , an atp binding protein (mejia et al. ; mauthe et al. ) . does cq have anti-viral effects? literature cites that cq exerts effects against a battery of different viruses such as flaviviruses, retroviruses and corona viruses. cq and hcq have demonstrated anti-viral potency against herpes simplex virus type , zika, hiv, mers, sars-cov, hcov-oc , chikungunya and hepatitis c (picot ). of the different mechanisms of anti-viral action known for cq, the most intriguing one is its ability to inhibit viruses at the entry point of host cell which happens in the case of borna disease virus, minute virus of mice mvmp and the avian leucosis virus (savarino et al. ). in the case hepatitis a virus, cq inhibits uncoating of the virus and, thus, blocking its entire replication cycle (bishop , devaux et al. . once inside the cell, cq works by inhibiting the replication of a virus by altering the ph of acidic organelles that are sites of viral dna replication (devaux et al. ) . apart from blocking viral entry and viral replication, cq can also block the final packaging process in certain viruses. eg, in the case of infection caused by enveloped viruses like mayaro viruses, cq administration causes accumulation of the base in endoplasmic and trans-golgi compartments of the cell. this affects the functioning of certain low ph-dependent proteases and glycosyl-transferases which are required for formation of viral envelopes (savarino et al. ) . in flaviviridae viruses, cq affects normal proteolytic processing of flavivirus prm protein to m protein (savarino et al. ) . for non-infectious retrovirus particles, as shown with the avian reticuloendotheliosis virus, cq inhibits glycosylation of envelope glycoproteins (savarino et al. ) . again in the case of hiv, cq reduces the production of the heavily glycosylated epitope g , which is located on the gp envelope glycoprotein surface which is fundamental for virus infectivity. again, these effects are likely to be attributed to the increased ph in trans-golgi network, which impairs the function of glycosyl-transferases involved in the post-translational processing of the hiv glycoproteins (savarino et al. ) . how effective can be cq against sars? in , nichol et al. reported the anti-viral effects against sars-cov infection in primate vero e cells (derived from the african green monkey kidney) (vincent et al. ) . thereafter, it was found that hydroxyl chloroquine (hcq, which has % lesser adverse effects than cq) has a better anti-sars-cov- activity than cq in vero cells liu et al. ; wang and cheng ). an interesting mode of action of both cq and hcq is their ability to inhibit binding of sars-cov- viral particles to ace without affecting the cellular level of ace expression. this was basically via an interference with the glycosylation on ace receptor, which is required for ligand recognition. this was ensured via inhibition of an enzyme called quinone reductase- which is vital for biosynthesis of sialic acids and thereby glycosylation process. ace when not in the glycosylated state is less efficient to interact with the sars-cov- spike protein and is one of the best ways to inhibit viral entry (vincent et al. ; wang and cheng ) . cq and hcq also have a track record of reducing over-production of cytokines during rheumatoid arthritis, lupus erythematosus, and sarcoidosis (rebecca et al. ; schrezenmeier and dörner ) . these immunomodulatory effects of cq and hcq need highlight since they may be useful for taming the cytokine storm in the covid- scenario (conti et al. ) . one of the proposed modulatory effect of hcq is its ability to inhibit the activity of the nucleic acid sensor cyclic gmp-amp (cgamp) synthase (cgas) by interfering with its binding to cytosolic dna, and by stopping tlr signaling and cgas-stimulator of interferon genes (sting) signaling (picot ) . at the cellular level, cq and hcq have been shown to inhibit immune activation by reducing signaling by pattern recognition receptors (toll-like receptor signaling) and cytokine production. both cq and hcq reduce the secretion of pro-inflammatory cytokines and, in particular, tnf-α in macrophages. different mechanisms have been proposed to explain the reduction in tnf-α by cq and hcq like (i) inhibition of tnf-α mrna expression, (ii) inhibition in posttranslational conversion of pro-tnfα to a soluble mature form, (iii) a pre-translational stage by a non-lysosomotropic mechanism and (iv) reduced surface expression of tnf-α receptors. apart from tnf-α, cq also suppresses cytokines like il- , il- and ifnγ in mononuclear cells, again by modulating tlr (totura et al. ; lester and li ; conti et al. ) . in light of the available references, the plausible interferences by cq or hcq are depicted in fig. . interference of virus binding to host cell: (i) the drug (cq or hcq) interferes with ace receptor glycosylation preventing sars-cov binding to target cells. (ii) drug limits the biosynthesis of sialic acids required for cell surface binding of sars-cov- (ii) interference in viral transport: drug modulates the acidification of endosomes thereby inhibiting transport and formation of the autophagosome (yang et al. ) (iii) interference in viral envelope formation: drug inhibits the cathepsin activity in early endosome of host by increasing endosomal ph which further blocks cleavage of virus envelops s protein leading to stop fusion of the viral envelope (wang et al. , cassell et al. ) (iv) interference to mitogen-activated protein (map) kinase pathway: by modulating cellular mitogenactivated protein (map) kinase activation, hcq may also inhibit virus replication (savarino et al. ) (v) modulating m protein: hcq may alter m protein maturation and interfere with virion assembly and budding (prajapat et al. ) (vi) interference to tlr signaling pathway: elevation of endosomal ph by cq interferes with tlr and tlr processing, hence pathogen recognition via toll-like receptor (tlr) and thereby viral infection (devaux et al. ) the mechanism of action as detailed above for cq or hcq is from literature but this warrants further investigations. now, the above-described abilities of cq or hcq to function as anti-viral and immunomodulatory agent highlight the rationale for usage of cq or its derivate as both prophylactic and therapeutic agents in clinically admissible concentrations (shah et al. ) . a small randomized clinical trial conducted in china with patients showed that cq had superior effects in inhibiting exacerbation of pneumonia, improving lung-imaging findings, promoting a virus-negative conversion and shortening the disease course when compared to a control drug (jie et al. ; gao et al. ) another clinical trial in marseille, france reported by gautret et al. ( ) demonstrated that hcq has efficacy in increasing viral clearance. in that non-randomized trial (n = ) conducted, hcq alone and in combination with azithromycin antibiotic effectively cleared viral nasopharyngeal carriage as evidenced by the gold standard pcr (polymerase chain reaction) test (versus control). the virus clearance at day post-inclusion hcq vs. control was . % versus . %, respectively (p = . ). a higher virus clearance effect was seen with a combinational therapy of azithromycin and cq. this combinational therapy was effective because azithromycin is an anti-viral agent that has shown efficacy against zika and ebola viruses (gautret et al. ) . however, all clinical results did not show the same picture. in a randomized study from china, patients with mild to moderate cases of covid- did not show recovery rates when treated with hcq ). likewise, another study conducted in france with hospitalized cases also failed to confirm anti-viral activity of hcq when administered with an azithromycin (yazdany and kim ) . a reason for the discrepancy in effect as noted among these clinical trials could be the dosage of cq or hcq selected; a higher dose of cq or hcq may be imparting untoward implications, especially if not monitored. the current doses tested may be with respect to the antimalarial effects of cq and hcq when the body encounters a deadly pathogen of plasmodium genus or even with respect to the other viral infections that it was tested with. since cq is effective at blocking viral entry, it would have better effects as a prophylactic or for patients with mild symptoms. we also see that trials were conducted during the different stages of the pandemic; at severe stages, perhaps, a different therapeutic regimen is required. upon further mutations with time (that is seen in the different stages of a pandemic), the adherence propensity of the virus towards host cells may change (increased) that cq or hq alone may not be not alone sufficient as an antiviral therapy. at the same time, a toxicity profile of cq or hcq in combination with azithromycin versus azithromycin and cq or hcq should be conducted to rule out the toxicity arising from combinational therapy (gautret et al. ) . additional research to carry out research on cq and hcq is emphasized because unlike other drugs, apart from the ability to block viral infection, these drugs also have immunomodulatory effects which is very much useful for countering the initiation of the cytokine storm seen in covid- patients (conti et al. ) . although this review focuses on the beneficial effects of cq, we do not overlook some of the precautions as evidenced from clinical studies (van den broek et al. ) . during the course of the prophylactic or therapeutic regimen involving cq or hcq, frequent monitoring of hematological parameters (rbc, wbc and platelet counts), measurement of serum electrolytes, blood glucose (owing to the hypoglycemic potential of hcq), liver function test, kidney function test and electrocardiography are warranted at time intervals to rule out any toxicity. combinational therapy of cq with any other allopathic drug be it azithromycin, lopinavir/ ritonavir and remdesivir is warranted only after testing in in vitro model systems and pre-clinical trials. clinical experience has shown that chloroquine is well absorbed in the body and distributes widely in the system. it has an apparent and terminal half-life of . days and weeks, respectively (smit et al. ) . the drug is metabolized by cytochrome p and renal clearance is responsible for one-third of total clearance of chloroquine (smit et al. ) . so with lower dose, it is probable that chloroquine can be used safely for an acute virus infection. but some of the potential side effects of hcq and cq, such as retinopathy, vomiting, diarrhea and increased risk of arrhythmia, should be also taken into account (conti et al. ) and we presume that these may be countered by a combinational therapy wherein the beneficial effect of cq may be hcq associated with azithromycin ( mg at day followed by mg once daily the next days) therapeutic hcq and azithromycin as a treatment of covid- : results of an open label nonrandomized clinical trial (gautret et al. ) cq ( mg) once weekly alone or in combination with proguanil mg daily prophylactic compliance and tolerability of mefloquine and chloroquine plus proguanil for longterm malaria chemoprophylaxis in groups at particular risk (the military) (peragallo et al. ) cq ( mg) daily or hcq ( mg) weekly prophylactic protecting chinese healthcare workers while combating the novel coronavirus (zhou et al. ) cq ( mg) once weekly prophylactic landscape analysis of therapeutics as st march (who a) cq ( mg) twice daily for days prophylactic expert consensus on chloroquine phosphate for the treatment of novel coronavirus pneumonia (jie et al. ) hcq ( mg) twice daily, followed by once weekly prophylactic indian council of medical research. advisory on the use of hydroxy-chloroquine as prophylaxis for sars-cov- infection (icmr , kalantri et al. ) cq ( mg) or hcq ( mg) prophylactic systematic review of hydroxychloroquine use in pregnant patients with autoimmune diseases (shah et al. ) hcq ( mg/kg/day (loading dose) followed by mg/kg/day), with a maximum limit of mg/day post exposure coalition: advocacy for prospective clinical trials to test the post exposure potential of hydroxychloroquine against covid- (picot ). retained and the toxicity of cq may be alleviated. table gives framework of some of the research work published using cq or hcq with suggested drug doses for treatment regimen of covid . while this article was under revision, we came across an extensive collaborative study by picot ( ) , which stated that in the current context of the severe worldwide health emergency, it is practical for clinicians to use hcq even if it can only reduce viral load by - %. they mentioned that these are effective measures in reducing covid- spread and improving clinical outcome in health care workers. they have stated that hcq at doses matching that of the standard treatment of systemic lupus erythematous [which has proven safety and efficacy in terms of hcq blood and tissue concentration adapted to bodyweight at mg/kg/day (loading dose) followed by mg/kg/day, with a maximum limit of mg/day in all cases] can be used as a drug for exposed people. the duration of hcq treatment can be as long as contact with a positive patient lasts. in the case of repeated exposure to virus, then treatment is suggested to be for a min of days so that there is a steady state of drug concentration in blood (picot ) . we also include in this article some details of our pilot study where we tested the effect of a combination of cq (sigma aldrich, usa) and betulinic acid (sigma aldrich, usa) on lysosomal function in mononuclear cells, in the absence of a pathogen. ba ( β-hydroxy-lup- ( )-en- -oic acid) is a triterpenoid belonging to lupane series which has a wide range of pharmacological properties like topoisomerase inhibitory potential (chowdhuri et al. ) , anti-cancer (ren et al. ), anti-malarial (de sa et al. ), antiviral (pavlova et al. ) and anti-inflammatory properties (chowdhuri et al. ; vijayan et al. ) . ba can be extracted from bark of birch tree and many other plant sources (moghaddami and ahmad ) . purified form of botanicals is a rich resource for novel antiviral drugs as these can interact with different stages of the viral life cycle, such as viral entry, replication, assembly, and release, as well as on the targeting of virus-host-specific interactions. cq is itself a synthetic form of quinine isolated from barks of barks of cinchona trees native to peru. so, it was of interest to check whether ba and cq exerted an additive effect on lysosomal function. briefly, mononuclear cells were isolated from healthy volunteers by density gradient centrifugation as detailed in vijayan et al. ( ) . blood collected on heparinized tubes were loaded on histopaque- (sigma aldrich, usa) and centrifuged at rpm for min. the buffy coat layer at interface was retrieved and plated on type i collagen coated cell culture dishes containing rpmi- media containing % fbs. after h, the media were carefully changed to remove the floating lymphocytes. the adherent mononuclear cells were cell cultured till plate reached - % confluency ( - days). cells were then shifted to media with autologous serum and exposed to a standardized concentration of chloroquine (cq, . micromolar) or betulinic acid (ba, ng/ml) and a combination of both (cq + ba). ba was pre-treated for min prior to cq addition that was for h. β-hexosaminidase activity was determined in cell lysates by colorimetric assay as previously described by lew and rarrazzi ( ) . lysed cell extracts prepared by repeated freeze-thaw cycles from basal, cq-, ba-and cq + ba-treated cell cultures were incubated with p-nitrophenyl- -deoxy-β-d-glucosaminide ( mm) in . -m citrate/phosphate buffer, ph . for h. the p-nitrophenol released by the enzyme-dependent hydrolysis of the substrate was quantified spectrophotometrically (shimadzu, japan) at -nm wavelength. units of activity were defined as nanomoles of substrate per hour per mg protein. results of the study demonstrate that a combination of ba and cq has greater effect in reducing lysosomal activity as compared to cq or ba (fig. ). this is a pilot experiment conducted in the absence of a cargo. cq is known for inhibiting autophagy by impairing autophagosome fusion with lysosomes rather than by affecting the acidity and/or degradative activity of this organelle (mauthe et al. ) . so, the reason for reduction in activity of lysosome enzyme by cq as seen in our pilot study requires investigation. we are assuming reduction in the count and activity of lysosomes with cq exposure. but whether ba is potentiating the effect or not requires more sophisticated experimentations which is a futuristic aspect. we are hoping that if we can lower the dose of cq by including ba, such combinational therapy basal c q c q+ba ba beta-hexosaminidase activity fig. the effect of a combination of betulinic acid and chloroquine on lysosomal function as evidenced by beta-hexosaminidase activity in mononuclear cells. units of activity were defined as nanomoles of substrate per hour per mg protein. the results are a mean of experiments where each experiment was conducted in duplicate. statistical analysis was conducted by anova followed by duncan test using spss software can be considered for improving efficacy of treatment in conditions of pathogen overload. cq has been in pharmaceutical industry for years now. it has low cost and is reasonably safe to use and widely available in countries where malaria is endemic, but cq usage warrants frequent monitoring of physiological parameters to avoid any adverse effects. cq has distinct ways of attacking a virus. cq interferes with the glycosylation of cellular receptor of sars-cov to block viral infection. cq inhibits the quinone reductase- , which is involved in sialic acid biosynthesis required for ligand recognition. cq changes the ph of lysosomes and likely inhibits cathepsins that lead to the formation of the autophagosome which cleaves sars-cov- spike protein. the potency of cq can be increased when used with an anti-viral agent. the government of india has put up a platform for identifying the use of botanicals to treat disorders. in that context, the effect of a botanical like betulinic acid was tested. this botanical has reported that anti-viral effect may aid to reduce the dose of cq for prophylactic or therapeutic regimen. pre-clinical and clinical studies are required to confirm this. recent progress and challenges in drug development against covid- coronavirus (sars-cov- )-an update on the status surviving sepsis campaign: guidelines on the management of critically ill adults with 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vitro against plasmodium falciparum and in vivo in p. bergheiinfected mice new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid- ? discovering drugs to treat coronavirus disease (covid- ) mount sinai health system treatment guidelines for sars-cov- infection (covid- novel coronavirus and severe acute respiratory syndrome breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial tissue distribution of ace protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis caution and clarity required in the use of chloroquine for covid- . www.thela ncet.com/rheum atolo gy advisory on use of hydroxy-chloroquine as prophylaxis for sars-cov- infection expert consensus on chloroquine phosphate for the treatment of novel coronavirus pneumonia hydroxychloroquine prophylaxis for covid- contacts in india molecular evolution analysis and geographic investigation of severe acute respiratory syndrome coronavirus-like virus in palm civets at an animal market and on farms toll-like receptors in antiviral innate immunity mitogenic effect of lysosomal hydrolases on bovine tracheal myocytes in culture therapeutic options for the novel coronavirus ( -ncov) viral metagenomics revealed sendai virus and corona virus infection of malayan pangolins (malaya javanica) hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion efficacy of chloroquine for the treatment of uncomplicated plasmodium falciparum malaria in honduras welfare and sport ( ) national institute for public health and the environment molecular virology various botanical sources of betulinic acid: a review antiviral activity of betulin, betulinic and betulonic acids against some enveloped and non-enveloped viruses compliance and tolerability of mefloquine and chloroquine plus proguanil for longterm malaria chemoprophylaxis in groups at particular risk (the military) coalition: advocacy for prospective clinical trials to test the post exposure potential of hydroxychloroquine against covid- drug targets for corona virus: a systematic review ppt promotes tumor growth and is the molecular target of chloroquine derivatives in cancer inhibition of betulinic acid to growth and angiogenesis of human colorectal cancer cell in nude mice effects of chloroquine on viral infections: an old drug against today's diseases? coronavirus envelope protein: current knowledge mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology a systematic review of the prophylactic role of chloroquine and hydroxychloroquine in coronavirus disease- (covid- ) chloroquine for sars-cov- : implications of its unique pharmacokinetic and safety properties into the eye of the cytokine storm toll-like receptor signaling via trif contributes to a protective innate immune response to severe acute respiratory syndrome coronavirus infection betulinic acid inhibits endotoxin-stimulated phosphorylation cascade and pro-inflammatory prostaglandin e( ) production in human peripheral blood mononuclear cells chloroquine is a potent inhibitor of sars coronavirus infection and spread david veesler d ( ) structure, function, and antigenicity of the sars-cov- spike glycoprotein increasing host cellular receptor-angiotensin-converting enzyme (ace ) expression by coronavirus may facilitate -ncov infection the pathogenesis and treatment of the cytokine storn in covid- sars coronavirus entry into host cells through a novel clathrin-and caveolae-independent endocytic pathway update -sars: chronology of a serial killer landscape analysis of therapeutics as st covid- ) pandemic. report of a who scientific group ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) use of hydroxychloroquine and chloroquine during the covid- pandemic: what every clinician should know probable pangolin origin of sars-cov- associated with the covid- outbreak protecting chinese healthcare workers while combating the novel coronavirus coronaviruses-drug discovery and therapeutic options publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - bdzut d authors: jha, ashish k.; kumar, ravikant; goenka, mahesh k.; dayal, vishwa m. title: emerging treatment and prevention strategies against covid- : a brief update date: - - journal: nan doi: . /s- - sha: doc_id: cord_uid: bdzut d patients with novel coronavirus disease (covid- ) are at significantly increased risk for mortality and morbidity. current management remains supportive care, ranging from symptomatic outpatient management to full–intensive care support, including intravenous fluids, invasive, and non-invasive oxygen supplementation. in patients with septic shock, treatment with antibiotics and vasopressors are recommended to keep mean arterial pressure (map) ≥ mm hg and lactate < mmol/l. because of the lack of effectiveness and possible adverse effects, routine corticosteroids should be avoided unless they are indicated for another reason (exacerbation of asthma or chronic obstructive pulmonary disease [copd], and septic shock in whom fluids and vasopressors do not restore hemodynamic stability). there is currently no sufficient evidence of efficacy of hydroxychloroquine/chloroquine, remdesivir, and other antivirals in the treatment or prevention of covid- . limited evidence shows that covid- convalescent plasma can be used as a treatment of covid- without the occurrence of severe adverse events. drug regulatory agencies granted an emergency-use authorization of chloroquine/hydroxychloroquine and remdesivir to treat patients when a clinical trial is not available or participation is not feasible. chloroquine and hydroxychloroquine are associated with qt interval prolongation and life-threatening cardiac arrhythmia in patients with pre-existing cardiovascular disease. guidelines are issued for use of convalescent plasma in patients with serious or immediately life-threatening covid- . data from several ongoing randomized controlled trials will provide further evidence regarding the safety and efficacy of these drugs for the treatment of covid- . the current novel coronavirus disease (covid- ) pandemic, caused by a novel severe acute respiratory syndrome-coronavirus- (sars-cov- ), is a rapidly spreading viral disease. patients with covid- are at significantly increased risk for mortality and morbidity. since the first report of sars-cov- infection, it has spread to more than countries around the world ( , , active cases and , death [till april , ]). to date, india has recorded , active cases and death (till april , ). there is currently no specific treatment of covid- . current management remains supportive care, ranging from symptomatic outpatient management to full intensive care support, including intravenous fluids, invasive and noninvasive oxygen supplementation, and antibiotics. the objective of this paper is to briefly review the literature and update the concept of prevention and treatment of covid- . we have highlighted here the potential therapeutic role of remdesivir, chloroquine/ hydroxychloroquine (hcq), lopinavir/ritonavir, and convalescent plasma in patients with sars-cov- infection. in the absence of a proven therapy for sars-cov , the cornerstone of therapy for patients with covid- remains supportive care. to suppress the inflammatory response which may lead to acute lung injury and ards. in a retrospective study (n = ), treatment with methylprednisolone was associated with a decreased risk of death ( % with steroids vs. % without). however, the authors noted that confounding bias may exist in this observational study. because of the lack of effectiveness and possible adverse effects, routine corticosteroids should be avoided unless they are indicated for another reason (exacerbation of asthma or copd and septic shock in whom fluids and vasopressors do not restore hemodynamic stability). , remdesivir is a nucleotide analogue prodrug that inhibits viral rna polymerases. it has broad-spectrum activity against rna viruses such as coronaviridae (e.g., sars-cov and middle east respiratory syndrome coronavirus [mers-cov]) and filoviruses (e.g., ebola). it has shown prophylactic and therapeutic efficacy in nonclinical models of these coronaviruses; however, there are currently only very limited data on the use of remdesivir in patients with covid- . in a recent multicentric study, patients with confirmed sars-cov- infection (n = ) who had an oxygen saturation of % or less with or without receiving oxygen support were enrolled. besides supportive care, patients received remdesivir, consisting of mg administered intravenously on day , followed by mg daily up to days. during a median follow-up of days, % had an improvement in oxygen-support class, including % patients receiving mechanical ventilation. however, interpretation of the result of this study is limited by the lack of a randomized control group, small sample size, exclusion of serious cases (creatinine clearance < ml/min and >five-time elevation of serum aminotransferase), variable duration of remdesivir administration, noncollection of viral load data, adverse effects, and short-term follow-up. a total of % patients reported adverse events during follow-up. the result of a study (simple trial; unpublished) demonstrated that patients receiving a -day course of remdesivir achieved similar clinical improvement compared with those taking a -day treatment course on day . the european medicines agency (ema) has published recommendations on compassionate use of remdesivir for covid- . data from several ongoing randomized controlled trials will provide further evidence regarding the safety and efficacy of remdesivir for covid- . the antimalarial and anti-inflammatory agents, chloroquine and hcq, appear to block viral entry into cells by inhibiting glycosylation of host receptors, proteolytic processing, and endosomal acidification. these agents also have immunomodulatory effects through attenuation of cytokine production and inhibition of autophagy and lysosomal activity in host cells. , both drugs have in vitro activity against sars-cov- , with hcq having relatively higher potency. early results obtained from more than patients enrolled in studies conducted in the china showed the superiority of chloroquine compared with the controls in terms of reduction of exacerbation of pneumonia, duration of symptoms, and delay of viral clearance, all in the absence of severe side effects. a recent open-label study of patients reported improved virologic clearance with hcq (n = ) compared with the controls (n = ). addition of azithromycin to hcq in six patients resulted in superior viral clearance ( %) compared with hcq monotherapy ( %). an uncontrolled observational study in a cohort of covid- inpatients (n = ) treated with a combination of hcq and azithromycin showed a rapid fall of nasopharyngeal viral load ( % at day ). in a randomized parallel-group trial, covid- patients (n = ) were assigned to receive an additional -day hcq ( mg/d) treatment. clinical recovery time was significantly shortened in the hcq treatment group. improvement in pneumonia was more in the hcq treatment group ( . %) compared with the controls ( . %). however, the patients had mild disease. in a prospective study (n = ), patients were randomized to hcq ( mg/day for days) plus standard of care and standard care alone groups. virologic clearance was similar in both the groups. a study of patients with covid- reported persistence of sars-cov- in the nasopharyngeal swab in of patients receiving hcq. in a randomized, double-blinded, phase-iib clinical trial (clorocovid- study), preliminary findings suggest that the higher chloroquine dosage is associated with higher rate of qtc prolongation and mortality. studies of chloroquine prophylaxis in health care workers (nct ) and hcq for postexposure prophylaxis after high-risk exposures (nct ) are planned or enrolling. some studies show no benefit at all, while others show a benefit of taking hcq/chloroquine. some of the aforementioned clinical studies showed hcq/chloroquine as promising agents for the treatment of mild-to-severe covid- . however, these trials have been criticized for their limitations, including small sample size, nonuniform study design, absence of control arm, and enrolment of less severe cases. , , , results from other clinical studies could not replicate these positive findings. , , therefore, there is currently no strong evidence of efficacy of hcq or chloroquine in the treatment or prevention of covid- . chinese and italian guidelines recommend early use of hcq or chloroquine for the treatment of covid- on case-to-case basis; however, this is based on weak evidence. the food and drug administration (fda) and ema has granted an emergency-use authorization for chloroquine and hcq to treat patients when a clinical trial is not available or participation is not feasible. , indian council of medical research (icmr) recommends the use of hcq for prophylaxis of sars cov- infection in high-risk cases. qt-prolongation and life-threatening cardiac arrhythmia is major concern in patients receiving hcq with or without azithromycin, especially in patients with preexisting cardiovascular disease. lopinavir/ritonavir demonstrated in vitro activity against other corona viruses via inhibition of -chymotrypsin-like protease. results of a few small case series showed clinical benefit of using with lopinavir/ritonavir in patients with covid- . however, an open-label randomized trial at a single hospital concluded that lopinavir/ritonavir recipients and those receiving standard care did not differ significantly in time to clinical improvement (median, days), duration of intensive care unit stay, and duration of mechanical ventilation/oxygen supplementation. at present, there is insufficient evidence to recommend the use of lopinavir/ritonavir for treatment of covid- . convalescent plasma from patients who have recovered from viral infections has been used as a treatment in previous virus outbreaks including sars and ebola virus. convalescent plasma or immunoglobulins were used in patients with sars whose condition continued to deteriorate despite treatment with intravenous corticosteroids. a systematic review and exploratory meta-analysis from studies of sars coronavirus infection and severe influenza showed a statistically significant reduction in the pooled odds of mortality following treatment with convalescent plasma compared with placebo (odds ratio = . ; % confidence interval [ci]: . - . ; i[ ] = %). study showed consistent evidence for a reduction in mortality, especially when convalescent plasma is administered early after symptom onset. no serious adverse events were seen. however, studies were commonly of low or very low quality, lacked control groups, and at moderate or high risk of bias. luke et al identified eight studies involving , patients with spanish influenza pneumonia treated with influenza-convalescent human blood products infusion, which showed a pooled absolute reduction of % (p < · ) in the overall crude case-fatality rate. mortality rate was lower in patients who received early treatment (after < days of complications) compared with late treatment group (after > days of complications; vs. % [pooled risk difference = % (ci: - %)]). convalescent plasma has also been tried in treatment of covid- . a few small preliminary case series showed clinical improvement after treatment with convalescent plasma; however, these studies had many limitations. , limited evidence shows that convalescent plasma can be used as a treatment of covid- without the occurrence of severe adverse events. convalescent plasma that contains antibodies to sars-cov- might suppress viremia. the patient usually develops a primary immune response by days to , which is followed by virus clearance. therefore, logically, it should be more effective to transfuse the convalescent plasma at the early course of disease. studies showed lower mortality rate in whom convalescent plasma is administered early after symptom onset. , the fda has issued guidance for use of covid- convalescent plasma in patients with serious covid- . about to weeks after a person recovers, blood can be drawn to look for the antibodies. the approach involves giving covid- patients an infusion of antibody-rich plasma (single dose of ml) from people who have recovered from an infection with sars-cov- . the rapidly expanding knowledge regarding sars-cov- virology provides a significant number of potential drug targets. potential mechanisms of action of antiviral agents are ( ) virus-based therapy, like monoclonal antibodies or antiviral peptides, targeting the various steps of viral machinery-like spike glycoprotein, viral enzyme inhibitors, and viral nucleic acid synthesis inhibitors, and ( ) host-based therapies to potentiate the interferon response, affect host signalling pathways, or host factors utilized by sars-cov- for replication. the world health organization (who) has started an international multicentric clinical trial ("solidarity"). this trial comprises of four treatment groups (remdesivir, chloroquine/hydroxychloroquine, lopinavir with ritonavir, and lopinavir with ritonavir plus interferon β- a) against standard of care, to assess their relative effectiveness against covid- . several therapies are currently being investigated globally, including nitazoxanide, stem cell therapy, intravenous immunoglobulin, interleukin- receptor antagonist (e.g., tocilizumab, sarilumab, and siltuximab), bacille calmette-guerin (bcg) vaccine, angiotensin-ii receptor antagonists, and other antivirals (e.g., oseltamivir, danoprevir, darunavir, ganciclovir, favipiravir, baloxavir marboxil, umifenovir, sofosbuvir, ribavirin, and interferon alfa/β). the most effective long-term strategy for prevention of future outbreaks of this virus would be the development of a vaccine providing protective active immunity. as of april , , the global covid- vaccine research and development landscape included vaccine candidates. out of confirmed active projects, are currently at preclinical stages. a few vaccine candidates have moved into clinical trial phase, including mrna- (moderna), ad -ncov (cansino biologicals), ino- (inovio), lv-smenp-dc, and pathogen-specific aapc (shenzhen geno-immune medical institute). unfortunately, a vaccine is unlikely to be ready for use before the end of or at the earliest. who and centers for disease control and prevention guidance emphasizes the role of supportive care based on severity of illness, ranging from symptomatic treatment for mild disease to evidence-based ventilator management for ards, and early recognition and treatment of bacterial infections and sepsis in critically ill patients. use of routine systematic corticosteroids should be avoided for the treatment of covid- . there is currently no strong evidence of efficacy of hcq/chloroquine and remdesivir or other antivirals in the treatment or prevention of covid- . the fda and ema has granted an emergency-use authorization for chloroquine/ hcq to treat patients when a clinical trial is not available or participation is not feasible. the icmr recommends the use of hcq for prophylaxis of sars-cov- infection in high-risk cases. the ema recommends use of remdesivir for covid- on a compassionate basis. guidelines are issued for use of convalescent plasma in patients with serious or immediately life-threatening covid- . data from several ongoing randomized controlled trials will provide further evidence regarding the safety and efficacy of these drugs for covid- . world health organization. country & technical guidance -coronavirus disease (covid- ) clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in covid- patients with at least a six-day follow up: a pilot observational study risk factors associated with acute respiratory distress syndrome and death in patients with corona virus disease pneumonia in wuhan, china compassionate use of remdesivir for patients with severe covid- covid- : a recommendation to examine the effect of hydroxychloroquine in preventing infection and progression new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid- ? breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial efficacy of hydroxychloroquine in patients with covid- : results of a randomized clinical trial a pilot study of hydroxychloroquine in treatment of patients with common coronavirus disease- (covid- ) treating covid- -off-label drug use, compassionate use, and randomized clinical trials during pandemics hydroxychloroquine in the management of critically ill patients with covid- : the need for an evidence base no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe covid- infection food and drug administrationrequest for emergency use authorization for use of chloroquine phosphate or hydroxychloroquine sulfate supplied from the strategic national stockpile for treatment of coronavirus disease a trial of lopinavir-ritonavir in adults hospitalized with severe covid- convalescent plasma study group. the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis meta-analysis: convalescent blood products for spanish influenza pneumonia: a future h n treatment? convalescent plasma as a potential therapy for covid- treatment of critically ill patients with covid- with convalescent plasma coronavirusesdrug discovery and therapeutic options the covid- vaccine development landscape none declared. key: cord- -kufs fxe authors: malviya, amit title: the continued dilemma about usage of hydroxychloroquine: respite is in randomized control trials date: - - journal: int j infect dis doi: . /j.ijid. . . sha: doc_id: cord_uid: kufs fxe nan is notable that in the present study, concomitant use of steroid in patients receiving hydroxychloroquine, was more than non-treated group. so, in absence of this critical information about the duration between the onset of symptoms and administration of first dose of hcq, it seems difficult to interpret that the positive effects were due to hcq or steroids. hcq is touted for treatment of covid primarily based on its anti-viral properties, thus the timing of administration becomes very important for a meaning full assessment of study results. secondly, hcq concentrates in lungs after initial doses and that is the time when its effect is supposed to be maximum. administration of hcq late in the course of disease may not be that effective. thirdly, they utilised maximal modified sequential organ failure assessment (msofa )scores for classifying patient severity of disease . recently it been shown that this score is not accurate for predicting severity of disease in covid patients . fourthly , a qtc interval-based algorithm specifically designed to ensure the safe use of hydroxychloroquine was utilised . this is a very safe practice but it might have resulted in exclusion of high cardiac risk patients . covid is a multisystem disease and the disease itself promotes proarrhythmic milieu with prolonged qt intervals at baseline . , risk assessment of hcq therapy is not complete if such patients are excluded. finally , mechanism of action of hcq against is a part of its broad anti-viral and immunomodulatory properties and no specific pharmacologic actions are described for sars-cov- infection. , majority of publications in the recent times on hcq , for usage in covid , are limited by low methodical quality and by and large have shown negative or neutral results . , weather hcq as initial anti-viral agent prevents progression to severe disease is not known clearly . in severe disease with multi system involvement and pro arrhythmic milieu covid- : immunopathology and its implications for therapy pharmacologic treatments for coronavirus disease (covid- ): a review mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology critically ill sars-cov -infected patients are not stratified as sepsis by the qsofa chloroquine and hydroxychloroquine in covid- ventricular arrhythmia risk due to chloroquine / hydroxychloroquine treatment for covid- : should it be given in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) effects of chloroquine on viral infections: an old drug against today's diseases? the lancet. infectious diseases the urgency of care during the covid- pandemic -learning as we go covid- coronavirus research has overall low methodological quality thus far: case in point for chloroquine/hydroxychloroquine key: cord- -et ekgdl authors: yazdany, jinoos; kim, alfred h.j. title: use of hydroxychloroquine and chloroquine during the covid- pandemic: what every clinician should know date: - - journal: ann intern med doi: . /m - sha: doc_id: cord_uid: et ekgdl two medications often used for treatment of immune-mediated conditions, hydroxychloroquine and chloroquine, have recently attracted widespread interest as potential therapies for coronavirus disease . the authors of this commentary provide guidance for clinical decision making for patients with coronavirus disease as well as for patients with rheumatologic conditions, such as systemic lupus erythematosus and rheumatoid arthritis i n the desperate search to find effective treatments for coronavirus disease (covid- ), generic drugs, used largely by rheumatologists and dermatologists to treat immune-mediated diseases, have entered the spotlight. the antimalarials hydroxychloroquine (hcq) and chloroquine (cq) have demonstrated antiviral activity against severe acute respiratory syndromecoronavirus (sars-cov- ) in vitro and in small, poorly controlled or uncontrolled clinical studies ( ) ( ) ( ) . normally, such research would be deemed hypothesisgenerating at best. a tweet by president trump on march claiming that the combination of hcq and azithromycin "ha[s] a real chance to be one of the biggest game changers in the history of medicine" accelerated a worldwide run on the drugs, with pharmacies reporting shortages within hours. here, we try to provide guidance regarding clinical decision making both for patients with covid- and those with immune-mediated conditions, such as systemic lupus erythematosus (sle) and rheumatoid arthritis (ra), and strategies to mitigate further harm to these patients. data to support the use of hcq and cq for covid- are limited and inconclusive. the drugs have some in vitro activity against several viruses, including coronaviruses and influenza, but previous randomized trials in patients with influenza have been negative ( , ) . in covid- , one small nonrandomized study from france ( ) (discussed elsewhere in annals of internal medicine [ ] ) demonstrated benefit but had serious methodological flaws, and a follow-up study still lacked a control group. yet, another very small, randomized study from china in patients with mild to moderate covid- found no difference in recovery rates ( ) . sadly, reports of adverse events have increased, with several countries reporting poisonings and at least death reported in a patient who drank fish tank cleaner because of its cq content. antimalarial drugs can cause ventricular arrhythmias, qt prolongation, and other cardiac toxicity, which may pose particular risk to critically ill persons. given these serious potential adverse effects, the hasty and inappropriate interpretation of the literature by public leaders has potential to do serious harm. at this time of crisis, it is our ethical obligation as physicians and researchers to organize and refer patients to expedited, well-performed randomized trials that can clarify if, when, and for whom antimalarial medications are helpful in covid- . as of this writing, such trials are under way, and information should be forthcoming within weeks. whereas the evidence supporting the use of antimalarial medications for covid- is equivocal, the evidence for the use of these drugs to treat immune-mediated diseases is not. for example, hcq is a cornerstone of therapy for sle. hydroxychloroquine can effectively treat disease manifestations, such as joint pain and rashes; reduce thrombotic events; and prolong survival. of note, landmark clinical trials have demonstrated that the withdrawal of hcq can lead to flares of disease, including life-threatening manifestations, such as lupus nephritis ( ) . the current shortages of hcq have therefore alarmed rheumatologists and patients. offices across the country report fielding calls from concerned patients who are having difficulty obtaining their medication. given the likelihood that shortages will continue in the near term, we propose that manufacturers, clinicians, pharmacies, health systems, and governmental health agencies continue to coordinate an aggressive response to ensure that antimalarial drug use is appropriately managed during the covid- pandemic. first, it is important to prioritize available supply for clinical trials evaluating important questions, such as dosing, prophylaxis, and treatment in covid- . second, treatment interruptions for those with sle and other rheumatic diseases must be prevented, because lapses in therapy can result in disease flares and strain already stretched health care resources. third, stakeholders should work together to see whether dispensation of remaining supply to patients with covid- makes sense as evidence rapidly changes. fourth, clear messages that reflect the proper interpretations of available data must be disseminated with high frequency to counteract misinformation, including misleading statements or articles with "clickbait" material. finally, safeguards should be put into place to discourage overutilization by health professionals who are depleting supply by prescribing antimalarials for preexposure prophylaxis. hoarding by health professionals for themselves and their friends or family is already occurring, but state governments and pharmacy boards have started to institute strict utilization policies to prevent further hcq overutilization. meanwhile, multiple manufacturers have already made critical commitments to initiate or increase production of hcq. what advice should clinicians give to patients with sle or ra who have difficulty securing hcq? the pharmacokinetics of hcq are an important consideration in answering this question. with long-term use of hcq, peak plasma levels occur to hours after each dose, with a terminal half-life of to days ( ) . the long half-life means that brief gaps in therapy, on the order of to weeks, are less concerning. however, longer treatment lapses put patients at risk for disease exacerbations, given studies showing that lower plasma con-this article was published at annals.org on march . centrations of hcq correlate with more sle disease activity ( ) . in addition, in a well-designed clinical trial, a higher incidence of sle flares was seen as soon as weeks after the drug was stopped ( ) . patients may also wonder whether rationing their supply by halving their current dose is a good approach. studies show significant heterogeneity in plasma concentrations of hcq, even when standard doses of approximately mg/kg are used ( ) . therefore, some patients may do better than others with this approach. the looming public health crisis for people with rheumatic diseases who will be unable to obtain hcq is the result of a perfect storm of fear and dissemination of overpromised data. however, there is still time to mitigate the damage. physicians should educate themselves about the strength of available data regarding hcq and cq in treating covid- . they should avoid misuse of hcq and cq for the prophylaxis of covid- , because there are absolutely no data to support this. public figures should refrain from promoting unproven therapies to the public, and instead provide clear messages around the uncertainties we face in testing and using experimental treatments during the current pandemic, including the risk for serious adverse events. well-done, randomized clinical trials should be performed urgently to test potential therapies, including hcq. in the meantime, physicians should remember that first, we must do no harm to the patients with rheumatic disease for whom high-quality evidence shows that hcq improves health. hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label nonrandomized clinical trial chloroquine for influenza prevention: a randomised, double-blind, placebo controlled trial in vitro inhibition of human influenza a virus replication by chloroquine covid- global rheumatology alliance. a rush to judgment? rapid reporting and dissemination of results and its consequences regarding the use of hydroxychloroquine for covid- a pilot study of hydroxychloroquine in treatment of patients with common coronavirus disease- (covid- ) a randomized study of the effect of withdrawing hydroxychloroquine sulfate in systemic lupus erythematosus mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology low blood concentration of hydroxychloroquine is a marker for and predictor of disease exacerbations in patients with systemic lupus erythematosus the authors thank the members of the key: cord- -fjvumaby authors: carvalho, alzira alves de siqueira title: side effects of chloroquine and hydroxychloroquine on skeletal muscle: a narrative review date: - - journal: curr pharmacol rep doi: . /s - - - sha: doc_id: cord_uid: fjvumaby purpose of review: concerning adverse neuromuscular effects, there are quite a few reports about the incidence and prevalence of chloroquine (cq) and hydroxychloroquine (hcq) myopathy. given the above, i decided to explore the relationships of these drugs with skeletal muscle in an attempt to clarify how they affect the muscle now and in the future, as millions of people are using cq and hcq. recent findings: the literature review identified publications about cq/hcq myopathy, totaling patients, from to . a compilation of all patients was carried out by computing demographic features, clinical aspects, laboratory exams, and clinical evolution. all articles but two represented a large series about incidence and prevalence of the myopathy. fifty-nine percent used qc, mean daily dose was mg per day, and mean duration of treatment was months. the predominant underlying diseases were rheumatoid arthritis ( . %) and lupus erythematosus ( . %). respiratory distress was present in . % in patients with proximal muscle weakness ( . %). dysphagia and cervical and axial weakness were observed in a smaller percentage. creatine kinase was elevated in . %, and emg showed a myopathic pattern in %. muscle biopsy showed a vacuolar pattern in . %, and curvilinear bodies (cb) were the predominant ultrastructural finding ( . %). after drug withdrawal, . % of patients improved, and . % died from other causes than myopathy. summary: cq and hcq myopathy has been known for a long time, but the incidence is low, being described only with long-term use. the use of these drugs for a short period has not been reported, although a prolonged elimination half-life of these drugs actually exists. the world faces the deadly covid- pandemic, the most challenging situation in a century that we have confronted. currently, emerging therapies and repurposing of old drugs have been considered therapeutic strategies, including chloroquine (cq) and hydroxychloroquine (hcq) [ , ] , both drugs showing activity against covid- in vitro [ ] ; however, the level of preclinical and clinical data is not strong and must be approved by a higher level of evidence [ , ] . specifically, at the beginning of , the incidental repurposing of antimalarial drugs (quinacrine and cq) was demonstrated after a crucial improvement of cutaneous rashes and arthritis in soldiers on malaria prophylaxis. sometime later, in , the first trial showed the efficacy of mepacrine in lupus erythematosus from cases. however, nine of the results were very dramatic, and the other nine showed goodto-slight improvement [ ] . cq and hcq are disease-modifying anti-rheumatic drugs (dmards) that suppress the clinical progression of several autoimmune diseases (e.g., systemic lupus erythematosus, rheumatoid arthritis, antiphospholipid syndrome, primary sjögren's syndrome, and sarcoidosis) [ ] . over the past few decades, these two compounds have also drawn attention as potential antiviral agents [ ] . the suggested mechanism of action is raising the ph of the cell membrane, thus making it difficult for the virus to enter cells and interfering in the final stages of virus replication [ ] . also, they have an immunomodulatory effect and block the this article is part of the topical collection on clinical pharmacology two articles presented large studies of the incidence and prevalence of antimalarial myopathy, the others being case reports. these findings are detailed in table and table . a previous and elegant study [ ] selected patients from a retrospective review of patients who initiated antimalarial therapy from january to april due to different rheumatic disorders. from these, only three patients presented with cq myopathy from a total of patient-years with an estimated incidence of in patient-years [ ] . after years, the second series of patients was published as a prospective study, which evaluated over years all patients with rheumatic diseases who were taking antimalarial drugs. a total of patients were included, being detected with cq or hcq myopathy. the annual incidence was . %, with a prevalence of . % [ ] . the mean age of affected patients was years (median , varying from to years), with a clear predominance of females ( . %). the preference for the use of cq or hcq was not explained in those previous reports: . % used cq and % used hcq. the median daily dose was mg per day (mean mg), and the mean duration of treatment was months (median months). the underlying diseases treated with these drugs were rheumatoid arthritis, . %; systemic and discoid lupus erythematosus (sle/dle), . %; other rheumatic disorders (progressive systemic sclerosis (pss), connective tissue disorder, psoriatic arthritis, polymyositis/ calcinosis/raynaud's phenomenon, esophageal dysmotility/ sclerodactyly/telangiectasia, sjögren's syndrome/primary biliary cirrhosis, pss/ interstitial lung disorder), . %; and a miscellaneous group (chronic graft-versus-host disease, shoulder pain, lumbar spondylosis, knee arthritis, morphea, polymyalgia rheumatica), . % (table ) . respiratory distress was present in . % of patients as an initial symptom and associated with proximal muscle weakness ( . %). dysphagia, cervical weakness, and axial weakness were also observed in smaller percentages: . %, . %, and . %, respectively. in laboratory tests, ( . %) patients underwent measurement of ck level that was elevated in . % (mean value, . iu; median value, iu; ref. value < to iu). emg was performed for patients with a predominance of a myopathic pattern ( %), followed by a neuromyopathic pattern in % and neurogenic in only one ( %). muscle biopsy, analyzed by optical microscopy (om), was performed in ( . %) patients showing a vacuolar pattern in . % and non-specific findings in . % (fig. ) . a total of ( . %) muscle samples were processed for electron microscopy (em); the predominant ultrastructural finding was the presence of cb in ( . %) patients, while myeloid bodies and non-specific findings were present only in three cases ( . %). from patients with a non-specific finding by om, ( . %) demonstrated the presence of cb and myeloid bodies on ultrastructural examination. after withdrawing the drug (cq or hcq), . % showed an improvement, and . %% died ( table and table ). the muscle toxicity of cq and hcq is often unrecognized and is likely to be more common than described in the literature since the diagnosis of muscle toxicity attributable to these drugs is quite challenging. this is mainly because the underlying diseases and associated medications mask the symptoms of possible harmful effects on skeletal, cardiac, and smooth muscle, and also because of a lack of classic symptoms and/or morphological abnormalities of muscle. cq has a large volume of distribution in the human body with an elimination half-life of - days and a tendency to accumulate in metabolically active tissues like the brain, muscle, skin, heart, and liver than in blood [ , ] . based on that, the recommended dose of mg twice per day can approach dangerous thresholds with prolonged treatment compared with a lethal dose of chloroquine ( g) in adults. the pathways of antimalarial drugs involved in muscle toxicity, besides being complex, are far from being fully explained, and available data are scanty with no long-term monitoring of experimental models or detailed molecular analysis. even now, it is difficult to relate the composite action mechanisms of these drugs to their efficacy in different autoimmune and infectious disorders. hcq and cq belong to a class of drugs known as aminoquinolines (containing an amino group attached to a quinoline ring). they are most notable for their roles as antimalarial drugs [ , ] . both drugs have a flat aromatic core structure. they are classified as weak bases due to the presence of a basic side chain, which contributes to the accumulation of these drugs in intracellular compartments, especially lysosomal organelles. their amphiphilic properties elevate intralysosomal ph causing specific lysosomal disarrangement and autophagic dysfunctions, which result in vacuolar changes in muscle. they also specifically inhibit the lysosomal proteinase, cathepsin b, responsible for intracellular proteolysis [ ] . the rimmed vacuolar changes found in muscle have been considered the most representative aspect in muscle biopsies of patients with myopathy induced by antimalarials; however, the absence of these vacuoles in some cases does not exclude the diagnosis [ ] . this study demonstrated by om the absence of vacuoles in half the sample ( . %), which could be partially explained by the interval between the interruption of therapy and timing of the muscle biopsy beyond the technical difficulties in interpreting the biopsies. on the other side, their presence has also been described in other neuromuscular disorders, such as sporadic and familial inclusion body myositis, myofibrillary myopathy, oculopharyngeal muscular dystrophy, and some other myopathies [ ] [ ] [ ] [ ] . considering the high percentage of cb ( . %) found on ultrastructural exam makes us think that em is more sensitive for the diagnosis of cq or hcq induced myopathy than om. also, two previous reports [ , ] observed the presence of coiled-and vermicular-shaped material very similar to cb on em. so, considering both cases, the percentage of cb would increase to % of cases. by contrast, the literature described cb as a rare finding on muscle tissue seen in some but not all patients who have received treatment with antimalarials [ , ] . cb has been described for the first time in different tissues, including muscle, of patients with late infantile and juvenile forms of batten disease. they appear as tightly packed, short, curved linear bodies under an electron microscope [ ] [ ] [ ] [ ] . another relevant aspect is that the consequences of using cq and hcq are not restricted to the peripheral organelles of the cell and seem to involve other pathways. nrf is a basic leucine zipper (bzip) protein that regulates the expression of antioxidant proteins that protect against oxidative damage triggered by injury and inflammation [ ] . in vacuolar muscle disorders, such as autophagic vacuolar myopathy induced by cq/hcq, nrf is persistently activated with negative consequences on organ functions. the chronic activation of nrf in skeletal muscle results in changes in cellular redox potential, a response that contributes to muscle pathologies [ ] [ ] [ ] . looking at some of the results, i was initially surprised not to find seemingly typical clinical symptoms and biopsy changes in this compilation. a study conducted by casado and colleagues suggested serial muscle enzyme screening of patients on these therapies as a way to identify patients at risk. all patients with myopathy presented increased levels, mild to moderate, of ck or lactic dehydrogenase. by contrast, kalajian and callen [ ] did not find an association between elevated serum muscle enzymes and underlying antimalarial-induced myopathy in patients taking cq or hcq. in this review, seven asymptomatic patients with mildly elevated muscle enzymes (ck or dhl) and normal emg presented curvilinear and myeloid bodies on em as a unique finding. the isolated presence of this specific ultrastructural finding in skeletal muscle may not always signify a muscle disorder. however, it could be a muscular accumulation of cq/hcq or their metabolites, as demonstrated by kumamoto et al., who observed dense membranous structures (cb) in soleus muscle fibers by em in cqtreated rats [ , ] . experimental studies have suggested that the absolute tissue levels of cq are . times higher than those of hcq. thus, the depositing of the drug in several tissues with subsequent enzyme inactivation, which is the proposed mechanism for toxicity, might be more likely to occur with cq [ ] . nevertheless, we did not observe crucial differences between compounds in terms of symptoms, morphological analyses, or clinical evolution. the number of patients using cq or hcq, . % and %, respectively, was not very different; specifically, in relation to morphological findings, we identified almost the same number of cases with vacuolar myopathy in the group using cq [ ] and hcq [ ] . concerning outcomes, prompt recovery was usual. improvement after discontinuation of therapy occurred in . % of cases, and seven deaths ( . %) were reported. apparently, five of the deaths were not related to the antimalarial drug (dose, treatment duration), but as a result of underlying disease complications, and the other two deaths occurred due to cardiac complications. despite major drug interactions of cq and hcq with other medications leading to a greater risk of arrhythmia, there were no reports of this as being as the cause of death (table ) . cq and hcq myopathy has been known for a long time, but the reports are sporadic, and the incidence is low, being described only with long-term use. the use of these drugs even for a short period requires attention as a prolonged elimination half-life of these drugs can be harmful. we declare that the supplementary material is available as a supplementary table. conflict of interest the author declares no conflicts of interest. human and animal rights and informed consent this article does not contain any studies with human or animal subjects performed by any of the authors. ethical publication statement we confirm that we have read the journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines and according to the declaration of helsinki. review of emerging pharmacotherapy for the treatment of coronavirus disease chloroquine and hydroxychloroquine in the treatment of covid- with or without diabetes: a systematic search and a narrative review with a 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but reversible cause of myopathy: hydroxychloroquine induced myopathy hydroxychloroquine-induced autophagic vacuolar myopathy with mitochondrial abnormalities pearls & oy-sters: hydroxychloroquine-induced toxic myopathy mimics pompe disease: critical role of genetic test inhibition of lysosomal function in red and white skeletal muscles by chloroquine chloroquine-induced cytosomes with curvilinear profiles in muscle mechanisms of activation of the transcription factor nrf by redox stressors, nutrient cues and energy status, and pathways through which it attenuates degenerative disease direct mechanism of action in toxic myopathies activation of the keap / nrf stress response pathway in autophagic vacuolar myopathies nrf -keap signaling in oxidative and reductive stress. (review). bba -molecular cell research myopathy induced by antimalarial agents: the relevance of screening muscle enzyme levels experimental chloroquine myopathy: morphological and biochemical studies publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -lq knxgf authors: takano, tomomi; satoh, kumi; doki, tomoyoshi; tanabe, taishi; hohdatsu, tsutomu title: antiviral effects of hydroxychloroquine and type i interferon on in vitro fatal feline coronavirus infection date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: lq knxgf feline infectious peritonitis (fip) is a viral disease with a high morbidity and mortality by the fip virus (fipv, virulent feline coronavirus). several antiviral drugs for fip have been identified, but many of these are expensive and not available in veterinary medicine. hydroxychloroquine (hcq) is a drug approved by several countries to treat malaria and immune-mediated diseases in humans, and its antiviral effects on other viral infections (e.g., sars-cov- , dengue virus) have been confirmed. we investigated whether hcq in association with interferon-ω (ifn-ω) is effective for fipv in vitro. a total of μm of hcq significantly inhibited the replication of types i and ii fipv. interestingly, the combination of μm of hcq and ( ) u/ml of recombinant feline ifn-ω (rfifn-ω, veterinary registered drug) increased its antiviral activity against type i fipv infection. our study suggested that hcq and rfifn-ω are applicable for treatment of fip. further clinical studies are needed to verify the combination of hcq and rifn-ω will be effective and safe treatment for cats with fip. coronaviruses are single-stranded positive-sense rna viruses in the subfamily orthocoronavirinae of the family coronaviridae [ ] . coronaviruses are important pathogens causing life-threatening infectious disease in mammals and birds [ ] . in humans, outbreaks of the novel coronavirus ( -ncov, official name is severe acute respiratory syndrome-related coronavirus : sars-cov- ) occurred worldwide [ ] . feline infectious peritonitis (fip) is a fatal, immune-mediated disease caused by feline coronavirus (fcov) [ ] . fcov is a member of the species alphacoronavirus- , genus alphacoronavirus, in the subfamily orthocoronavirinae. it is divided into two serotypes based on the amino acid sequence of the spike (s) protein, serotype i fcov, and serotype ii fcov [ ] . the majority of fcov infections are subclinical (avirulent fcov is known as feline enteric coronavirus: fecv; type i and type ii fecv) [ ] . however, several mutations occurred in the s protein, leading to development of the virulent type called feline infectious peritonitis virus (fip virus, fipv; type i and type ii fipv) [ , ] . fipv infection typically causes a fatal disease in cats known as fip. the hallmark pathological findings of fip in cats are serous fluid in peritoneal and pleural cavities, and pyogranulomatous lesions in the internal organs and brain [ ] . the absorbance of formazan produced was measured at nm using a -well spectrophotometric plate reader, as described by the manufacturer. percentage cell viability was calculated using the following formula: cell viability (%) = [(od of compound-untreated cells -compound-treated cells)/(od of compound-untreated cells)] × . the % cytotoxicity concentration (cc ) was defined as the cytotoxic concentration of each compound that reduced the absorbance of treated cells to % when compared with that of the untreated cells. confluent fcwf- cell monolayers were cultured in medium with or without compounds at the indicated concentrations in -well multi-plates at • c for h or h. cells were washed and the virus (moi . ) was adsorbed into the cells at • c for h. after washing, cells were cultured in . % carboxymethyl cellulose (cmc)-mem or mem with or without compounds. in the case of cells cultured in cmc-mem, the cell monolayers were incubated at ºc for h, fixed, and stained with % crystal violet solution containing % buffered formalin, and the resulting plaques were then counted. the percentage of the decrease or increase in plaques was calculated using the following formula: percentage of the plaque reduction (%) = [(plaque number of compound-treated cells)/(plaque number of compound-untreated cells)] × . the ec was defined as the effective concentration of compounds that reduced the virus titer in the culture supernatant of infected cells to % when compared with that of the virus control. in the case of cells cultured in mem, the culture supernatants were collected h post-infection, and virus titers were measured by the tcid assay. the nucleocapsid (n) protein levels of fipv-infected fcwf- cells were determined by an immunofluorescence assay (ifa), as described previously [ ] . briefly, fipv-infected cells were washed pbs and fixed with % paraformaldehyde at rt for min. the cells were incubated with mab yn- (fipv n protein-specific mab) at • c for min. after washing, the cells were incubated with goat anti-mouse-igg conjugated to fluorescein (jackson immunoresearch, pa, usa) at • c for min. after washing, the cells were stained with , -diamidino- -phenylindole (dapi; dojindo laboratories, kumamoto, japan) at rt for min. the stained cells were analyzed using leica dm b microscope and las x integrated imaging system (leica microsystems, wetzlar, germany). data from only two groups were analyzed using the student's t-test (welch's t-test) and those of multiple groups were analyzed by one-way anova followed by tukey's test using microsoft excel software and open-source statistical graphpad prism (graphpad software, ca, usa). a p-value of < . was considered statically significant. cytotoxicity assay was performed to clarify the non-toxic concentration of cq and hcq against fcwf- cells (figure ). the cc of cq and hcq was . µm ( figure a ) and . µm ( figure b) , respectively. hcq was less toxic ( . %) than cq in feline cells. the antiviral activity of compounds against both serotypes of fipv was evaluated using plaque inhibition assay. the ec of cq and hcq against fipv-i ku was . µm ( figure a ) and . µm ( figure b ), respectively. the ec of cq and hcq against fipv-ii - was . µm ( figure a ) and . µm ( figure b) , respectively. therefore, the antiviral effects of hcq on fipv were comparable to those of cq. based on these results, hcq was suggested to be a safer anti-fipv drug than cq. the antiviral activity of hcq and rfifn-ω in fcwf- cells for a prolonged time ( h) was investigated. to evaluate and assess the antiviral effects of hcq ( μm) and rfifn-ω ( u/ml), we used strains of serotype i fipv (fipv-i ucd , fipv-i ucd , and fipv-i ku ) and strain of serotype ii fipv (fipv-ii - ). the virus titers of both serotypes of fipv significantly decreased in the culture supernatant of cells pretreated with hcq or rfifn-ω ( figure ). we evaluated the antiviral effects of the combination of hcq and rfifn-ω. the combination of these drugs strongly suppressed the replication of viruses in fcwf- cells. when both μm hcq and u/ml of rfifnω were added, fcwf- cell viability was . ± . %. the antiviral activity of hcq and rfifn-ω in fcwf- cells for a prolonged time ( h) was investigated. to evaluate and assess the antiviral effects of hcq ( µm) and rfifn-ω ( u/ml), we used strains of serotype i fipv (fipv-i ucd , fipv-i ucd , and fipv-i ku ) and strain of serotype ii fipv (fipv-ii - ). the virus titers of both serotypes of fipv significantly decreased in the culture supernatant of cells pretreated with hcq or rfifn-ω ( figure ). we evaluated the antiviral effects of the combination of hcq and rfifn-ω. the combination of these drugs strongly suppressed the replication of viruses in fcwf- cells. when both µm hcq and u/ml of rfifn-ω were added, fcwf- cell viability was . ± . %. the antiviral activity of hcq and rfifn-ω in fcwf- cells for a prolonged time ( h) was investigated. to evaluate and assess the antiviral effects of hcq ( μm) and rfifn-ω ( u/ml), we used strains of serotype i fipv (fipv-i ucd , fipv-i ucd , and fipv-i ku ) and strain of serotype ii fipv (fipv-ii - ). the virus titers of both serotypes of fipv significantly decreased in the culture supernatant of cells pretreated with hcq or rfifn-ω ( figure ). we evaluated the antiviral effects of the combination of hcq and rfifn-ω. the combination of these drugs strongly suppressed the replication of viruses in fcwf- cells. when both μm hcq and u/ml of rfifnω were added, fcwf- cell viability was . ± . %. we investigated whether hcq and rfifn-ω, which acted on fipv in fcwf- cells for a short time ( h), exhibit antiviral activity. as shown in figure , type i fipv and type ii fipv replication was significantly inhibited by hcq and rfifn-ω. interestingly, the combination of these drugs strongly decreased the replication of type i fipvs in fcwf- cells, but not type ii fipv. viruses , , x for peer review of we investigated whether hcq and rfifn-ω, which acted on fipv in fcwf- cells for a short time ( h), exhibit antiviral activity. as shown in figure , type i fipv and type ii fipv replication was significantly inhibited by hcq and rfifn-ω. interestingly, the combination of these drugs strongly decreased the replication of type i fipvs in fcwf- cells, but not type ii fipv. we evaluated the antiviral activity of hcq and rfifn-ω against type i fipv after viral infection. hcq and rfifn-ω were added to the cells h after inoculation. as shown in figure , type i fipv replication was significantly inhibited by hcq and rfifn-ω, and the combination of these drugs strongly decreased the replication of virus. we investigated the expression of viral proteins in order to evaluate the antiviral effects of the combination of hcq and rfifn-ω on fipv. the n protein levels of fipv-i ku- were specifically decreased in fcwf- cells pre-treated (short-time exposure) and post-treated with hcq and rfifn-ω we evaluated the antiviral activity of hcq and rfifn-ω against type i fipv after viral infection. hcq and rfifn-ω were added to the cells h after inoculation. as shown in figure , type i fipv replication was significantly inhibited by hcq and rfifn-ω, and the combination of these drugs strongly decreased the replication of virus. viruses , , x for peer review of we investigated whether hcq and rfifn-ω, which acted on fipv in fcwf- cells for a short time ( h), exhibit antiviral activity. as shown in figure , type i fipv and type ii fipv replication was significantly inhibited by hcq and rfifn-ω. interestingly, the combination of these drugs strongly decreased the replication of type i fipvs in fcwf- cells, but not type ii fipv. we evaluated the antiviral activity of hcq and rfifn-ω against type i fipv after viral infection. hcq and rfifn-ω were added to the cells h after inoculation. as shown in figure , type i fipv replication was significantly inhibited by hcq and rfifn-ω, and the combination of these drugs strongly decreased the replication of virus. we investigated the expression of viral proteins in order to evaluate the antiviral effects of the combination of hcq and rfifn-ω on fipv. the n protein levels of fipv-i ku- were specifically decreased in fcwf- cells pre-treated (short-time exposure) and post-treated with hcq and rfifn-ω we investigated the expression of viral proteins in order to evaluate the antiviral effects of the combination of hcq and rfifn-ω on fipv. the n protein levels of fipv-i ku- were specifically decreased in fcwf- cells pre-treated (short-time exposure) and post-treated with hcq and rfifn-ω ( figure ). in contrast, post-treatment with hcq and rfifn-ω slightly affected the protein levels of fipv-ii - in fcwf- cells. viruses , , x for peer review of ( figure ). in contrast, post-treatment with hcq and rfifn-ω slightly affected the protein levels of fipv-ii - in fcwf- cells. fip is a fatal coronaviral infection of cats. several drugs have been identified aiming at the treatment of fip, but no commercial drugs can be used to treat fip by veterinarians. we have searched for a drug applicable to treat fip among commercial drugs [ , ] . cq is an antimalarial drug and improved symptoms of cats with fip [ ] . however, increased liver enzymes were observed in some cats treated with cq. increased liver enzymes are observed in cats with fip, but the possibility of cq-induced liver disorder was also suggested. if there is a drug with cytotoxicity weaker than that of cq that exhibits comparable antiviral effects, it may be applicable as a therapeutic drug for fip. we focused on hcq, which is -aminoquinoline similar to cq [ ] . the cytotoxicity of hcq has been reported to be lower than that of cq in mouse, rat, and dog [ ] . in addition, hcq has been demonstrated to have antiviral effects on sars-cov- infection equivalent to those of cq in vitro [ ] . we confirmed that hcq has anti-fipv activity equivalent to that of cq. moreover, cytotoxicity of hcq setting the criterion to cc was one-third or lower than that of cq. accordingly, hcq is applicable to fip treatment as a substitute for cq. hcq at μm significantly inhibited the replication of both serotypes of fipv. to our knowledge, the pharmacokinetics of hcq in cats have not been analyzed. thus, it is necessary to refer to pharmacokinetic data of hcq in dogs. the tolerated dose of intramuscular injection of hcq is mg/kg [ ] . in dogs treated with mg/kg of hcq, the plasma hcq level reaches . μm ( μg/l) [ ] , i.e., it is difficult to make the plasma hcq level reach μm in dogs. however, it has been reported that the tissue hcq levels in the liver, spleen, kidney, and lung increased to a level several hundred-times higher than the plasma level [ ] . therefore, hcq administration to cats with fip within the low dosage may be expected to yield sufficient therapeutic effects. on the other hand, it is unclear whether the pharmacokinetics described above can apply to cats. cytochrome p (cyps) are involved in the metabolism of hcq [ ] . generally, cyp activities could be lower in cats than in dogs [ ] . on the basis of this fact, the blood concentrations of hcq in cats will be higher than those in dogs. therefore, pharmacokinetic studies are still needed to use hcq in cats. the antiviral agent rfifn-ω has a wide safety range and is practically used to treat feline viral infection in veterinary practice. many points are unclear as to whether rfifn-ω is effective as a therapeutic drug for fip. fip is a fatal coronaviral infection of cats. several drugs have been identified aiming at the treatment of fip, but no commercial drugs can be used to treat fip by veterinarians. we have searched for a drug applicable to treat fip among commercial drugs [ , ] . cq is an antimalarial drug and improved symptoms of cats with fip [ ] . however, increased liver enzymes were observed in some cats treated with cq. increased liver enzymes are observed in cats with fip, but the possibility of cq-induced liver disorder was also suggested. if there is a drug with cytotoxicity weaker than that of cq that exhibits comparable antiviral effects, it may be applicable as a therapeutic drug for fip. we focused on hcq, which is -aminoquinoline similar to cq [ ] . the cytotoxicity of hcq has been reported to be lower than that of cq in mouse, rat, and dog [ ] . in addition, hcq has been demonstrated to have antiviral effects on sars-cov- infection equivalent to those of cq in vitro [ ] . we confirmed that hcq has anti-fipv activity equivalent to that of cq. moreover, cytotoxicity of hcq setting the criterion to cc was one-third or lower than that of cq. accordingly, hcq is applicable to fip treatment as a substitute for cq. hcq at µm significantly inhibited the replication of both serotypes of fipv. to our knowledge, the pharmacokinetics of hcq in cats have not been analyzed. thus, it is necessary to refer to pharmacokinetic data of hcq in dogs. the tolerated dose of intramuscular injection of hcq is mg/kg [ ] . in dogs treated with mg/kg of hcq, the plasma hcq level reaches . µm ( µg/l) [ ] , i.e., it is difficult to make the plasma hcq level reach µm in dogs. however, it has been reported that the tissue hcq levels in the liver, spleen, kidney, and lung increased to a level several hundred-times higher than the plasma level [ ] . therefore, hcq administration to cats with fip within the low dosage may be expected to yield sufficient therapeutic effects. on the other hand, it is unclear whether the pharmacokinetics described above can apply to cats. cytochrome p (cyps) are involved in the metabolism of hcq [ ] . generally, cyp activities could be lower in cats than in dogs [ ] . on the basis of this fact, the blood concentrations of hcq in cats will be higher than those in dogs. therefore, pharmacokinetic studies are still needed to use hcq in cats. the antiviral agent rfifn-ω has a wide safety range and is practically used to treat feline viral infection in veterinary practice. many points are unclear as to whether rfifn-ω is effective as a therapeutic drug for fip. the combination of hcq and rfifn-ω blocked virus production in type i fipv-infected cells, but although the duration of activity was only h, the antiviral activity of these drugs decreased in type ii fipv-infected cells. we previously demonstrated that types i and ii fipv enter the cytosol through late and early endosomes, respectively [ ] . we also reported that type ii fipv strongly inhibited type i ifn expression [ ] . based on this knowledge and our current study, type ii fipv may show less effect on the antiviral activity of hcq and type i ifn, compared to type i fipv. there are some reports about the relationship between hcq and type i ifn. wang et al. reported that hcq inhibited dengue virus infection in all serotypes in vitro [ ] . they suggested that the induction of interferon or related protein is an antiviral activity mechanism of hcq. on the other hand, inhibition of type i ifn production in hcq-treated cells has been reported [ ] , being contradictory to other findings. we confirmed that potent antiviral activity was induced by the combination of hcq and rfifn-ω (type i ifn). although negative action on type i ifn may have been induced by hcq, type i ifn added at the same time may have canceled this. to demonstrate this, further investigation is necessary. in this study, we confirmed that hcq is a safer anti-fipv drug than cq. in addition, we demonstrated that the combination of hcq and rfifn-ω increases the antiviral activity. our study revealed that these fip therapeutic drugs are applicable to veterinary practice. it should be noted that in vitro data do not always translate into in vivo efficacy. therefore, a deeper understanding of pharmacokinetics of the combination between hcq and rfifn-ω will be needed in cats. an overview of their replication and pathogenesis the species severe 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is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors are grateful to their colleagues. the authors declare no conflict of interest. key: cord- -epqqd n authors: chen, jun; lu, hongzhou; melino, gerry; boccia, stefania; piacentini, mauro; ricciardi, walter; wang, ying; shi, yufang; zhu, tongyu title: covid- infection: the china and italy perspectives date: - - journal: cell death dis doi: . /s - - - sha: doc_id: cord_uid: epqqd n the severe acute respiratory syndrome coronavirus (sars-cov- ) is responsible for the covid- pandemic. since its first report in december , despite great efforts made in almost every country worldwide, this disease continues to spread globally, especially in most parts of europe, iran, and the united states. here, we update the recent understanding in clinical characteristics, diagnosis strategies, as well as clinical management of covid- in china as compared to italy, with the purpose to integrate the china experience with the global efforts to outline references for prevention, basic research, treatment as well as final control of the disease. being the first two countries we feel appropriate to evaluate the evolution of the disease as well as the early result of the treatment, in order to offer a different baseline to other countries. it is also interesting to compare two countries, with a very significant difference in population, where the morbidity and mortality has been so different, and unrelated to the size of the country. the covid- pandemic exploded first in china and subsequently in italy. we therefore compared these two countries with the earliest and strongest impact on the population. • with over , , cases, including , , recovered and , deaths worldwide as of may , the spread of infection still seems difficult to contain. • both the availability of intensive care and the local medical heath system on the territory may be responsible for the vast heterogeneity in infection containment and mortality. • the therapy, especially in the advanced stages, seems complicated, and we are waiting the outcome of several hundreds of clinical trials, often duplicated and non-coordinated. major drugs are mostly nonviral-specific: hydroxychloroquine, lopinavir/ ritonavir, remdesivir, convalescent plasma/ monoclonal antibody, camostat, and ivermectin. new drug development is clearly the first priority. • the role of immunity is crucial both to overcame the acute phase and for establishing the vaccine. • exit from the acute phase and establishing the new protocol for the post-acute phase is of utter priority. the severe acute respiratory syndrome coronavirus (sars-cov- ) is responsible for the covid- disease as originally shown in wuhan, china, as early as documented from december (ref. ). on march , the who announced covid- as a worldwide pandemic, months after the official disclosure from the chinese government of the actual cluster epidemics in whuan . as of may , there were over , , confirmed covid- cases worldwide, of which more than % cases in the eu and uk, with more than , deaths worldwide . figures and show the current set of data of the pandemic, with a special attention to china and italy. with the great efforts mostly based on strict containment measures, china has successfully emerged out of the first wave of the epidemic at the beginning of april , when the social order slowly returned to the norms. although different precautionary measures have been taken at national level in the eu to limit and to monitor the entrance of potential covid- cases from china, the first symptomatic cases have been reported before the end of february in a number of eu countries including italy, spain, germany, and, just weeks later, uk. in this context, italy is one of the first and hardest hit country in europe, with over , cases and , deaths reported. the sars-cov- virus is structurally and functionally closely related to the other coronaviruses causing middle-east respiratory syndrome coronavirus (mers-cov) and severe acute respiratory syndrome coronavirus (sars-cov). in particular, it is the external protein of the virus, the glycoprotein spike (s) (fig. ) , that is able to bind and recognize the human receptor angiotensin-converting enzyme (ace ), primed by the transmembrane protease serine type (tmprss ), and/or cd (extracellular matrix metalloproteinase inducer), and therefore infect the host cells [ ] [ ] [ ] (fig. ) . the crucial recent elucidation of these structures at the fine biochemical level will pave the way for future potential therapeutic interventions. the viral genomic sequences assembled from samples in shanghai together with uploaded sequences in the global initiative on sharing all influenza data (gisaid, www.gisaid.org) also showed a stable evolution (h.l., unpublished data). based on current data, sars-cov- seems to have a mutation rate of around mutations per year, which is much lower than that of influenza virus . therefore, development of effective longlasting vaccines against the virus is possible. in this review, we update the latest clinical characteristics, diagnosis strategies, as well as clinical management of the covid- in china. we decided to compare the data of china and italy because these were the first two countries hit by the pandemic at the highest level; therefore, this experience may be of relevance for other countries. being the first two countries we feel appropriate to evaluate the evolution of the disease as well as the early result of the treatment, in order to offer a different baseline to other countries. it is also interesting to compare two countries, with a very significant difference in population, where the morbidity and mortality has been so different and unrelated to the size of the country. the covid- clinical features in adults are reported in table . the median incubation period (interquartile range, - ) is around days . fever is one of the most common symptoms occurring in as high as % of the patients reported . a relatively high proportion of patients develop fever although they are afebrile at the onset of the disease . the predominant symptom is dry cough, reported in around % of the patients . gastrointestinal symptoms including nausea, vomiting, and diarrhea are less frequently developed, with an incidence lower than % of the patients , , . other symptoms including rhinorrhoea, sore throat, fatigue, dyspnea, muscle weakness, dizziness, and headache are also often reported in covid- patients. lymphopenia and leukopenia has been observed in the majority of patients, together with elevated level of c-reactive protein, lactate dehydrogenase, d-dimers, and other inflammatory biomarkers, including tumor necrosis factor-α (tnfα), interleukin- β (il- β), il- , granulocyte-macrophage colony-stimulating factor as well as il- (refs. , [ ] [ ] [ ] [ ] ). t cell exhaustion, especially cd + t cells is a hall marker of infected patients, paralleling with the severity of the illness . the vast majority only suffered from mild symptoms. however, the early report in chinese involving , confirmed cases, % patients are mild cases, and . % patients are severe, while only % patients are critically ill , . numbers of comorbidities were associated with poorer outcomes . the median onset time from early symptoms to dyspnea is around days, while acute respiratory distress syndrome (ards) developed around days . the median days of fever in survivals is - days and cough persisted for days , . the severity of the diseases varies in different age groups, with older patients at higher risk of mortality compared to those of younger age. in children, the symptoms are often mild and the prognosis of pediatric patients is largely more favorable than adults . table compares the major characteristics of covid- in china versus italy. patients in italy were more older compared to patients in china, with more numbers of comorbidities. the number and severity of these co-morbidity has been a major factor influencing the outcome; this was particularly evident where the virus diffused into old pension homes. indeed, while the mortality was . % in italy, with the exception of the milan area (fig. b) where it was . %, within the residences for old people it peaked to % (https://www. epicentro.iss.it/). in this case, out of death, only were confirmed by swabs, while had all symptoms but they were not tested. disease severity was strongly age dependent, primarily due to the presence of comorbidities. therefore, the proportions of severe and critical patients in italy were higher than that in china, partially leading to a higher mortality. a proportion of the patients showed no symptoms at enrollment as they were at very early stage of the diseases. these patients could either recover without developing symptom or would continue to develop symptoms. however, the former group of patients never have any symptoms or signs, but their respiratory tract specimens are pcr positive for the virus. the exact number of the proportion of asymptomatic patients requires longitudinal study with repeated pcr tests. in a study that followed patients in wuhan, china, % of them never developed symptoms . in another study performed on the diamond princess cruise ship, repeated pcr testing of quarantined passengers and crew members showed that asymptomatic proportion is around % . more recently, the proportion of infected people have mild or asymptomatic were estimated to represent some % of all infections . notably, asymptomatic and symptomatic patients show comparable viral load, suggesting that these patients have strong transmission potentials . indeed, viral transmission from asymptomatic carriers have been reported . in a recent study from china, chen et al. cases was . % comparing with . % in asymptomatic patients, indicating the importance of identification and isolation of asymptomatic patients in the effort of containment the spread of the virus . importantly, for largescale screening, antibody testing should be combined with pcr to avoid asymptomatic viral spreading. nucleic acid tests the definitive diagnosis of the disease relies on the identification of viral genomic rna using either pcrbased technology or deep sequencing. the detailed pcrbased methods have been reviewed elsewhere . the presence of sufficient viral genome for amplification at the site of sample collection is the precondition of the tests. therefore, collecting the appropriate specimen from patients at the right time using the adequate protocol is a key in diagnosis of the infection. besides in the respiratory tract, viral particles have also been detected in the blood, lacrimal fluid, urine, and feces , . in a recent study, the virus was detected in different types of samples, such as bronchoalveolar lavage fluid (balf) ( % positive), pharyngeal swabs ( %), fibrobronchoscope brush ( %), nasal swabs ( %), feces ( %), sputum ( %), and blood ( %) . however, the samples were not from the same patients at the same time, making it impossible to compare the sensitivity of the pcr test in different types of specimens. as a viral pneumonia, respiratory tract specimens from covid- patients are first of choice to collect for the detection of viral nuclear acid. collecting and testing upper respiratory specimens would be ideal considering the feasibility in clinical practice. nasopharyngeal and oropharyngeal swab are the optimal manner for swab viral testing. these samples can even be collected by patients themselves. self-collected saliva specimens had been test and yield positive results in most of the infected patients, indicating that it is an adequate non-invasive test for monitoring and diagnosis of the infections . to increase the sensitivity of the diagnosis, it is preferably to collect multiple different types of specimens. however, negative naso-oro-pharyngeal swab could not completely rule out covid- (ref. ). cases with repeated negative pcr for the virus in naso-oro-pharyngeal tests but positive in bronchoalveolar lavage fluid (balf) samples have been reported . it is therefore necessary to collect balf via bronchoscopy in patients highly suspected of covid- , although this procedure could significantly increase the safety danger to healthcare personnel through the creation of aerosol droplets. this is an important issue, ace receptor binding the sars-cov- virus. the initial step of sars-cov- viral entry during the infection is the binding of the viral trimeric spike protein (cleaved into s and s subunits, the former of which contains the receptor-binding domain, rbd) to the dimeric human receptor angiotensin-converting enzyme (ace ) which is here represented in the complex with the membrane protein that it chaperones, boat . ace is formed by an n-terminal peptidase domain (pd) and the c-terminal collectrin-like domain (cld). ace shows a closed and an open (depicted) conformation at the pd level of contact; however, only the closed conformation binds the rbd of sars-cov- . the structure shown was obtained by cryo-em at . Å resolution (pbd = m ) was released on march from yan et al. . considering that in italy alone, over doctors have died so far! as soon as possible on the earliest days of symptoms, swabs should be collected from the upper respiratory tract. recent studies showed that the viral load in the upper airway samples peaks within the first week and the virus was cleared at a median of days from infection [ ] [ ] [ ] . similar shedding patterns were seen for mers-cov and sars-cov: rna swab positivity was evident within the first week of mers infection, while it peaked at - days after symptom onset in sars. the dynamic of sars-cov- shedding in the lower respiratory tract specimens is still unclear. in mers, mers-cov viral load peaked between week and in lower airways specimens, while the sars-cov rna-positive rates in lower airways samples remained higher for weeks after beginning of illness in sars , . it is likely that sars-cov- also shares a comparable viral shedding pattern in the lower respiratory tract specimens. this may explain why some patients progressed despite pcr turned negative in the upper respiratory specimens . despite the high accuracy of the pcr tests, these nucleic acid tests highly depend on the procedures of sample collecting, in addition to high labor and time costing. the test protocol is generally expensive and complex, and therefore difficult to be broadly applied in resource limited locations. transferring samples to a central laboratory not only decrease the accuracy of the tests either by sample decay due to incorrect storage or contamination but also further delay the test results. a false-positive test could result in an unnecessary quarantine and therapy while a false-negative diagnosis may allow infected patients to spread the virus. therefore, other methods are also needed to serve as a supplement. most importantly, as the virus spreading many healthcare personnel have caused wanton community transmission before correct testing allowed isolation and tracking. therefore, tests that are able to be scaled up quickly are urgently needed. serological assays detect both sars-cov- -specific antibodies igm, iga, and igg, which are produced both at the early and later phases of the disease, respectively. unlike pcr tests, antibody tests could be less accurate and require longer time to be established as routine tests. however, serological tests are portable, user-friendly, and can produce results in - min at least at the qualitative level. moreover, they request very limited sample volume: one drop of blood from a pinprick is sufficient to detect the antibodies. therefore, they can be established as decentralized point-of-care (poc) tests. in a cohort comprised of confirmed and probable (qpcr test is negative despite other indications of covid- including symptoms and epidemiology) covid- cases, the positive detection rate is extremely high ( . %) when pcr is performed in conjunction with igm elisa for every patient as compared to qpcr analysis alone ( . %) . therefore, the serological tests could be used together with nuclei acid detection to increase the sensitivity of tests. in china, some of these kits have received fast-track approval from the national medical products administration (nmpa). in the usa, only one serological test has been approved by the date of submission. these tests are of good accuracy with sensitivity range from . % to . % and specificity range from . % to %. the detailed serological tests has been reviewed elsewhere , . however, the sensitivity of serological assays is influenced by both the time of sample collection as well as human immune response status. in the study by guo et al., as high as . % ( / ) of the pcr confirmed cases were found to be negative by an igm antibody tests. most of the covid- patients were able to generate antibody , if nucleic acid tests are not available. in addition, the serological assays can also provide historic information about viral exposure, which are important for epidemiology studies. interestingly, early presence of iga might be protective and a surrogate marker of good outcome, detecting of which may help identifying patients warrant medical attention , . therefore, the speed and versatility of serological assays make them invaluable tools for covid- diagnosis and efforts to produce antibody detection kits on a huge scale have begun to be produced . clearly, serological determination is the future both for diagnostic as well for epidemiological purposed. the covid- patients presented some typical characteristics on the chest computed tomography (ct) image. ground-glass opacity was the most frequent, with subsequent consolidations, air bronchogram, irregular or smooth interlobular septal thickening, and thickening of the nearby pleura, with predominantly lower and peripheral lobe involvement , (fig. ) . a recent study by ai et al. involved patients in china reported that the sensitivity of toracic ct in suggesting covid- was %. in the early february, , some experts in china suggested ct as one of the best clinical diagnostic criteria together with epidemiology, clinical, and laboratory characteristics to pronounce the clinically diagnosed covid- when pcr tests were not available. nonetheless, not all the patients had the typical chest ct images (e.g., asymptomatic covid- cases). absence of ct or radiographic abnormality was observed in as high as . % patients with non-severe disease and in . % cases with severe disease . notably, the typical ct image sometimes could not distinguish covid- from influenza infections. in the study by ai et al. , % of patients with negative rt-pcr swabs showed positive thoracic ct results. given that the misdiagnosis of covid- in cases has grave consequences as mentioned above, ct should not be used alone to make the diagnosis of covid- . at present, there are no specific treatment with confirmed efficacy for covid- and the therapeutic protocol is as per best supportive care for any respiratory disease. however, both antivirals and immune modulators warrant further investigation especially considering the biphasic pattern of immune responses during the diseases course . several drugs with potential antiviral activities are under active investigation. more recently, the who also launched multinational trial to validate four drugs especially in combinations, including ritonavir/lopinavir, lopinavir and ritonavir with interferon-beta, chloroquine and/or remdesivir. several clinical trials, over , have been registered worldwide and are listed at www. clinicaltrials.gov (specifically see: https://clinicaltrials. gov/ct /results?cond=covid- ). a selected clinical trials with possible significance are listed in table . chloroquine (cq) and its related drug hydroxychloroquine (hcq), both of which are well-known for their effectiveness in treating malarial and autoimmune diseases, have also recently been repurposed to treat covid- . in vitro studies showed that both cq and hcq were of excellent antiviral activity against sar-cov- with an ec of . and . μm, respectively, which are reachable in humans when admitted , . in fact, cq and hcq have been shown to have antiviral activities to different viruses including dengue virus, chikungunya virus, ebola virus, etc. any effect found has been limited to in vitro culture. there is no in vivo efficacy reported. many randomized controlled trials are undergoing to evaluate the efficacy of cq and hcq in treating or even in preventing covid- . we completed the first randomized, control, open-label trial to evaluate the antiviral activity and safety of hcq in treating covid- . unfortunately, in this pilot study with patients, we failed to observe any benefit of adding hcq on top of the standard of care . however, more recently, another study from france showed that hcq was effective against sars-cov- , especially when combined with azithromycin . these two studies were both limited by the small sample size. the u. s. food and drug administration (fda) has launched a trial to investigate the effectiveness of cq in covid- (https://www.fda.gov/news-events/press-announcements/ coronavirus-covid- -update-fda-continues-facilitatedevelopment-treatments). more recently, a study was completed in renmin hospital of wuhan university with covid- patients . this study was based on a follow-up survey, showing that none of the sle patients who took long-term oral hcq showed sars-cov- infection or appeared to have related symptoms. the study found that the cough remission time and the body temperature recovery time were strongly reduced in the hcq treatment group. over % ( of ) of the patients in hcq treatment group showed improvement in pneumonia compared with the matched controls ( . %, of ). importantly, . % of patients in the hcq treated group showed significant pneumonia absorption. among the patients in the hcq treated group, of them had mild adverse reactions, one developed rash and one patient experienced a headache. clearly, these results warrant further randomized, controlled studies. remarkably, hcq is a strong immune modulator and has been used widely for the therapy of systemic lupus erythematosus or rheumatoid arthritis. it exerts its effects likely through inhibiting the secretion of il- β, tnf-α, and il- from macrophages/monocytes, which are a key cell population in ards development in covid- patients. thus hcq application should be limited to patients who have started to develop or already developed ards. since immune response at the early stage is critical for controlling infection and the antiviral effect of hcq is still in question. in addition, the safety of cq/hcq should also be taken into consideration. cq/hcq are associated with cardiac arrhythmias, hypoglycemia, and neuropsychiatric effects . therefore, these compounds should not be recommended for mild patients or for prevention purpose, at least for now. moreover, the combination of hcq with antibiotic drug azithromycin has been reported to have added benefit. since azithromycin is known to reversibly binds to the s ribosomal subunit of the s ribosome to inhibit the translocation step of protein synthesis, whether it has antiviral effect is not known. it is also not known whether azithromycin synergize with hcq to provide better antiviral activity. a recent prospective study failed to find antiviral activity or clinical benefit of this combination for the treatment of our hospitalized patients with severe covid- (ref. ). further investigations are needed. needless to say, at late stage of ards, controlling secondary bacterial infection should not take lightly. in addition to treatment, cq/hcq has also been suggested for prophylaxis of covid- (ref. ). when awaiting more evidence from clinical trials, we currently do not recommend use cq/hcq for prophylaxis of covid- . lopinavir/ritonavir (lpv/r) is a mix of protease inhibitors commonly used for the therapy of hiv- infection, often known as highly active anti-retroviral therapy (haart). the combination of lpv/r with additional ribavirin was reported to be able to reduce the risk of acute respiratory distress syndrome and mortality in sars patients . meanwhile, lpv/r and interferon β were found to have better efficacy compared with controls for mers-cov infection in animal experiments and case reports [ ] [ ] [ ] [ ] . lpv/r was therefore repurposed to treat covid- , as sars-cov- was genetically close to mers-cov and sars-cov. however, in a retrospective study with patients (more than % patients were mild cases), we recently find that administration of lpv/r along with interferon alpha inhaling was not associated with faster virological clearance or clinical improvement . more recently, a randomized, controlled study conducted in wuhan, china also failed to identify beneficial effect of lpv/r beyond standard therapy in hospitalized patients with severe covid- (ref. ). more importantly, in another randomized control study with a small sample size, lpv/r was associated with adverse events not only failed to show antiviral activity . based on these available data, it is likely that lpv/r and other hiv- protease inhibitors including darunavir are not effective in treating covid- . another promising drug is the adenosine analog remdesivir (gs- ) that is incorporated into nascent viral rna chains where it causes a pre-mature termination . remdesivir is also an experimental drug that was generated for the therapy of the ebola viral infection. notably, remdesivir has shown antiviral efficacy in treating sars and mers in animal models , . recently, in vitro study showed that, in vero e cells, ec value of remdesivir versus sars-cov- was . μm, indicating its active concentration could likely be obtained in vivo . in sars-cov- -infected rhesus macaques, therapeutic remdesivir treatment was found to reduce viral load when given early . in the usa, the first covid- patient was treated with intravenous remdesivir and recovered . in a recent cohort of hospitalized patients with severe covid- , compassionate-use remdesivir was associated with clinical improvement in of patients ( %) . in a randomized, double-blind, placebo-controlled, multicentre trial, remdesivir use was not associated with a difference in time to clinical improvement. however, when given with symptom duration of days or less, patients receiving remdesivir had a numerically faster time to clinical improvement than those receiving placebo . this study was underpowered as it failed to enroll the estimated number of participants. however, preliminary results from another nih clinical trial shows remdesivir was associated with % faster time to recovery from advanced covid- and a marginal survival benefit, with a mortality rate of . % for the group receiving remdesivir versus . % for the placebo group (p = . ) . as an emergent therapeutic approach, fda recently issued emergency-use authorization for remdesivir to treat hospitalized patients with severe covid- . several clinical trials are currently ongoing, and the results regarding effectiveness and safety are still being awaited for. the recent work elucidating the structure and molecular mechanism of viral entry [ ] [ ] [ ] showed the specificity of the serine protease inhibitor camostat mesylate as an active compond against tmprss which is required for viral infection. camostat mesylate seems rather specific, since sars-cov- infection requires tmprss for the priming of the viral s protein. clinical trial of camostat mesylate or its derivatives are therefore required - . the recent work on the fda-approved anti-parasitic compound ivermectin seems extremely powerful even though so far it is only in vitro . clinical trial to repurpose this approved drug is urgently needed. convalescent plasma is also a potentially promising strategy to treat covid- . in a recent case study, the clinical status of all the five critically ill covid- patients receiving convalescent plasma showed a significant improvement within week following the infusion, normalization of body temperature, as well as scores of the sequential organ failure assessment. moreover, within - days following the infusion, the neutralizing antibody titers of the patients improved and the respiratory samples tested negative for sars-cov- (ref. ). in another study of severe cases, the viral titers were undetectable following the infusion in seven patients who had previously high viremia . previous studies on other respiratory viral diseases provided some evidences on the efficacy of convalescent plasma on treating severe and critical viral diseases. several studies in sars patients reported that the use of convalescent plasma was linked to reduced hospital stay and reduced mortality , . clinical trials also showed that in patients with severe h n influenza a, in the pandemic, therapy with convalescent plasma from patients who recovered, especially within days of symptom onset, resulted in a lower viral load and lower mortality , . subsequent analysis showed that the mortality of patients with severe acute viral respiratory infections was reduced after therapy with convalescent plasma, while absence of adverse events or complications were observed . nevertheless, there are still issues we need to tackle. the first question is when to collect plasma from recovered covid- patients. recent work by to et al. showed that, day after symptom onset, both igg and igm antibodies increased in the majority of patients, while seroconversion was observed within the first weeks. importantly, the anti-sars-cov- igg and igm antibody levels against the internal nucleoprotein and the spike s domain correlated with neutralizing activity. therefore, it would be ideally to collect convalescent plasma from week after symptom onset. despite hundreds of patients had recovered from covid- , eligible convalescent plasma is quite limited as the donors have to pass physical and laboratory examination, and plasma should also be tested for sars-cov- nuclear acid, hiv- , hbv, and hcv, as well as antibody titers, to list a few. the second question is deciding which patients and when should receive the convalescent plasma. the effects of convalescent plasma are difficult to observed when used in critical patients with multiple organ failure, as the viral load in this kind of population is quite high. hence it is preferably to use convalescent plasma in mild patients whose diseases was deteriorating in their early phase of diseases. normally, in covid- , the viral load peaked at the first week of illness, and then slowly decline during the subsequent week . accordingly, in principle, the most effective to administer the convalescent plasma is at the early phases of the disease. the biggest challenge is that it is quite difficult to identify which patient will deteriorate in the early stage. several risk factors including older age, male, multiple comorbidities, elevated il- , and elevation in ddimer levels that are associated with bad outcomes may be used as surrogate markers . provided further studies demonstrate its efficacy in appropriately selected patients, the next step would be the production of humanized antibody at biotechnological level. it is still not clear why some patients progressed while others recovered, which underlying biological marker would be of essential benefit for the management of the patients. indeed, several reports are still in wide contrast. the duration from onset of symptoms to viral clearance is significantly longer in severe and critical ill sars-cov- infected patients compared with that in the mild cases . notably, elevated level of a bundle of pro-inflammatory cytokines was observed in severe and critical ill patients, which include interferon-γ inducible protein , interleukin (il)− , il- , il- , macrophage inflammatory protein -α, granulocyte-colony stimulating factor, tumor necrosis factor-α, and monocyte chemoattractant protein (refs. , ). more importantly, in a recent retrospective, multicenter study conducted in wuhan, china, increased plasma levels of ferritin and il- were identified as predictors of fatality . therefore, it is reasonable to speculate that the persistence of sars-cov- induced excessive and abnormal non-effective response that leads to organ dysfunction , . the inflammation persists in some patients despite the viral clearance , . taken together, these evidences support the importance of dampening the overly exuberant immune responses besides antiviral therapy in reducing the mortality , as we discussed previously . the dual role of the immune system is crucial and still under investigation to clarify the strength and duration of the immune response both for the perspective of postinfection protection and re-infection, but also, and most relevant, for the efficacy of the future vaccine. many therapeutic drugs are now available to suppress immune response. among these drugs, corticosteroid is the most widely used. however, the utility of corticosteroid in treating covid- is still debating. most guidelines on covid- currently do not recommend application of corticosteroid as data are very limited. on the one hand, corticosteroid apparently delay clearance of mers-cov from respiratory tract and sars-cov from blood, respectively , . on the other hand, clinical studies reported that low or physiologic dose of corticosteroids treatment could have clinical benefits to earlier reversal of shock, shorter stay in icu, and less mechanical ventilation although it did not reduce mortality caused by primary lung infections . in patients with severe h n -illness, low dose of corticosteroids also lead to lower mortality . several clinical trials are now in progress to evaluate the benefit of corticosteroids in treating covid- . when waiting for the results, proper use of low-dose corticosteroids at the right time might has survival advantages for severe/critically ill patients with covid- . cq and hcq might also be able to modulate the over activated immune response. they alter the ph and disrupt autophagy and lysosomal activity, destabilize membrane, and disrupt signaling pathways and transcriptional activity, subsequently inhibiting antigen presentation, immune activation, mhc class ii expression, reduced proinflammatory cytokines, and deregulation of costimulatory molecules . however, whether its suppression on the immune response may lead to delay of sars-cov- clearance is still unclear. in some viral diseases including hiv infection, hcq administration was associated with increased viral replication . other therapeutic drugs that under investigation include the il- inhibitor, il- inhibitor, and jak-stat signal pathway inhibitors. treatment with il- blockade, in a phase randomized controlled trial in sepsis patients with macrophage activation syndrome, resulted in a drastic improvement in the -day survival rate . il- inhibitor, which was used as a treatment for rheumatoid arthritis and crs associated with car-t cell therapies for cancer, has now repurposed to treating covid- in severe and critical patients as recommended by a chinese government guideline . interestingly, preliminary results from the two available il- inhibitors (tocilizumab and sarilumab) with different study population showed contrasting results, indicating that choose appropriate population is essential for anti-il- treatment . upstream of that, the treatment with inhibitors of tnf-β could be applied, but there are no data available so far. inhibition of janus kinase (jak) in covid- patients target both inflammation and cellular viral entry . recently, a pilot study suggest that jak inhibitor is active, well tolerated in patients with secondary hemophagocytic lymphohistiocytosis indicating these drug maybe apply to covid- treatment . most of the patients can be discharged after archive negative pcr results from two continues respiratory tract specimens, together with defervescence and improvement in radiological image . the median duration from symptoms onset to the first negative pcr results was days in non-intensive care units group, as compared with days in the icu groups. in critical ill patients, the time of viral shedding can be as long as days. the slower clearance of the virus in the critical ill patients might be explained by both higher viral load at baseline and impaired immune responses. in a recent study, the mean sars-cov- viral load of critical ill patients was circa times higher than that of moderate cases, indicating that higher viral loads is linked to severe clinical outcomes . in addition, the critical ill patients also showed impaired immunity characterized by lymphopenia and lower cd t cell counts, which were associated with the duration of viral shedding , . our cohort in shanghai showed the median duration of hospital stay was days , , . however, studies have shown that viral shedding is longer in the feces than in the respiratory tract specimens . more importantly, sars-cov- isolated from feces was of replicability. despite the uncertainty of transmissibility of sars-cov- in feces, it indeed challenged the quarantine given the high infectivity of the virus. notably, pcr turned to be positive again in a small proportion of patients, although no infection cases of these close contactors had been reported. therefore, we recommend that patients recovered and discharged should be continue to quarantine for another days. some models had attempted to quantify the impact of different strategies to prevent the potential spread of covid- that should be carefully considered for policy makers in planning the next steps. in interpreting these models, we should keep in mind that they assume that variations in the replication rate of sars-cov- -an evaluation of transmission -are an immediate response to the interventions implemented, rather than broader gradual behavioral changes. while first models, however, addressed a set of variables before locking down, on the number of cases and avoided deaths, more recently new models are struggling to predict the effect of gradual normalization of public life of different strategies. in fact, despite the fact that in italy, spain, and germany the increase in number of new sars-cov- infections has been reducing in recent days, the political measures (on early april ) for restricting contacts at national level continue to be in place. in a recent report from the leopoldina nationale akademia der wissenchaften , authors concluded that in addition to the current recommendations (hygiene, physical distancing), also nose and mouth protection, testing, use of digital data and targeted quarantine should be reinforced, see fig. and also ref. . assuming, if optimally implemented, that the rate of new infections caused by an distinct person could be lower than . , the aforementioned measures might after several months' lockdown, china is now reopening, facing the risk of second wave of covd- . in italy, despite lots of patients had already been infected, second wave of diseases is also challenging should they reopen the cities, as there is currently no evidence that people who have recovered are protected from a second infection. while waiting for an effective vaccine for covid- and for a clear specific treatment, current management is highly depended on supportive therapy. the best way to reduce the mortality is therefore related first of all to risk reduction of sars-cov- infection. this might be achieved by combining six main actions: social distance, masks, hygiene recommendations, contact tracing, extensive use of early diagnosis tools, and confirmed and suspected cases quarantine. we learnt from countries like south korea and singapore that such approaches can be feasible, even in absence of very strict lockdown. secondly, strengthen health systems capacity in order to be fully equipped 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retrospective study baseline characteristics and outcomes of patients infected with sars-cov- admitted to icus of the lombardy region the authors would like to thank dr. e. carafoli for critical comments and suggestions. the work has been partially supported by grants from the national key key: cord- - gn zhvt authors: huybrechts, krista f.; bateman, brian t.; zhu, yanmin; straub, loreen; mogun, helen; kim, seoyoung c.; desai, rishi j.; hernandez-diaz, sonia title: hydroxychloroquine early in pregnancy and risk of birth defects date: - - journal: am j obstet gynecol doi: . /j.ajog. . . sha: doc_id: cord_uid: gn zhvt background hydroxychloroquine (hcq) is generally considered safe in pregnancy for the treatment of rheumatic conditions, but studies have been too small to evaluate teratogenicity. quantifying the risk of congenital malformations associated with early pregnancy exposure to hcq is important both in the context of its ongoing use for rheumatological disorders as well as its potential future use for covid- prophylaxis, for which a number of clinical trials are ongoing despite initial trials for covid- treatment having been negative. objective the study objective was to evaluate the risk of major congenital malformations associated with exposure to hcq during the first trimester, the period of organogenesis. study design we performed a population-based cohort study nested in the medicaid analytic extract (max, - ) and ibm marketscan research database (marketscan, - ). the source cohort included , hcq exposed and , , unexposed pregnancies continuously enrolled in their respective insurance program from months before the last menstrual period through at least one month after delivery; infants were enrolled for at least months after birth. we compared the risk of congenital malformations in women with hcq use during the first trimester versus no use, restricting the cohort to women with rheumatic disorders and using propensity score matching to control for indication, demographics, medical comorbidities, and concomitant medications (n= , hcq exposed; , unexposed pregnancies). the outcomes considered included major congenital malformations diagnosed during the first days after delivery, and specific malformation types for which there were at least exposed events: oral clefts, cardiac, respiratory, gastrointestinal, genital, urinary, musculoskeletal, and limb defects. results overall, . per , infants exposed to hcq were born with a major congenital malformation versus . per , unexposed infants, corresponding to an unadjusted relative risk of . ( % ci, . – . ). patient characteristics were balanced in the restricted, propensity score matched cohort. the adjusted relative risk was . ( . – . ); it was . ( . - . ) for a daily dose ≥ mg and . ( . - . ) for < mg. among the different malformation groups considered, more substantial increases in the risk for oral clefts, respiratory anomalies and urinary defects were observed, although estimates were imprecise. no pattern of malformations was identified. conclusions our findings suggest a small increase in the risk of malformations associated with first trimester hcq use. for most patients with autoimmune rheumatic disorders, the benefits of treatment during pregnancy will likely outweigh this risk. if hcq were shown to be effective for covid- prophylaxis in ongoing trials, the risk of malformations would need to be balanced against such benefits. hydroxychloroquine (hcq) is an antimalarial drug widely used in the treatment of systemic lupus erythematosus (sle) and other rheumatic disorders. it is generally considered to be safe for treatment of autoimmune rheumatic conditions during pregnancy and continuation of hcq during pregnancy is commonly recommended to improve disease management and pregnancy outcomes. - studies have been too small, however, to evaluate teratogenicity. over the last several months, there has been heightened interest in hcq due to it being a candidate drug for the treatment and/or prophylaxis of coronavirus disease . health organization has discontinued the hcq arm of the solidarity trial evaluating its efficacy for the treatment of hospitalized patients, numerous randomized controlled studies are still ongoing in particular to evaluate its effects for pre-exposure prophylaxis and including a trial in pregnant women. most studies regarding the safety of hcq when used for malaria and for rheumatic disorders like sle suggest no increase in the risk of common adverse obstetrical outcomes such as spontaneous abortion, prematurity, and intrauterine growth restriction. [ ] [ ] [ ] [ ] however, data regarding the risk of major congenital malformations associated with early pregnancy exposure is very limited, with the largest published cohort study including fewer than exposed pregnancies (supplementary table ). quantification of the risk of congenital malformations associated with early pregnancy exposure to hcq is therefore important both in the context of its ongoing use for rheumatological disorders as well as its potential future use for j o u r n a l p r e -p r o o f although based on the results of initial trials, its usefulness in this clinical context remains highly uncertain. given the limited data currently available, we evaluated the risk for major congenital malformations associated with hcq using two large healthcare utilization databases. include demographic and insurance enrollment information, medical visits and hospitalizations, diagnoses and procedures received as an in-or outpatient, and prescriptions filled on an outpatient basis. the development of the linked mother-infant pregnancy cohorts has been described previously. , briefly, we identified all completed pregnancies in women to years of age and linked these pregnancies to liveborn infants by state, family identification number and delivery/birth dates. using a validated algorithm, we estimated the date of the last menstrual period on the basis of the delivery date and diagnostic codes indicative of preterm delivery. mothers were required to be continuously insured from months before the start of pregnancy until month after delivery. infants were required to be insured from birth until months thereafter, unless they died sooner. these restrictions did not affect the age or race distribution in max, but resulted in a decrease in the proportion of who become medicaid eligible due to the occurrence of pregnancy and a corresponding increase in the proportion who become eligible based on other criteria. pregnancies with exposure to a known teratogenic medication (i.e., warfarin, antineoplastic agents, lithium, isotretinoin, misoprostol, thalidomide) during the first trimester and pregnancies with a chromosomal abnormality were excluded. j o u r n a l p r e -p r o o f exposure. women were considered exposed if they filled a prescription for hcq during the first trimester of pregnancy (defined as date of the last menstrual period through day of pregnancy), the etiologically relevant exposure window for congenital malformations. the reference group consisted of women without a prescription for hcq from months before the start of pregnancy through the end of the first trimester given hcq's long half-life and to reduce the probability of exposure during early pregnancy from use of hcq dispensed at an earlier time point. outcomes. the outcome of interest was major congenital malformations overall. in secondary analyses we also evaluated specific malformation types for which we observed at least exposed events across the two cohorts: oral clefts, cardiac, respiratory, gastrointestinal, genital, urinary, musculoskeletal, and limb defects. the presence of malformations was defined using validated algorithms based on inpatient or outpatient diagnoses and procedures, which have been shown to identify the outcomes with high specificity (i.e., more than one date with the respective diagnostic codes recorded, or one diagnostic code and a code for a procedure/surgery or infant death). isolated congenital heart block was not included in the definition for cardiac malformations because its risk is increased in babies born to women with sle. (supplementary table covariates. potential confounders and proxies for confounders considered included sociodemographic information (e.g., state of residence, age, race/ethnicity (max only)), autoimmune rheumatic disorders (e.g., rheumatoid arthritis, sle, ankylosing spondylitis, psoriatic arthritis), other maternal conditions (e.g., diabetes, hypertension, psychiatric conditions, renal disease, neurological conditions, chronic respiratory conditions, anemia, infections), concomitant medication use (e.g., systemic steroids, non-biologic and biologic disease modifying antirheumatic drugs (dmards), psychiatric medications, nsaids, suspected teratogens), and general markers of the burden of illness (e.g., maternal comorbidity index, healthcare utilization measures) (table , supplementary table and ) . analyses. baseline characteristics were compared between women exposed to hcq and the reference group of unexposed women. relative risks (rr) with their % confidence intervals (ci) were estimated using generalized linear models. as a first level of adjustment, the reference group was restricted to women with a recorded diagnosis of autoimmune rheumatic disorders commonly treated with hcq ('restricted cohort'). in fully adjusted analyses, exposed and unexposed women in the restricted cohort were matched on their propensity score (ps), using a : variable ratio matching and a . caliper ('restricted matched cohort'). the ps, which reflects the probability of being treated with hcq, was estimated using a logistic regression model including all (> ) pre-specified covariates. when evaluating the balance in baseline characteristics in the restricted matched cohort, the counts for the unexposed group were weighted to account for the variable ratio matching. we conducted analyses stratified by dose, using the highest daily dose dispensed during the first trimester (< mg and ≥ mg daily) as well as duration of exposure (≤ days and > days). in a sensitivity analysis, both the exposed and the reference group were restricted to women with a recorded diagnosis of autoimmune rheumatic disorders. estimates from both cohorts were combined using a meta- analytic approach with random effects. for all analyses presented, results were described as similar or different from the reference group based on the magnitude of the point estimates, taking into account the precision of each estimate as reflected in the width of its % ci. we focused on estimating magnitude of effects in preference to dichotomizing the results as statistically significant or not. the research was approved by the institutional review board of brigham and women's hospital, which waived the need for informed consent. the combined cohort included , pregnancies exposed to hcq during the first trimester ( in max and , in ms), and , , unexposed pregnancies ( , , in max and , , in ms). the mean daily dose of hcq was mg (standard deviation, mg) and % of women used a daily dose of mg. among exposed women, . % were exposed for ≤ days, . % for to days, and . % for > days during the first trimester. women exposed to hcq tended to be older, had more comorbid conditions, took more concomitant medications (especially pain medications, steroids, nsaids, and dmards) and had greater health care utilization. after cohort restriction and adjustment through ps matching, all covariates -including treatment indications -were well balanced ( table the pooled risk of any congenital malformation was . per , hcq exposed infants (n= events) and . per , unexposed infants in the general population (n= , events), corresponding to a pooled unadjusted rr of . ( % ci, . - . ). restricting the reference group to women with rheumatic disorders resulted in an absolute risk of . per , unexposed infants (n= events out of , ) and a rr of . ( % ci, . - . ). adjusting for all potential cofounding variables through ps matching did not result in further attenuation of the association (rr= . ; % ci, . - . ). estimates were consistent between the two cohorts. the risk of malformations among the hcq exposed was the same regardless of whether women had concomitant exposure to steroids. the adjusted rr was . ( % ci, . - . ) for a daily dose of ≥ mg and . ( % ci, . - . ) for < mg. the risk was not affected by the duration of exposure (figure ) , and results were similar when restricting both the exposed and the reference group to women with a recorded diagnosis of autoimmune rheumatic disorders (supplementary table ) . j o u r n a l p r e -p r o o f in the context of few events, risk estimates for the specific malformation types considered were relatively imprecise ( figure ). the point estimates indicated an approximately to -fold increase in the risks for oral clefts (rr= . ; % ci, . - . ), respiratory defects (rr= . ; % ci, . - . ), and urinary defects (rr= . ; % ci, . - . ), which were consistent between the two cohorts. none of the hcq exposed cases of oral clefts had concomitant exposure to steroids. the upper limit of the % ci for the pooled estimates suggested no more than a -fold increase in risk for other specific malformation types with the exception of genital defects (upper limit % ci= . ). among the exposed infants with malformations, utilizing data from health plans that provide coverage for large populations of both commercially and publicly insured individuals in the us, we identified a cohort of pregnant women with chronic autoimmune rheumatic diseases and assessed the relative prevalence of major congenital malformations in their newborns following exposure to hcq during early pregnancy. women who filled prescriptions for hcq during the first trimester had a higher risk of malformations in their newborn than the general population. upon restriction to women with the indication (mainly sle and rheumatoid arthritis), the relative risk attenuated but was still elevated. in utero exposed newborns had an adjusted risk of major congenital malformations % higher than unexposed overall, and % higher for daily doses of mg or greater (while no increased risk was observed for lower doses based on the point estimate, reflecting the estimate most consistent with the data). a more substantial increase in the risk for oral clefts, respiratory anomalies and urinary defects was observed, although confidence intervals for specific malformations were wide. no pattern of malformations was identified. prior studies evaluating the safety of hcq in pregnancy included between and women and overall suggested no increased risk of pregnancy losses, prematurity, intrauterine growth retardation, pre-eclampsia, fetal distress or induction of delivery compared to reference groups. , , [ ] [ ] [ ] [ ] since flares are associated with these and other complications and hcq is effective at controlling them, drug use in pregnancy may improve pregnancy outcomes for women with rheumatic disorders, as well as reduce the risk for congenital heart block in the neonate. moreover, hcq use reduces the dose of prednisone needed during pregnancy. however, most of these studies were too small to assess the risk of major malformations, and many based their conclusion on the statistical significance of underpowered comparisons. given that hcq crosses the placenta and inhibits cell division and dna synthesis, and that initial reports suggested an increased risk of chromosomal damage attributable to chloroquine, concerns regarding effects on the rapidly dividing embryonic cells remain. specific malformations reported among exposed newborns included cleft lip and palate ( out of ) and pulmonary hypoplasia in a preterm infant ( out of ). moreover, two of the largest studies found a meaningful, though not statistically significant, increased risk of malformations overall. in one study, malformations were more common in the hcq exposed ( . %) than in the reference ( . %) group (adjusted rr . ; % ci, . - . ), with no clear pattern. in another study, the hcq exposed had a prevalence of malformations of . % ( / ) and the reference group of . % ( / ) with a p-value of . , again with no clear pattern. for pregnant women with malaria or rheumatic disorders the benefits of hcq may still outweigh the potential risk, especially given that discontinuation of hcq after conception would not necessarily prevent birth defects because the half-life is more than a month, and would increase the risk for flares and their complications. therefore, our findings of a potential small increase in the risk of malformations -while important for prescribers to be aware of -should not necessarily alter the treatment recommendation for a given woman with malaria or rheumatic disorders. for covid- , it will depend on whether the currently ongoing clinical trials demonstrate meaningful benefits of hcq in reducing covid- infection or its severity. while initial trials using hcq to treat covid- have failed to demonstrate efficacy, trials regarding its use for preexposure prophylaxis have not yet been reported. in addition to several strengths (including a large and nationally representative population, as well as robust control for confounding through restriction and matching), our study is also subject to certain limitations, most of which would bias the results towards the null. first, we included only women with a live-born delivery because abortions and stillbirths are incompletely recorded in healthcare utilization data. this approach may have resulted in the exclusion of pregnancies with the outcome, as fetuses with malformations are more likely to experience fetal death or termination. therefore, the incidence of major malformations reported in this study could underestimate the risk in pregnant women. if a higher proportion of women on hcq had lethal malformations, more prenatal screening or a higher propensity to terminate an affected pregnancy, this study would also underestimate the relative risk. however, differential terminations have been shown to be an unlikely source of selection bias. second, identification of major congenital malformations was based on diagnosis and/or procedure codes recorded in claims. misclassification would tend to bias relative risks towards the null unless a higher proportion of malformation diagnoses were identified in women exposed to hcq. while others and we have shown a high positive predictive value for malformations , , , the potential for some misclassification remains. third, information on hcq exposure is obtained from claims of filled prescriptions. since some women may fill prescriptions for medications but not use them, our study may misclassify unexposed pregnancies into the hcq group, thus underestimating any potential effect; however, a large fraction of our cohort filled prescriptions for hcq throughout the first trimester. fourth, it is possible that some women in the reference group were taking immunomodulatory agents in lieu of hcq. if these agents were teratogenic, we would be underestimating the effect of hcq. however, their use is negligible during pregnancy and upon restriction and propensity score matching our exposed and reference groups were balanced in the use of these medications. fifth, disease flares in women with rheumatic disorders such as sle have been associated with poor pregnancy outcomes and hcq use during pregnancy improves disease activity and reduces the antiphospholipid syndrome. , therefore, the reference group of women with the disease and without hcq could have a higher risk, thus potentially underestimating the relative risk for flare-related adverse pregnancy outcomes including fetal loss, fetal growth retardation and prematurity. alternatively, it is possible that there is misclassification of the unexposed with respect to the presence of underlying rheumatic disease or that women being treated with hcq have more severe underlying disease than women without hcq. although neither rheumatic disorders nor flares have been associated with congenital malformations, it is conceivable that women with more severe disease receive higher doses of steroids and this may not be fully captured in our data. however, recent studies , have refuted initial reports of strong associations between steroids and oral clefts. more directly, in the current study, the absolute risk of malformations was the same among hcq exposed pregnancies with and without concomitant exposure to systemic steroids, and none of the cases of oral clefts in the hcq exposed were exposed to steroids. together, this suggests that steroid exposure is not a major threat to the validity of our analyses. sixth, the max cohort included data through -the most recent data available at the time of study conduct -and marketscan included data through , to avoid the use of icd- based algorithms for cohort creation and outcome identification that have not yet been validated. however, the biological association between hcq exposure and malformations should not change over time. finally, despite being the largest exposed cohort to date, the numbers were small for specific malformation groups and confidence intervals were wide; specific individual defects could not be examined. however, there is enough information to suggest that the magnitude of a potential risk of malformations would not be on the order of that associated with major teratogens. in this study, there was no evidence of a large increase in prevalence of major congenital malformations in the newborn from first-trimester maternal exposure to hcq. however, it is the third study to suggest a moderate increased risk. for most patients with autoimmune rheumatic disorders, the benefits of treatment during pregnancy will likely outweigh this risk. if proven effective for covid- prophylaxis in ongoing randomized trials, the benefits of hcq would need to be weighed against the potential risk in pregnancy. rheumatic disorders, the number of outcome events and the total number of deliveries in both the hydroxychloroquine exposed and the unexposed group are not meaningful for absolute risk estimation; therefore, counts after restriction to deliveries with rheumatic disorders but before the propensity score matching are reported in figure . table selected patient characteristics for hydroxychloroquine exposed and unexposed pregnancies miscarriage risk was % in exposed and % in unexposed; and stillbirth % and %, respectively. among the liveborn exposed, had cleft lip and palate. in the unexposed fetuses had fatal congenital anomalies and had an abdominal hernia. limited sample to estimate the relative risk of malformations. controlled study. in-and outpatient claims in the infant record between date of birth (dob) and dob+ days and/or in the maternal record between delivery and delivery+ days are considered. • ≥ dates with a code for a malformation within the group o exception: codes . x, . , . x, . x require ≥ dates with a code for a malformation of which at least one code is documented at ≥ weeks after dob • ≥ date with a code for a malformation within the subgroup and cardiac procedure o exception: code . • ≥ date with a code for a malformation within the subgroup and infant died • if codes identified in the maternal record between lmp and lmp+ days and there are no codes in the infant record between dob and dob+ days (i.e., only maternal codes between delivery and delivery+ ), the defect is considered a pre-existing maternal defect. • any of the subgroups of cardiovascular anomalies is present • ≥ dates with a code for any of the cardiac malformations (regardless of the subgroup [ ]) • ≥ date with a code for any of the cardiac malformations [ ] and cardiac procedure • ≥ date with a code for any of the cardiac malformations [ ] and infant died • if codes identified in the maternal record between lmp and lmp+ days and there are no codes in the infant record between dob and dob+ days (i.e., only maternal codes between delivery and delivery+ ), the defect is considered a pre-existing maternal defect. • ≥ dates with a code for the malformation group/subgroup o exception: for gastroschisis: if code . was used (before / ), require ≥ date with a code and icd- procedure . • ≥ date with a code for the malformation group/subgroup and malformation-specific procedure • ≥ date with a code for the malformation group/subgroup and infant died • if codes identified in the maternal record between lmp and lmp+ days and there are no codes in the infant record between dob and dob+ days (i.e., only maternal codes between delivery and delivery+ ), the defect is considered a pre-existing maternal defect. • any of the malformation groups/subgroups mentioned above is present. [ ( ) given the variable ratio matching, the counts for the unexposed group are weighted counts to demonstrate the balance in baseline covariates. ( ) autoimmune rheumatic disorders were measured from months before the start through the end of pregnancy ( ) maternal conditions and concomitant medication use were measured from months before the start of pregnancy through the end of the first trimester ( ) women exposed to known teratogens have been excluded (i.e., warfarin, antineoplastic agents, lithium, isotretinoin, misoprostol, thalidomide). suspected teratogens considered include danazol, methimazole, propylthiouracil, aminoglycosides, trimethoprim, triamterene, sulfasalazine, spasmophen, cholestyramine, potassium iodide, tetracycline, fluconazole ( ) general markers of the burden of illness were measured during the months before but not during pregnancy, as these measures may be affected by early detection of pregnancy complications cell size < for max cohort are suppressed in accord with the cms cell size suppression policy. abbreviations: hcq, hydroxychloroquine; n, number; nsaids, nonsteroidal anti-inflammatory drugs; dmards, disease modifying antirheumatic drugs; sd, standard deviation; stand diff, standardized difference american college of rheumatology guideline for the management of reproductive health in rheumatic and musculoskeletal diseases bsr and bhpr guideline on prescribing drugs in pregnancy and breastfeeding-part i: standard and biologic disease modifying anti- rheumatic drugs and corticosteroids trends in use of hydroxychloroquine during pregnancy in systemic lupus erythematosus patients from observational study of hydroxychloroquine in hospitalized patients with covid- association of treatment with hydroxychloroquine or azithromycin with in-hospital mortality in patients with covid- in new york state a randomized trial of hydroxychloroquine as postexposure prophylaxis for covid- drug evaluation during the covid- pandemic. the new england journal of medicine hydroxychloroquine efficacy and safety in preventing sars-cov- infection and covid- disease severity during pregnancy (covid-preg). nct available at clinicaltrials.gov safety of hydroxychloroquine in pregnant patients with connective tissue diseases: a study of one hundred thirty-three cases compared with a control group brief report: risk of adverse fetal outcomes associated with immunosuppressive medications for chronic immune-mediated diseases in pregnancy harnessing the medicaid analytic extract (max) to evaluate medications in pregnancy: design considerations identifying pregnancies in insurance claims data: methods and application to retinoid teratogenic surveillance algorithms to estimate the beginning of pregnancy in administrative databases pregnancy outcome following first trimester exposure to chloroquine hydroxychloroquine and lupus pregnancy: review of a series of cases hydroxychloroquine in pregnant patients with systemic lupus erythematosus hydroxychloroquine (hcq) in lupus pregnancy: double-blind and placebo-controlled study safety of hydroxychloroquine in pregnant patients with connective tissue diseases: a study of one hundred thirty-three cases compared with a control group follow-up of infants exposed to hydroxychloroquine given to mothers during pregnancy and lactation hydroxychloroquine in lupus pregnancy outcomes after anti-rheumatic drug use before and during pregnancy: a cohort study among , pregnant women and expectant fathers pregnancy outcome following in utero exposure to hydroxychloroquine: a prospective comparative observational study brief report: risk of adverse fetal outcomes associated with immunosuppressive medications for chronic immune-mediated diseases in pregnancy longterm outcomes of children born to mothers with sle exposed to hydroxychloroquine in pregnancy hydroxychloroquine and pregnancy on lupus flares in korean patients with systemic lupus erythematosus key: cord- - q un rl authors: giri, allan; das, ankita; sarkar, ajoy k.; giri, ashok k. title: mutagenic, genotoxic and immunomodulatory effects of hydroxychloroquine and chloroquine: a review to evaluate its potential to use as a prophylactic drug against covid- date: - - journal: genes environ doi: . /s - - - sha: doc_id: cord_uid: q un rl hydroxychloroquine (hcq) and chloroquine (cq) are two anti-malarial drugs that are now being extensively used by front-line healthcare workers and other common people as a prophylactic drug against the corona virus disease − (covid- ) in india and as well as in many parts of the world. while only a few in vitro studies have pointed to some efficacy of these drugs as a prophylactic against covid- , to date, there are no clinical studies that have established any clinical efficacy of these drugs as a prophylactic. these drugs are commonly used for the treatment of rheumatoid arthritis (ra) and systemic lupus erythematosus (sle) because of its immunomodulatory effects. previously, we have evaluated the genetic toxicology of different drugs and chemicals including antimalarial drug cq both in vitro and in vivo. thus, we recognize the need to critically review the mutagenic, genotoxic, and immunomodulatory effects of these drugs, to find out whether it is safe to use as a prophylactic drug against covid- . existing literature suggests that cq can induce mutagenic and genotoxic effects in multiple test systems and both the drugs have immunomodulatory effects. there was no data available to evaluate the mutagenicity and genotoxicity for hcq. however, during metabolism about % of both the drugs remain unchanged and about % of the drugs are metabolized into two metabolites, desethylchloroquine and bisdesethylchloroquine by the action of the cytochrome p (cyp) enzymes in the liver. both hcq and cq are immunomodulatory drugs and have the potential to suppress normal immune system activation. in this review, we have elucidated the mechanism of immunomodulation by both hcq and cq and highlighted the mutagenic and genotoxic effects from the available literature. this article is written with the sole objective that the reader will be able to recognize the adverse effects of these drugs when consumed by healthy individuals as a prophylactic. current literature indicates that healthy individuals should refrain from the use of these drugs until further investigation. the novel severe acute respiratory syndrome corona virus (covid- ) pandemic has now become a nightmare throughout the world. this pandemic has caused serious health crisis not only among the poor nations but also across the world's advanced countries. researches all across the globe are trying to find an effective drug that would show promising results to prevent or to treat and control the covid- . recently, scientists have pointed out that the novel covid- was transmissible in aerosol [ ] . thus, it puts the healthcare workers at risk who works in close proximity with covid- patients. this demanded the need for a prophylactic drug against covid- amongst healthcare workers. hydroxychloroquine (hcq) and chloroquine (cq) are two antimalarial drugs that remain the universally accepted drugs for the treatment of rheumatoid arthritis and systemic lupus erythematosus [ ] [ ] [ ] [ ] . these two drugs have shown some results in inhibiting the novel covid- in vitro [ , ] . a recent study demonstrated that certain cell types when treated with hcq or cq and then exposed to the novel covid- strain, presented antiviral activity and that hcq was more potent than cq [ ] . on the other hand, another in vitro study found out that cq was potent than hcq at all four different multiplicities of infection to act as antiviral when exposed to covid- post-incubation with these drugs [ ] . additionally, cq was able to act as anti-viral both pre and post-infection against the covid- in vitro [ ] . these findings may have led to the proposal and optimistic use of hcq and cq as prophylactics. yet historically, we have seen that in vitro studies don't always translate in vivo or human subjects. for instance, despite the strong evidence of cq as a prophylactic against influenza a and b in vitro, cq was not effective to prevent either influenza a or b in the human subjects [ ] . rather, dizziness, nausea, and diarrhea were more common in the cq group compared to the control (placebo) group. another study showed that ebola virus replication was successfully inhibited in vitro by cq, however, it failed in guinea pig models in vivo [ ] . yet another study showed cq enhanced chikungunya virus replication in vivo when in fact cq had been shown to have an effective inhibitory effect in vitro [ ] . thus, to date, with the lack of any controlled clinical trials, the clinical effectiveness of these drugs as prophylactics against covid- in vivo remains unanswered. like any other drugs, these drugs also comes with certain risks. so, we mustn't overlook the toxicological risks while making a rational decision of using these drugs as prophylactics. previously, we have extensively reviewed and evaluated the genetic toxicology of antimalarial, analgesics, antipyretic drugs including cq [ ] [ ] [ ] . cq and hcq are both water-soluble drugs that are absorbed rapidly in the gut and have a long elimination half-life in the plasma of and h respectively [ ] . multiple authors, including us, have reported in vitro and in vivo evidence of cq-induced genotoxicity in the mammalian system. these drugs also possess immunomodulatory roles that have the potential to suppress the activation of the immune system in healthy individuals [ ] [ ] [ ] . considering the current situation, there is an urgent need for clinical studies to evaluate the clinical efficacy of hcq and cq as a prophylactic drug against covid- . we have not included the long-term side effects of these drugs since it is unlikely that prophylactic use of these drugs would be for a long time. here in this review, we mainly aim to critically review and discuss the mutagenic, genotoxic, and immunomodulatory aspects of hcq and cq using the available literature. mutagenic and genotoxic effect table summarizes the mutagenic, genotoxic and carcinogenic effects of cq in multiple test systems. it is interesting to note that there is almost no report on the mutagenic and genotoxic effects of hcq both in vitro and in vivo. however, both hcq and cq have a very similar, flat aromatic core structure with a basic side chain. the only difference is the presence of an additional hydroxyl (−oh) group in hcq. during metabolism, about % of both the drugs remain unchanged and about % of both the drugs are metabolized into two common metabolites desethylchloroquine and bisdesethylchloroquine by the action of the cytochrome p (cyp) enzymes in the liver [ ] . despite cq being recognized as more toxic than hcq, the tissue and plasma distribution of these two drugs were reported to be nearly identical when administered in equivalent dosage in humans [ ] . figure shows the comparison of structures and metabolism of hcq and cq as described by schrezenmeier and dorner [ ] . hcq produces two first-stage metabolites instead of one. one being desthylhyoxychloroquine and the other desthylchloroquine. desthylchloroquine is also the firststage metabolite product of chloroquine. both the firststage metabolites are further metabolized to a common product, bisdesthylchloroquine [ ] . while many authors have reported cq to induce mutagenic effects in bacterial systems [ - , , - , ] , few authors had found weak or no mutagenic associations in certain bacterial strains [ , , , , [ ] [ ] [ ] . positive mutagenic effects (either weak positive or positive) reported by several authors showed that cq is capable of inducing mutation in the salmonella strains ta , ta a, ta and ta , which are used to detects the frameshift mutations. during the mutagenicity assay, most studies did not find any significant differences in the revertant numbers either with or without metabolic activation [ , [ ] [ ] [ ] . cq also can interact with dna and produce an intercalated complex that may induced frameshift mutation by shifting the reading frame [ , ] . this also indicates it's dna damage and inhibition of dna repair potentials reported by several authors [ , , , , ] . cq is further reported to induce sex-linked recessive lethal mutation in drosophila melanogaster [ ] . these results indicate the mutagenic potentials of cq in bacterial systems. in a broad genotoxic review on several antimalarial drugs, cumulative pieces of evidence pointed out that cq is also a genotoxic drug [ ] . our study on cq has demonstrated genotoxic effect as measured by chromosomal aberrations (ca), sister chromatid exchange (sce), and micronuclei (mn) formations in vivo in mice [ ] . these results are in agreement with several other authors who have reported cq to be a genotoxic drug in both in vitro and in vivo systems [ - , , ] . cq has also been reported to induce oxidative stress in animal models [ ] . for instance, when cq administered intraperitoneally in rats, it induced dna breaks in the kidney within to weeks and in the liver within to weeks [ ] . chromosomal aberrations (ca) have also been long considered to be a predictor for cancer. rossner et al., [ ] reported a strong association between increased frequencies of ca in cells and an increased risk for cancer using a cohort of , subjects. ca along with other genotoxic effects like sce and mn as reported here, suggests that long-term use of cq can induce significant chromosomal damages which may lead to an increased risk of cancer in humans. cq is not considered carcinogenic due to inadequate evidence pointing to carcinogenicity in humans. yet, a well-controlled study by el-mofty et al., [ ] in egyptian toad showed that separately cq and primaquine can induced tumor formation in and % of the toads respectively. they further showed that cq and primaquine when used together the incidence of tumor rose to . % [ ] . this type of co-tumorigenic effect of cq was also observed in another study by reyes et al., [ ] where cq promoted the carcinogenic effect of a drug called ethynitrosourea on ependymal cells of rodents in vivo. the only report of cq induced aplasis and leukemia was observed in a patient treated with long term cq therapy [ ] . brambilla and martelli, [ ] showed that n-nitroso compounds, which are capable of inducing genotoxic effects and tumor formation in animal models, can be formed in the gastric environment when cq is used with nitrite drugs. so, the genotoxiccarcinogenic effect may be induced when nitrite drugs are taken along with amine drugs like cq and hcq. results presented in the table indicate that cq is mutagenic and genotoxic drug in both bacterial systems, and in vitro and in vivo on mammalian systems. table summarizes the available reports on the immunomodulatory aspects of hcq and cq in multiple test systems. both hcq and cq has been reported to inhibit the activation of the immune system in many ways. lysosomotropic drugs (like hcq and cq) can accumulate inside lysosomes and being basic they can increase the ph inside the lysosomes and prevent its normal functions [ , ] . these drugs can also cause lysosomal membrane destabilization and thus the release of lysosomal contents and enzymes inside the cells [ ] . lysosomes have an essential role in the exogenous (lysosomal) pathways of antigen presentation and therefore proper lysosome functions are essential for mhc class ii antigen processing and presentation. the intervention of hcq and cq in the exogenous pathway of antigen presentation has been presented in fig. [ ] . autophagy is an implied concept in immunity development. besides the degradation and recycling of endogenous substrates, the process of autophagy is a key mechanism used by cells to tackle intracellular pathogens [ ] . hcq and cq both can potentially inhibit the normal autophagy processes. autophagosomes require fusion with the lysosomes to start the process of degradation. the increased ph of lysosomes, due to hcq and cq intervention, inhibits the maturation of the autolysosome. the inhibition of autophagy has been further linked to the induction of apoptosis of memory t-cells, which is the basis of the mechanism of immunomodulation by these drugs in several autoimmune diseases [ , ] . mechanism of the inhibition of autophagy by hcq and cq which triggers apoptosis has been presented in fig. [ ] . hcq can further block endosomal activation of nadph oxidase (nox ) that normally generates the reactive oxygen species and involved in the proinflammatory response of the immune system [ ] . with a decreased activity of nadph oxidase, cells can phagocyte pathogens but can't degrade them inside the phagocytic vesicle. hcq can also function as an immunosuppressant by blocking steps in the t-cell activation pathway. hcq has been shown to inhibit transcription factor nfat (nuclear factor of activated t-cells) upon t-cell activation in vivo and block expression of co-stimulatory ligand cd i.e. cd l, which initiates t-cell dependent bcell proliferation and antibody formation [ ] . the probable mechanism of the interference in the t-cell activation by hcq has been presented in fig. [ , ] . hcq intervention can further down-regulate the cd marker in healthy controls by inhibiting calcium mobilization and dephosphorylation of nfat [ ] . cd functions as a costimulatory molecule for t-cell activation and proliferation. the same study showed that b-cell antigen receptor calcium signaling was also reduced by hcq intervention [ ] . patients on antimalarial drugs like hcq and cq have lower levels of il- , soluble cd , and il- receptors which is beneficial for those suffering from autoimmune diseases like sle and ra [ ] . studies have shown that cq, at doses that are expected in the serum of treated patients, was able to interfere with mitogenic-response of monocytes and this diminished mitogenic response was determined to be irreversible [ ] . furthermore, cq inhibited the generation of immunoglobulin-secreting cells by preventing the capacity of monocytes to secrete factors like il- [ ] . normal secretion of tnf-α, il- b, and il- by monocytes or macrophages was also inhibited by cq [ ] . hcq and cq can potentially inhibit toll-like receptor (tlr) signaling of tlr and tlr in antigen presenting cells (apcs) including dendritic cells, macrophages and b-cells. normally, upon activation of by nucleic acids, endolysosomal tlr and tlr are cleaved which in turn activates myd and triggers an innate immune response in the downstream cascade. this proteolytic cleavage is inhibited by the changes in the endosomal ph as a result of hcq and cq interference [ ] . the mechanism of the inhibition of endosomal tlr and tlr by hcq and cq has been presented in fig. [ ] . hcq and cq have further been shown to directly bind to nucleic acid, inhibiting tlr-nucleic acid interaction and preventing tlr activation [ ] , and cq has been shown to inhibit rna-mediated tlr activation [ ] . hcq treatment in vivo caused a significant reduction of the production of inf-α and tnf-α by the plasmacytoid dendritic cells by suppressing the activation of tlr and tlr [ ] . innate tlr signaling leads to the production of cytokines such as il- , tnf-α, and il- that eventually triggers the adaptive immune response. at clinical concentration, hcq can efficiently block tlr ligation and have an inhibitory effect on classswitched memory b-cells [ ] . fig. intervention of hcq and cq in the exogenous pathway of antigen presentation: exogenous antigens that are taken up either by endocytosis or phagocytosis requires fusion with lysosomes for the process of degradation. finally, the degraded antigen fragments are loaded onto the mhc class ii molecules for antigen presentation. lysosomotropic drugs like hcq and cq accumulate inside the lysosome and these drugs potentially increase the ph inside and disrupt lysosomal functions the cgas-sting is another pathway that is involved in the type i interferon response of innate immunity. cyclic gmp-amp synthase (cgas) is a nucleotidyltransferase that is activated to cgamp ( ′, ′ -cyclic gmp-amp dinucleotide) when dsdna, usually from a viral or bacterial origin, binds to it. cgmap then activates an endoplasmic reticulum membrane-associated protein known as the sting (stimulator of interferon genes). activation of sting leads to the activation of the transcription factor irf and nf-kb, which then can migrate to the nucleus to activate the type i ifns and cytokines [ ] . evidence suggests one-way hcq and cq can achieve the immunomodulatory effect is because of its ability to suppress the activation of this pathway by inhibiting ligand binding [ ] . the mechanism of the inhibition of cgas-sting pathway by hcq and cq has been presented in fig. [ ] . given all the above mechanisms, it is now clear that these drugs can effectively modulate cellular signaling. sometimes, this can be helpful but only when used by patients infected with covid- . for instance, covid- patients have usually higher levels of inflammatory cytokines which ultimately result in collateral damage to the host tissues. the use of hcq and cq has been reported to reduce the overproduction of il- b, il- , and granulocyte-colony stimulating factor (gm-csf). the reduction in endosomal acidification as a result of hcq and cq accumulation is believed to halt or reduce the disruption of viral particles and thus the release of infectious nucleic acid. the ace receptors of the lungs are required for sars-cov entry into the host cells. glycosylation of ace receptors is required for the translocation of ace receptors to the cell membrane. hcq has fig. inhibition of autophagy by hcq and cq triggers apoptosis: autophagy is often utilized by cells as a survival mechanism to prevent apoptosis. normal autophagy process includes the fusion of autophagosomes with the lysosome, followed by the degradation and recycling of the internal components like damaged organelles. accumulation of hcq and cq increases the ph inside the lysosomes and prevents the normal autophagy process. this prevention of autophagy triggers apoptosis in memory t-cells and this is one of the fundamental mechanisms of immunomodulation by hcq and cq. also, not shown in the diagram, inhibition of autophagy can also reduce antigen presentation via mhc class ii molecules fig. hcq interference in the t-cell activation pathway and transcription of cd : when t-cell receptor (tcr) is stimulated by antigen via mhc, a series of events leads to the activation of the phospholipase c, which then generates inositol triphosphate (ip ). ip induces the release of calcium from the endoplasmic reticulum (er). calcium acts as a secondary messenger to activate calcium-release-activated calcium channel (crac) for a steady influx of extracellular calcium. intracellular calcium binds to calmodulin and activates the phosphatase calcineurin (not shown in the diagram). calcineurin dephosphorylates and activates transcription factor nfatc . nfatc migrates the nucleus and triggers the transcription of nfatc . nfatc mrna is exported outside the nucleus where de novo synthesis of transcription factor nfatc occurs. nfatc then migrates back into the nucleus to triggers the transcription of cd . hcq can potentially interfere with intracellular calcium signaling and prevent dephosphorylation and activation of the transcription factor nfat. this is one of the mechanisms by which hcq interfere in t-cell activation and cd transcription also been shown to reduce this glycosylation step [ ] . these studies have together motivated healthcare workers to use these drugs as a prophylactic, postexposure prophylactic, and as a curative drug. unfortunately, these results were only successful in vitro. as we will discuss later, large clinical trials have found no evidence of the benefit of using these drugs. it is important to remember the metabolism of these drugs in vivo. both hcq and cq are metabolized in the liver by an enzyme known as cytochrome p (cyp) and the gene expression of this enzyme varies between different individuals as a result of the difference in nucleotide polymorphisms [ ] . furthermore, these polymorphisms have been associated with the formation of unstable enzymes and thus a decreased in its activity [ ] . some ethnicities have a complete absence of certain functional cyp enzymes like the cyp d , which is one of the important cyp enzymes that is actively involved in the metabolism of these drugs [ ] . an individual's p polymorphisms should be taken into account when considering to prescribe hcq since there is a clear association between different polymorphisms in cyp d and the blood concentration of hcq in sle patients [ ] . these polymorphisms have also been linked to the toxic accumulation of these drugs in the blood of the patients treated with hcq and cq [ ] . to date, there is still no evidence that points to any benefit of using these drugs against the covid- as a prophylactic. moreover, the potentials of these drugs as fig. endosomal tlr and inhibition by hcq and cq: mammalian toll-like receptors (tlr) and initiate immune response when it encounters microbial nucleic acids (only shown here is a viral particle). the ectodomain of tlr and are cleaved in the endolysosome (not shown) which then recruits myd , followed by the activation of irak, traf , and nf-kb. nf-kb then migrates to the nucleus and triggers the transcription of inflammatory cytokines. both hcq and cq can increase the ph of the endolysosome and interfere with the tlr and tlr cleavage and processing. furthermore, hcq and cq can directly bind to the microbial nucleic acids and prevent tlr-ligand interaction a post-exposure prophylactic or curative drug has also been called into question. recently, the food and drug administration (fda) of the united states (us) has revoked their emergency use authorization (eua) for emergency use of these drugs [ ] , and the world health organization (who) [ ] , along with national institute of health (nih) [ ] in the us had also stopped conducting further clinical studies because of sufficient evidence that these drugs provided no benefit. initially, a few in vitro evidence and small studies suggested that these medications might be helpful, however, large scale studies have found no such significance. the first clinical evidence of a positive result stems from a small study in china where subjects treated with cq were found to have a superior benefit over the control group suffering from covid- pneumonia [ ] . another small study in france with only subjects, who received hcq, showed a significant reduction in the viral load compared to the control group [ ] . the time to clinical recovery was also shown to be reduced in those under hcq treatment in a small randomized trial of just covid- patients [ ] . clearly, the small sample size was a major drawback in these studies. however, larger clinical randomized controlled trial has now been conducted and results have provided us with a different outcome. for instance, a recent study in brazil fig. cgas-sting pathway inhibition by hcq and cq: dna sensor cgas (cyclic gmp-amp synthase) can recognize dna of viral/bacterial origin and synthesize a dinucleotide molecule, cgamp ( ′, ′-cyclic gmp-amp dinucleotide). newly synthesized cgamp can activate an endoplasmic reticulum (er)-associated protein sting (stimulator of interferon genes). sting potentially activates the transcription factors irf and nf-kb, which can then migrate to the nucleus and lead to the transcription of type i ifns and cytokines. although hcq/cq doesn't directly bind to the active site of cgas, these molecules can occupy the minor grooves of dna molecule and prevent association with cgas with confirmed covid- patients, hcq treatment had no significant benefit over no treatment group [ ] . furthermore, chloroquine at high doses ( mg, twice daily) for consecutive days was associated with higher lethality and is now not recommended for critically ill patients [ ] . a large randomized clinical study investigating the post-exposure prophylactic role of hcq found that a higher dose of hcq did not prevent infection when treatment was initiated within days after exposure [ ] . another study showed us that in covid- patients those taking hcq only or hcq plus azithromycin had a death rate of . and . % respectively when compared to . % in the no-treatment group [ ] . most pathogenic microorganisms that infect humans are tackled satisfactorily by the innate and adaptive immune system of our body. healthy humans have an extraordinary capability to fight off infections caused by pathogenic microorganisms. the activation of innate immunity initiates the first line of defense until the more specific adaptive immunity develops. existing literature suggests that hcq and cq can potentially interfere with both innate and adaptive immune responses in multiple ways. here in this review, we have highlighted the known pathways where hcq and cq can intervene to achieve its immunomodulatory effects and also provided systematic diagrams for a better understanding of the affected pathways. furthermore, cq has been recognized to induce genomic instability by inducing mutagenic, genotoxic, and dna damages both in vivo and in vitro systems. most authors, including us, found weak to moderately strong mutagenic effects in different salmonella strains routinely used for mutagenicity screening of drugs and chemicals. moreover, almost all authors who worked on genotoxicity assays have reported positive genotoxic effects of cq in multiple test systems. this indicates that cq is a mutagenic and genotoxic drug. however, with a lack of sufficient studies on the genetic toxicology of hcq both in vitro and in vivo, the mutagenic and genotoxic effects of hcq remains inconclusive. current evidence cumulatively demonstrates that both hcq and cq are not effective against the covid- infection either as post-exposure prophylaxis or as a curative drug. no studies on the prophylactic role of these drugs have been evaluated to date. the world is going through tremendous turmoil because of the covid- pandemic. thus, we do recognize the importance of optimism and the implementation of any advancement in science during this emergency. thus, the use of these drugs post-infection might be useful, but this discussion is beyond the scope of this review. here, we are mainly concerned with the use of these drugs by healthy individuals as a prophylactic without any evidence. so, without any clinical or in vivo evidence, current literature suggests that healthy individuals should refrain from the use of these drugs as prophylactics until further investigation. aerosol and surface stability 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new mechanism to explain its immunomodulatory properties inhibition by chloroquine of uv repair in e. coli b mutagenicity evaluation of the two antimalarial agents chloroquine and mefloquine, using a bacterial fluctuation test feasibility of testing dna repair inhibitors for mutagenicity by a simple method mutagenicity of antiamebic and anthelmintic drugs in the salmonella typhimurium microsomal test system mutagenicity examination of several non-steroidal anti-inflammatory drugs in bacterial systems the role of chloroquine supplementation in liquid holding recovery and ultraviolet lethality of escherichia coli strains the comparative responses of salmonella typhimurium ta and ta a to a range of reference mutagens and novel compounds testing of chloroquine and quinacnne for mutagenicity in drosophila melanogaster comparison of the ames assay and the induction of sister chromatid exchanges: results with ten pharmaceuticals and five selected agents aspects of chloroquine mutagenicity frameshift mutagenesis by chloroquine in escherichia coli and salmonella typhimurium cortinas de nava c. influence of the uvr repair system on the mutagenicity of antiparasitic drugs genetic toxicology testing of the antimalarial drugs chloroquine and a new analog, aq- study of the evaluation of mutagenic effects of antimalarial drug chloroquine in ames salmonella assay choloroquine inhibition of repair of dna damage induced in mammalian cells by methyl methanesulfonate cytogenetic effects of chloroquine in human lymphocyte cultures simultaneous detection of chromosomal aberrations and sister-chromatid exchanges: experience with dna intercalating agents putative identification of functional interactions between dna intercalating agents and topoisomerase ii using the v in vitro micronucleus assay anti-malarial chloroquine stimulate p -apoptotic pathway in rat hepatocytes genotoxicity of chloroquine in rat liver cells: protective role of free radical scavengers action of chloroquine phosphate in rheumatoid arthritis. ii. chromosome damaging effect aplasia and leukaemia following chloroquine therapy the antimalarials quinacrine and chloroquine potentiate the transplacental carcinogenic effect of ethylnitrosourea on ependymal cells effects of low dose radiation and vitamin c treatment on chloroquine-induced genotoxicity in mice the carcinogenicity of some antimalarial drugs using the egyptian toad bufo regularisas a biological test animal effects of primaquine and chloroquine on oxidative stress parameters in rats animal toxicity and pharmacokinetics of hydroxychloroquine sulfate mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology spectrophotometer studies of the interaction of chloroquine with deoxyribonucleic acid variation of the supercoils in closed circular dna by binding of antibiotics and drugs evidence for molecular models involving interaction carcinogenicity of antibacterial, antiviral, antimalarial and antifungal drugs effects of chloroquine treatment on antioxidant enzymes in rat liver and kidney chromosomal aberrations in lymphocytes of healthy subjects and risk of cancer genotoxic and carcinogenic risk to human of drugnitrite interaction product immunosuppressive potential of antimalarials the relevance of antimalarial therapy with regard to thrombosis, hypercholesterolemia and cytokines in sle immune stimulation mediated by autoantigen binding sites within small nuclear rnas involves toll-like receptors and chloroquine inhibits production of tnf-alpha, il-beta and il- from lipopolysaccharide-stimulated human monocytes/macrophages by different modes the ectodomain of toll-like receptor is cleaved to generate a functional receptor hydroxychloroquine preferentially induces apoptosis of cd ro+ effector t cells by inhibiting autophagy: a possible mechanism for therapeutic modulation of t cells hydroxychloroquine inhibits proinflammatory signalling pathways by targeting endosomal nadph oxidase hydroxychloroquine efficiently suppresses inflammatory responses of human class-switched memory b cells via toll-like receptor inhibition cutting edge: antimalarial drugs inhibit ifn-b production through blockade of cyclic gmp-amp synthase-dna interaction modulating lysosomal function through lysosome membrane permeabilization or autophagy suppression restores sensitivity to cisplatin in refractory nonsmall-cell lung cancer cells mechanism of action of hydroxychloroquine as an antirheumatic drug autophagy in immunity and inflammation inhibition of macroautophagy triggers apoptosis dna sensing by the cgas-sting pathway in health and disease smooth or risky revisit of an old malaria drug for covid- ? genetic polymorphism in cytochrome p d (cyp d ): population distribution of cyp d activity association of polymorphisms of cytochrome p d with blood hydroxychloroquine levels in patients with systemic lupus erythematosus guimarães l sp. and hutz m h. the effect of snps in cyp in chloroquine/ primaquine plasmodium vivax malaria treatment food & drug administration. letter revoking eua for chloroquine phosphate and hydroxychloroquine sulfate q&a: hydroxychloroquine and covid- national institutes of health. nih halts clinical trial of hydroxychloroquine. study shows treatment does no harm, but provides no benefit breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial efficacy of hydroxychloroquine in patients with covid- : results of a randomized clinical trial hydroxychloroquine with or without azithromycin in mild-to-moderate covid- effect of high vs lowdoses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus (sars-cov- ) infection a randomized clinical trial a randomized trial of hydroxychloroquine as postexposure prophylaxis for covid- outcomes of hydroxychloroquine usage in united states veterans hospitalized with covid- springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors are grateful to the indian national science academy to provide the insa-senior scientist position to akg. authors are also grateful to the u.s. federal government for providing financial support to ag for graduate studies in kansas city university of medicine and biosciences and to the council of scientific and industrial research, new delhi for providing csir-net (jrf) to ad. our deep gratitude also to dr. pritha bhattacharjee, department of environmental sciences, university of calcutta for allowing her student ms. ankita das to work in this review and also for her valuable suggestions. all the figures in this review have been created with biorender.com there is no funding for this work.availability of data and materials not applicable.ethics approval and consent to participate not applicable. not applicabe. the authors declares that he have no competing interests. key: cord- -cjfglili authors: fteiha, bashar; karameh, hani; kurd, ramzi; ziff‐werman, batsheva; feldman, itamar; bnaya, alon; einav, sharon; orlev, amir; ben‐chetrit, eli title: qtc prolongation among hydroxychloroquine sulfate‐treated covid‐ patients: an observational study date: - - journal: int j clin pract doi: . /ijcp. sha: doc_id: cord_uid: cjfglili background: the liberal administration of hydroxychloroquine‐sulphate (hcq) to covid‐ patients has raised concern regarding the risk of qtc prolongation and cardiac arrhythmias, particularly when prescribed with azithromycin. we evaluated the incidence of qtc prolongation among moderately and severely ill covid‐ patients treated with hcq and of the existence of concomitant alternative causes. methods: all covid‐ patients treated with hcq (between mar and apr , ) in a tertiary medical center were included. clinical characteristics and relevant risk factors were collected from the electronic medical records. individual patient qtc intervals were determined before and after treatment with hcq. the primary outcome measure sought was a composite endpoint comprised of either an increase ≥ milliseconds (ms) in the qtc interval compared with pretreatment qtc, and/or a maximal qtc interval > ms. results: ninety patients were included. median age was years (iqr ‐ ) and ( %) were male. thirty‐nine patients ( %) were severely or critically ill. hypertension and obesity were common (n= each, %). qtc prolongation evolved in fourteen patients ( %). age > years, congestive heart failure, severity of disease, c‐reactive protein level, hypokalemia and furosemide treatment, were all associated with qtc prolongation. adjusted analysis showed that qtc prolongation was five times more likely with hypokalemia [or , ( % ci, . ‐ )], and three times more likely with furosemide treatment [ or ( % ci, . ‐ . )]. conclusion: in patients treated with hcq, qtc prolongation was associated with the presence of traditional risk factors such as hypokalemia and furosemide treatment. the emerging outbreak of corona virus disease (covid- ) due to the novel beta corona virus sars-cov- has spread globally at an alarming rate. in search of effective treatments, one strategy was drug repurposing of an approved drug for a different disease than that for which it was originally developed. among other options, the anti-malarial drugs chloroquine (cq) and hydroxychloroquine (hcq) have resurfaced as promising drugs for the treatment of patients with covid- disease , .the accumulated long-term experience with these drugs with regards to dosage, safety, adverse effects and drug interactions provided the leeway required for off-label use in the setting of the new pandemic. pressured to provide antiviral treatment options in the midst of the pandemia, the fda had approved the use of cq and hcq for treating covid- disease . an open-label nonrandomized (nowadays controversial) trial also showed that hcq treatment was associated with a significant reduction in viral shedding, an effect reportedly accentuated by concomitant treatment with azithromycin in six patients . despite the small sample studied and the major methodological limitations of the study, it had garnered substantial attention in the media and scientific community. this led to liberal use of hcq in hospitals overwhelmed with the sars-cov- pandemic despite the paucity of evidence. in the wake of the widespread use of hcq, several authors have raised concerns regarding the potential for inducing cardiac arrhythmias. significant qtc prolongation was attributed to treatment with hcq in several papers, albeit mainly in patients receiving combined hcqazithromycin treatment - . resultantly it has been proposed that frequent electrocardiographic (ecg) monitoring be mandatory for patients with covid- who receive treatment with hcq. this article is protected by copyright. all rights reserved as similar monitoring has not been previously recommended in other chronic illnesses such as systemic lupus erythematosus (sle) or rheumatoid arthritis (ra) where hcq is prescribed for months, this raises a major question regarding the actual risk of arrhythmias in this patient cohort. this study aimed to quantify the evolution of ecg changes potentially attributable to hcq among moderate and severely ill covid- patients and to seek concomitant causes. we hypothesized that qtc prolongation may be induced by the combination of severe acute illness and severe inflammation (reported in in addition to medications. following receipt of institutional review board approval ( - -szmc) with waiver of informed consent, data collection was performed during patient admission for the purpose of this observational study. clinical setting: the shaare zedek medical center (szmc) is a tertiary -bed teaching hospital. as the sars-cov- pandemic reached jerusalem in march , six departments were established to treat patients with covid in the szmc. these departments were isolated and set up with telemedicine and monitoring technologies to enable constant observation and monitoring of patients. patients were admitted to these departments only after testing positive in a polymerase-chain reaction (pcr) nasopharyngeal swab specimen for sars-cov- . patients were classified as having mild to moderate illness defined as mild respiratory symptoms (no oxygen support) up to mild pneumonia. severe disease was defined as hypoxia, diffuse bilateral infiltrates on imaging, requirement of high-flow nasal cannula or non-invasive ventilation. critically ill patients were those who required mechanical ventilation with or without multi-organ failure. treating physicians were allowed to administer hcq to any patient with confirmed covid- of moderate severity or worse unless contraindicated (qtc > ms, known allergy to hcq, significant liver disease). the treatment dose of hcq was mg twice daily on day , and mg twice daily thereafter for to days. the dose was reduced by % in patients with creatinine clearance less than ml/min. in cases where qtc interval increased by more than ms or prolonged > ms, hcq was withdrawn. inclusion/exclusion criteria: the emrs of all adult patients admitted for treatment of confirmed covid- and treated with hcq were screened (mar- - to apr - ). inclusion was confirmed following manual verification of a positive pcr nasopharyngeal swab specimen tested this article is protected by copyright. all rights reserved for sars-cov- and receipt of > days of hcq and at least one follow-up ecg post-treatment ( figure ). patients with known arrhythmias such as atrial fibrillation or conduction blocks were not excluded from the analysis. data collection and variables: all relevant demographic, clinical and laboratory data (including additional medications which could potentially prolong qtc interval, prescribed throughout hospitalization and overlapped with hcq treatment) were collected from the electronic medical record (emr). hcq was defined as a qt prolonging agent. in-line with the american heart association/american college of cardiology scientific statement on prevention of tdp in hospital settings, a qtc of > ms for men, and > ms for women was considered prolonged. a qtc > ms was considered highly abnormal for both men and women . drug-induced qt prolongation was defined as a qtc of ms or greater or an increase of ms or greater in the qt interval compared with the premedication baseline interval qtc calculation -the calculation of qtc in all patients was performed in accordance with a special qtc calculation protocol written by a team of senior electrophysiologists based on the guidelines recommended in the literature. the qt was measured manually using the "tangent" method, looking mainly at leads ii and v . a tangent was drawn to the steepest last limb of the presumed t wave to define the end of the t wave as the intersection of this tangent with the baseline. the qtc interval was calculated from the qt and r-r intervals using bazett's formula. the qrs interval was measured from the onset of the q wave, or the r wave if no q wave was visible, to the j point. the jtc interval was calculated by subtracting the qrs duration from the qtc interval (qtc interval -qrs duration). the protocol also accounts for the two special cases of atrial fibrillation and patients with wide complexes such as clbbb -in cases of atrial fibrillation the average of qt interval and rr intervals for to beats was calculated, and qtc was then calculated according to bazett's formula. in case of clbbb or pacemaker rhythm, qtc was calculated by subtracting ms from the corrected qt when qrs width was more than ms, or subtracting ms from the original value in a pacemaker provided the pacing rate is around beats per second. ecg was repeated at the convenience of the treating physician in order to reduce unnecessary contact with patients. the maximal qtc measurement observed in all follow-up electrocardiograms was documented for each patient. this article is protected by copyright. all rights reserved outcome measures: the primary outcome measure was a composite endpoint consisting of either an increase  ms of the qtc interval post hcq treatment (as compared with baseline qtc prior to treatment) and/or a maximal qtc interval longer than ms. the secondary outcome was the adjusted association of the occurrence of qt prolongation (yes/no) with hcq treatment combinations. all data were inserted to -a microsoft excel (version two hundred and ninety-seven patients with confirmed covid- disease were screened during the -day study period. among these patients, overall were treated with hcq during admission. one patient was initially treated with cq but this treatment was replaced with hcq three days later due to hallucinations. overall ninety patients met inclusion criteria. the rest were excluded due to lack of serial ecg recordings, missing data or completion of less than days of treatment ( figure ). hcq was prescribed for a median of days (iqr - ). the demographic and clinical characteristics of the study cohort are presented in table . the median age of the patients was (iqr - ) years and ( %) were male. fifty-one patients ( %) had mild to moderate disease and ( %) had severe or critical illness. the mean crp level was (± ) mg/dl. twenty-six patients ( %) were treated with hcq alone and patients ( %) were treated with hcq and azithromycin. thirty-three patients were treated with this article is protected by copyright. all rights reserved hcq and other qtc prolonging medications including patients who were treated with levofloxacin ( table ). the mortality rate in the study cohort was . % ( patients), all were critically ill. nine patients had atrial fibrillation. two had a pacemaker and one had clbbb. the table ) . a significant qtc prolongation of more than ms was noted in ( %) patients. qtc prolongation greater than ms was identified in ( . %) patients. the overall composite endpoint was identified in overall patients ( %). the increase in the qtc interval seemed somewhat higher among patients treated with combination therapy as compared to hcq treatment only but the difference was not found to be statistically significant (supplemental figure) . overall, patients died. the median age of non-survivors was (iqr - ). median bmi and crp was (iqr - ) and ( - ), respectively. five had chf, had atrial fibrillation. nine were treated with multiple qtc prolonging agents including azithromycin, ciprofloxacin, and antipsychotics. six had met the composite endpoint. two had an increase of more than ms in the qtc interval, three had a maximal qtc longer than ms, and in one patient both endpoints were reached. in all patients hcq was withdrawn. three of the patients had hypokalemia. five were treated with multiple qt prolonging drugs. sudden clinical deterioration and asystole was documented in a -year-old morbidly obese male patient with diabetes. a -year-old female patient with congestive heart failure, atrial fibrillation and hyperkalemia had ventricular tachycardia. ventricular fibrillation occurred in one -year-old patient with multiple co-morbidities (ihd, diabetes, end-stage kidney disease) not meeting the composite endpoint. this article is protected by copyright. all rights reserved univariate analysis revealed that in covid- patients treated with hcq, age above years, severe or critical illness, congestive heart failure, hypokalemia, furosemide treatment and increased crp level were all significantly associated with the composite endpoint (table ) . hypomagnesemia was not detected in any of the patients. co-linearity testing showed a strong interaction between furosemide treatment, crp and severe or critical disease. although crp had a strong correlation with meeting the composite endpoint (p= . ), the or was not as significant as that of furosemide -or . ( % ci . - . ), vs. multivariate analysis revealed that hypokalemia and furosemide therapy were strongly associated with qtc prolongation (table ). the current study reports significant qtc interval prolongation in % of the patients treated with hcq with/without other agents. however, multivariate analysis in this small dataset also suggested that in covid- patients treated with hcq, concomitant hypokalemia and furosemide treatment were strongly associated with qtc prolongation. our study findings support those of previous studies that showed a mild increase in the qtc interval among patients treated with hcq and azithromycin and to a higher degree with quinolones . in our cohort qtc prolongation was not significantly higher in patients receiving hcq in combination with other drugs than with hcq treatment alone but our study was not powered to seek this outcome. similarly, a recent multicenter randomized controlled trial conducted in brazil reported more frequent events of qtc interval prolongation among patients who received hcq, either with azithromycin or alone, than patients who did not receive either agent . this article is protected by copyright. all rights reserved the presence of hypokalemia did not correlate with furosemide treatment. hypokalemia may be a common finding in covid- patients, a finding that has been mainly attributed to urinary potassium loss secondary to angiotensin converting enzyme (ace ) degradation in recent reports . gastrointestinal loss of potassium may also play and important role in inducing hypokalemia as diarrhea may occur in % to % of covid- patients . the use of loop diuretics is an independent risk factor for qtc prolongation . it is also one of the variables comprising the tisdale risk score which predicts the risk of qt prolongation > ms in hospitalized patients . a similar finding was demonstrated in a recent report . furosemide was often administered to patients in our cohort, most often to those who were severely ill. diuretics may be appropriate in ards and are the mainstay of treatment of heart failure. the pulmonary infiltrates that are almost pathognomonic of covid- disease tempt clinicians treating these complex patients, particularly those who have been trained to treat these diseases, to administer diuretics. however, our findings suggest that treatment with loop diuretics is not harmless in this scenario and that administration of such treatment, particularly when combined with hcq, should not be empirical. testing for beta natriuretic factor levels and performing an echocardiograph may be of value. treatment should be accompanied by appropriate monitoring of blood potassium and magnesium levels as well as periodic ecgs. severely ill patients are at increased risk of qtc prolongation due to multiple risk factors [ ]. these patients are often treated with qtc prolonging drugs other than loop diuretics (e.g. amiodarone, anti-emetics and anti-psychotics). acute delirium is common among patients with covid- disease .in our cohort patients ( %) received anti-psychotic medications (table , footnote). although we did not identify an association between such treatment and qtc prolongation, our cohort may have been too small to detect it mercuro et al. also showed an association between qtc prolongation > ms and severe covid- infection with more than two inflammatory systemic response syndrome criteria other studies have also suggested that critically-ill patients with increased levels of il- , il- , tnf-ɑ and crp are prone to arrhythmia - . we also found that severely ill patients had a higher mean crp than did mild to moderately ill patients ( ± . versus ± . , respectively, p value < . ) and higher values were significantly associated with the composite endpoint (table ) . this article is protected by copyright. all rights reserved among the study patients eleven died. all were elderly, severely ill patients with markedly elevated crp and a high bmi. it is difficult to delineate the cause of death as in most cases it is multi-factorial (multiple co-morbidities, respiratory insufficiency, renal failure, secondary infections) and this was not the aim of our study. however, qtc prolongation is a risk factor for cardiac death and indeed, six patients who did not survive had met the composite endpoint of the study. although fatal arrhythmia was documented in only two, most of the cohort patients were not continuously monitored, thus, the likelihood of under-diagnosis of severe arrhythmias was high. our study has several limitations, including the inherent limitations of a retrospective design, a single center study, and its small sample size. however, similar observations have been reported elsewhere , . some patients had only one follow-up ecg reflecting the initial technical difficulties of many medical teams that were learning to manage mass isolation at the outset of the pandemic. in addition, patients excluded from the study due to missing serial ecgs may have had mild cardiac disease and this may have contributed to selection bias. finally, the lack of a control group of patients without hcq treatment makes it difficult to ascertain whether the qtc prolongation is related to treatment with hcq or is a consequence of the disease itself. nevertheless, serial electrocardiograms with successive qt measurements are not routinely performed for covid- patients which makes creating a control group a rather difficult task. in conclusion, our study shows that qtc prolongation among hcq-treated patients was associated with traditional, modifiable risk factors such as hypokalemia and furosemide treatment which are both commonly observed in covid- patients. importantly, the present study was based on data from patients admitted during the first weeks of the pandemia in israel, when treatment guidelines were based on preliminary reports from china and italy suggesting hcq to be beneficial. since then, accumulating data based on better-designed studies, do not support the use of this drug , . in our institute, hcq is no longer recommended in covid- . table . univariate analysis and adjusted multivariate regression analysis of variables associated with qt prolongation > ms and/or qt prolongation > ms post-treatment with hcq (the composite endpoint). this article is protected by copyright. all rights reserved in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies request for emergency use authorization for use of chloroquine phosphate or hydroxychloroquine sulfate supplied from the strategic national stockpile for treatment of coronavirus disease united states food and drug administration hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label nonrandomized clinical trial ventricular arrhythmia risk due to hydroxychloroquine-azithromycin treatment for covid- interval prolongation associated with use of hydroxychloroquine with or without concomitant azithromycin among hospitalized patients testing positive for coronavirus disease (covid- ) prevention of torsade de pointes in hospital settings: a scientific statement from the american heart association and the american college of cardiology foundation determination and interpretation of the qt interval hydroxychloroquine with or without azithromycin in mild-to-moderate covid- hypokalemia and clinical implications in patients with coronavirus disease infection: pathogenesis, epidemiology, prevention and management risk factors for qtc interval prolongation development and validation of a risk score to predict qt interval prolongation in hospitalized patients qtc interval prolongation in critically ill patients: prevalence, risk factors and associated medications accepted article qt interval and inflammatory cytokines in rheumatoid arthritis an emerging role for inflammation and immunity considerations for drug interactions on qtc in exploratory covid- (coronavirus disease ) treatment covid- and the cardiovascular system hydroxychloroquine in hospitalized patients with covid- electrocardiographic features, median (iqr) highest crp during hcq treatment, mg/dl . ( . ) key: cord- - lxhvyyb authors: chen, li; chen, haiyan; dong, shan; huang, wei; chen, li; wei, yuan; shi, liping; li, jinying; zhu, fengfeng; zhu, zhu; wang, yiyang; lv, xiuxiu; yu, xiaohui; li, hongmei; wei, wei; zhang, keke; zhu, lihong; qu, chen; hong, jian; hu, chaofeng; dong, jun; qi, renbin; lu, daxiang; wang, huadong; peng, shuang; hao, guang title: the effects of chloroquine and hydroxychloroquine on ace related coronavirus pathology and the cardiovascular system: an evidence based review date: - - journal: function (oxf) doi: . /function/zqaa sha: doc_id: cord_uid: lxhvyyb the ongoing pandemic of coronavirus disease (covid- ) caused by the severe acute respiratory syndrome coronavirus (sars-cov- ) poses a serious threat to global public health and there is currently no effective antiviral therapy. it has been suggested that chloroquine (cq) and hydroxychloroquine (hcq), which were primarily employed as prophylaxis and treatment for malaria, could be used to treat covid- . cq and hcq may be potential inhibitors of sars-cov- entry into host cells, which is mediated via the angiotensin-converting enzyme (ace ), and may also inhibit subsequent intracellular processes which lead to covid- , including damage to the cardiovascular system. however, paradoxically, cq and hcq have also been reported to cause damage to the cardiovascular system. in this review, we provide a critical examination of the published evidence. cq and hcq could potentially be useful drugs in the treatment of covid- and other ace involved virus infections, but the antiviral effects of cq and hcq need to be tested in more well-designed clinical randomized studies and their actions on the cardiovascular system need to be further elucidated. however, even if it were to turn out that cq and hcq are not useful drugs in practice, further studies of their mechanism of action could be helpful in improving our understanding of covid- pathology. the coronavirus disease (covid- ) is due to infection by the severe acute respiratory syndrome coronavirus (sars-cov- ). [ ] [ ] [ ] the most common symptoms of covid- are fever and cough. , both human-to-human and asymptomatic transmission have been reported. the covid- pandemic has rapidly evolved into a global health crisis as there is currently no proven drug for treating coronavirus patients. however, the strategy of drug repurposing may offer hope for a new approach to covid- treatment. among the myriad existing drugs that are potential repurposing candidates for treating covid- , the immunomodulatory agents chloroquine (cq) and hydroxychloroquine (hcq) have captured great attention. cq and its more soluble and less toxic metabolite hcq are primarily used for prophylaxis and treatment of malaria, but they have also been reported to effectively inhibit the effects of certain viruses, such as severe acute respiratory syndrome coronavirus (sars-cov) and influenza a h n. [ ] [ ] [ ] [ ] recently, the possible use of cq/hcq as a repurposed therapeutic agent against covid- has been explored. angiotensin-converting enzyme (ace ), a new homolog of ace, can convert angiotensin ii (ang ii) to ang( - ) , . ang( - ) binds and activates the g-protein coupled receptor mas (masr) and acts as a natural damping mechanism for the activation of the classical renin-angiotensin system (ras), , , , which plays a critical role in maintaining normal cardiovascular (cv) functions. apart from its crucial role in cv disease, ace has also been shown to be a functional host cellular entry receptor for coronavirus that directly binds the viral spike (s) protein, which is primed by the transmembrane serine protease (tmprss ). [ ] [ ] [ ] [ ] the ongoing covid- pandemic, caused by sars-cov- , poses a serious threat to global public health, and cross-sectional data suggest that sars-cov- infected patients have a high prevalence of cv disease. , recent data indicated that cq and hcq (cq/hcq) may have a promising ability to inhibit sars-cov- and other ace related viral diseases [ ] [ ] [ ] , but the effects of cq/hcq on the cv system seem paradoxical. cq/hcq shows cv benefits, including a reduction in the risk of developing hyperlipidemia and diabetes mellitus, but cv disorder has also been reported as one of the rare but severe side effects of cq/hcq. in this review, we summarize and evaluate the published evidence concerning the actions and mechanisms of action of cq/hcq in treating sars-cov- and other ace related viral infections. we conclude that further mechanistic studies as well as well-designed clinical randomized trials are needed to investigate the molecular pathogenesis of sars-cov- infection and to examine the antiviral efficacy of cq/hcq against covid- . furthermore, the effects and mechanisms of action of cq/hcq on the cv system should be further investigated. the ras is a humoral regulation cascade that elegantly orchestrates key vascular physiology in humans. sars-cov- infection has been proposed to interfere with ras through the ace receptor for host cell entry (figure ) . , severe covid- infection has many clinical characteristics which are strikingly similar to the effects of overactivation of the ras. it has been reported that coagulation is activated and accelerated in patients with sars-cov- . the complex entry process of coronavirus into susceptible cells requires multistep actions of receptor-binding and proteolytic processing of the s protein to promote virus-cell fusion. s protein cleavage occurs at the boundary between the s and s subunits, and s is further cleaved at the s ' site by host proteases to facilitate the fusion of viral and cellular membranes via extensive irreversible conformational changes. [ ] [ ] [ ] [ ] a recent study provided fresh evidence that sars-cov- exploits ace and tmprss for host cell entry. like sars-cov entry into host cells, the s glycoprotein domain b (s b ) of sars-cov- binds to the human ace (hace ) receptor and is subsequently primed by tmprss . , moreover, sars-cov- s has a similar or even higher (~ -to -fold) affinity for binding to hace as compared to sars-cov s. , however, a novel and very important feature of sars-cov- s is that it harbors a furin cleavage site at the s /s boundary, which is processed during biosynthesis. therefore, the presence of the polybasic cleavage site in sars-cov- s, processed by furin-like proteases, may modulate tropism, transmissibility and pathogenicity of sars-cov- , making it a highly pathogenic virus, like avian influenza viruses. the relationship between the expression level of ace and susceptibly to sars-cov- infection still remains elusive. it will thus be interesting to determine whether sars-cov- interferes with ace expression and activity as well as to evaluate the functional consequence of the potential cleavage site used in sars-cov- and its impact on transmissibility and pathogenesis in animal models. in order to better understand the initial step of sars-cov- infection, elucidation of the interactional mechanism between the receptor-binding domain (rbd) of sars-cov- s and ace appears to be particularly important. two recent independent studies have reported the cryo-em structure of the sars-cov- spike trimer. , moreover, another study presented the cryo-em structures of the full-length hace -b at (the neutral amino acid transporter) complex and a complex between the rbd of sars-cov- and the hace -b at complex as well as the hace -rbd interface. in addition, analytical modelling of structure predicted the potential residues of sars-cov- rbd that are recognized by ace . furthermore, x-ray crystallography data at a higher resolution showed the interaction between sars-cov- rbd and ace , demonstrating that sars-cov- and sars-cov rbd share high structural similarity. it remains to be investigated how sars-cov- alters the conformations of s glycoprotein trimers and the interactions between ace and s proteins in receptor-mediated endocytosis. interestingly, single-cell rna-sequencing data from multiple healthy human tissues discovered that the sars-cov- entry receptor ace and the viral entry-associated protease tmprss are highly expressed in nasal goblet and ciliated cells. these new insights indicate that the primary viral sars-cov- transmission occurs through infectious droplets. although tmprss activity is essential for viral transmission, it still needs to be determined whether the endosomal cysteine proteases cathepsin b and l or other proteases, as reported in sars-cov and middle east respiratory syndrome coronavirus (mers-cov), , , [ ] [ ] [ ] are involved in priming sars-cov- s. hence, further mechanistic studies are needed to elucidate the underlying detailed mechanism of sars-cov- entry into host cell and to test the potential of sars-cov- neutralizing antibodies. , several studies have reported that % to % of covid- patients develop acute respiratory distress syndrome (ards) which is a common complication and cause of death as a result of sars-cov- infection. , , , although the pathophysiology of covid- has not been completely unraveled, the potential main mechanism of covid- -asscoiated ards would appear to be the immune-pathological event of the so-called cytokine storm. laboratory tests showed that patients infected with sars-cov- express high amounts of pro-inflammatory cytokines and chemokines, including interleukin (il)- ꞵ, tumor necrosis factor α (tnfα), interferon-γ (ifn-γ), c-x-c motif chemokine ligand (cxcl)- , and monocyte chemoattractant protein (mcp ). the evidence obtained from the postmortem biopsy study of a -year-old male patient suggested that the severe immune injury in covid- -associated ards is related to over-activation of t cells, manifested by the elevation of t-helper- (th ) and high cytotoxicity of cd t cells. sars-cov- , sars-cov and mers-cov cause acute lethal disease characterized by dysregulated and excessive immune responses and lung damage during viral infection. it was reported that relative delayed type i interferon (ifn-i) signaling promoted inflammatory monocyte-macrophage accumulation in balb/c mice infected with sars-cov. consequently, these accumulated mononuclear macrophages produce more monocyte chemoattractants through activating the ifn-α/β receptors and mononuclear macrophage-derived proinflammatory cytokines, such as tnfα, il- β and il- , induce apoptosis of t cells. signaling or tumor necrosis factor-related apoptosis-inducing ligand (trail)-death receptor (dr ) signaling. this eventually results in the apoptosis of airway and alveolar epithelial cells. [ ] [ ] [ ] the apoptosis of these endothelial and epithelial cells could potentially lead to vascular leakage and alveolar edema, which is regarded as playing a key role in the pathogenesis of virus infection-associated ards. it seems to be this deadly uncontrolled cytokine storm that triggers the frantic attack on the body by the immune system causing ards and finally unprecedented mortality in severe cases of sars-cov- infection. future work needs to investigate the details of the ifn signaling involved in sars-cov- infection, how the inflammatory response is triggered as well as the type of cell death that occurs during covid- . also, further autopsy or biopsy studies, including more patients of different ages and backgrounds, would be needed to examine the histopathological changes and ace levels in different tissues. the acid milieu in endosomes and lysosomes (ph between and ) is due to a bafilomycin-sensitive pump that concentrates h + in the lumen of endosomes/lysosomes. this low ph is essential for virus/cell membrane fusion. , [ ] [ ] [ ] [ ] . cq was reported to cause an increase in the intra-lysosomal ph of macrophages. however, there is still no evidence showing the effect of hcq on the ph dynamics of endosomes/lysosomes. nevertheless, both cq and hcq are weak bases so they should both be able to elevate endosomal ph and could thereby inhibit virus/cell membrane fusion. it has recently been reported that cq is highly effective in the control of sars-cov- infection in vitro. compared to remdesivir (gs- ), the time-of-addition assay showed that cq functioned at entry as well as at post entry stages of the sars-cov- infection in vero e cells. similarly, another in vitro study also found that hcq can efficiently inhibit sars-cov- infection via the same routes. the therapeutic effect of both cq and hcq may be due to blockade of the transport of sars-cov- from early endosomes (ees) to endo-lysosomes (els), which seems to be the same viral genome releasing mechanism that operates in the case of sars-cov. however, the mode of actions of cq and hcq showed discrepancy in certain aspects such as in the morphology and ph values of endosomes/lysosomes. another recent study found that hcq exhibited a smaller ec than cq in in vitro anti-sars-cov- activity and physiologically-based pharmacokinetic models indicated that hcq is likely to be more effective than cq in the treatment of sars-cov- infection. however, there are controversies about the effect of cq/hcq in altering endosomal /lysosomal ph and treating viral infection. it was reported that cq/hcq could directly bind to nucleic acids and inhibit the activation of the endosomal toll-like receptor (tlr) by masking tlr ligand-binding epitopes rather than increase the endosomal ph. in addition, cq was shown to inhibit autophagy mainly through impairing autophagosome fusion with lysosomes rather than by increasing ph in this organelle. furthermore, there was a study showing that cq could enhance porcine circovirus infection of porcine epithelial cells via inhibition of endosome-lysosome system acidification. the effect of cq/hcq may differ from cell type to cell type and between virus species. nevertheless, whether cq/hcq are able to affect the acidity of ees and els in sars-cov- infection should be examined carefully in the future. the progressive acidification that normally occurs from ees to els depends on a high ca + concentration in the ees. the ph in the lumen of these organelles decreases in line with a decrease in the ca + concentration. ca + signaling has been demonstrated to be involved in viral fusion into host cells of many viruses such as ebola virus (ebov), mers-cov and sars-cov. ca + release from intracellular stores within the endolysosomal system, via two-pore channels (tpc , tpc ) and channels belonging to the mucolipin family (e.g. trpml ) can be evoked by nicotinic acid adenine dinucleotide phosphate (naadp) and phosphatidylinositol , -bisphosphate (pip ). , in pancreatic acinar cells, antibodies against tpc are very effective, and much more effective than antibodies against tpc , in reducing naadp-elicited ca + release from acidic stores. in this context it is of particular interest that it has very recently been shown that blocking tpc activity by tetrandrine decreases entry of sars-cov- s pseudovirions. in contrast, a trpml inhibitor had no effect. the endo-lysosomal ca + level and ph, altered by tpc activity, - regulate the activity of furin required for proteolytic activation of the s protein and viral fusion. , it was reported that inhibition of tpcs rather than trpml could block mers-cov infectivity. moreover, it has been demonstrated that the endosomal calcium channels tpc and tpc are necessary for ebov infection. interestingly, tetrandrine has been identified as a highly potent and low cytotoxic tpcs inhibitor. because of its ability to disrupt tpcs function, tetrandrine can prevent ebov from escaping the endosomal network into the cell cytoplasm and thus block ebov infection. some other ca + channel blockers such as amiodarone, verapamil, nimodipine, diltiazem, bepridil and lomerizine could effectively protect against filoviral entry into target cells. there is much evidence indicating that sars-cov and sars-cov- infect host cells through ace . , , , , furthermore, cq could block sars-cov fusion with and entry into the host cell through interfering with the glycosylation of the ace receptor and the s protein. cq/hcq have promising ability to inhibit sars-cov- and ace related viral infection. it has been shown that cq is an effective inhibitor of the replication of the sars-cov in vero e cell culture. another study further confirmed that cq is effective against sars-cov in frankfurt and urbani strains. in addition, this study found that cq impaired the terminal glycosylation of ace , suggesting that the variations in its glycosylation status might result in the ace -sars-cov interaction being less efficient and therefore inhibit virus entry when the cells are treated with cq. also, it was shown that the recombinant sars-cov s protein downregulates ace expression. in experimental mouse models, infection with avian influenza a h n virus resulted in down-regulation of ace expression in the lung. genetic inactivation of ace caused severe lung injury in h n -challenged mice, suggesting a role for ace in h n -induced lung pathologies. cq was found to effectively inhibit autophagy in the lungs of avian influenza h n mice and to ameliorate the acute lung injury and further, significantly improve the survival rate in mice infected with live avian influenza a h n virus. there is also evidence that cq had an inhibitory effect against the replication of human influenza a virus h n and h n in vitro. ace could mediate the severe acute lung injury induced by influenza a (h n ) virus infection in an experimental mouse model. moreover, ace deficiency worsened the disease pathogenesis markedly, mainly by targeting the angiotensin ii receptor type (at receptor, at r). therefore, the potential effects and mechanism of cq/hcq against ace related viruses appears worth further investigation. cq/hcq shows cv benefits, including reductions in the risks of developing hyperlipidemia, diabetes mellitus, and thrombosis, as well as improving insulin sensitivity, glucose profiles, and hba c, and decreasing cholesterol, triglycerides, and low-density lipoprotein-cholesterol (ldl-c). , cq/hcq is extensively used in the treatment of rheumatic diseases, the patients of which are at higher risk of cv disease. , a retrospective study of a cohort of , patients with rheumatoid arthritis (ra) found that hcq was associated with an approximately % reduction in the risk of cv disease. another longitudinal registry showed that, compared to non-users, ra patients with hcq treatments had significantly lower levels of total and low-density cholesterol. a longitudinal cohort study of systemic lupus erythematosus patients found lowered serum cholesterol levels associated with hcq treatment. a prospective, multicenter observational study of adults with ra also reported that use of hcq is associated with a % reduction of diabetes risk. hcq is also found to reduce blood pressure variability among systemic lupus erythematosus patients. there are a few small clinical trials studying the effects of cq/hcq on cv risks in humans. a randomized controlled trial (rct) carried out among patients with metabolic syndrome found that a one-year cq treatment decreased blood pressure, lipids, and the activation of c-jun n-terminal kinase (jnk). another randomized, double-blinded, placebo-controlled crossover study found that a -week hcq treatment decreased insulin resistance, total cholesterol, and ldl-c among ra patients. a small open-label clinical trial administered hcq to obese participants for six weeks, which significantly increased the insulin sensitivity index. another rct with patients with sulfonylurea-refractory type diabetes proved that hcq could decrease glycated hemoglobin and improve glucose tolerance. in animal studies, cq could lower blood pressure through toll-like receptor signaling and prevent the subsequent recruitment of immune cells to the vasculature in spontaneously hypertensive rats. in rat hepatocytes, cq was shown to be an effective inhibitor of cholesterol synthesis. it has also been reported that cq improved the cardiac diastolic function by inhibiting autophagy in streptozotocin-induced heart failure with preserved ejection fraction in mice. evidence was also found that activation of ataxia telangiectasia mutated with low-dose cq decreased features of the metabolic syndrome including atherosclerosis in mice. taken together, the claimed cv benefits of cq/hcq are mostly generated from animal studies or observational studies in humans. in rare cases, cq/hcq treatment presents cardiotoxicity including hypotension, arrhythmia, atrioventricular block , cardiomyopathy , and heart failure , , which could be serious. , the cardiotoxicity of cq/hcq may be under-recognized . among the episodes of intentional cq overdosage, % died, and % had cardiac arrest. ex vivo acute cq treatment decreased heart function, and in vivo chronic lowdose cq treatment significantly decreased aortic output and total work in hearts. the mechanisms underlying the effects of cq/hcq on the cv system are not fully understood (figure ) . cq could improve insulin sensitivity by increasing the affinity of insulin receptors, inhibiting insulin degradation, and increasing insulin secretion. cq/hcq could also increase the lipid clearance rate and expression of ldl receptors. hcq is thought to protect against accelerated atherosclerosis, targeting toll-like receptor signaling, cytokine production, t-cell and monocyte activation, oxidative stress, and endothelial dysfunction. hcq can also reduce the induction of endosomal nadph oxidase (nox) by tnfα, il- β and antiphospholipid antibodies (apl) through the inhibition of the translocation of the catalytic subunit of nox into the endosome, which is involved in many inflammatory and pro-thrombotic signaling pathways. however, chronic use of cq/hcq can result in an acquired lysosomal storage disorder, leading to cardiomyopathy characterized by concentric hypertrophy and conduction abnormalities associated with increased adverse clinical outcomes and mortality. hcq is structurally and mechanistically similar to the class ia antiarrhythmic quinidine, and may therefore inhibit voltage-gated sodium and potassium channels, prolonging the qt interval and increasing the risk of 'torsades de pointes' (a specific type of abnormal heart rhythm) and sudden cardiac death. an animal study found that high-dose cq significantly impaired mitochondrial antioxidant buffering capacity and accentuated oxidative stress and mitochondrial dysfunction in pressure-overload hypertrophy. in addition, cq may increase cv risk by impairing the terminal glycosylation of ace , which possibly amplifies ace/angii/at axis signaling and depresses ace /ang - /masr axis signaling. the role of ace in the action of cq/hcq needs to be further studied. finally, it is challenging to interpret the extensive amount of covid- related research that has been published within a very short space of time. this is a highly unusual situation in the routine life cycles of any research topic. we therefore need to maintain a degree of healthy skepticism when interpreting the covid- related scientific literature. we carried out electronic searches using pubmed, web of science, researchgate and google. the search terms were "virus", "coronavirus", "angiotensin-converting enzyme ", "chloroquine", "hydroxychloroquine", "cardiovascular system", and others, alone and in combination. many firstly identified references were investigated further to find the original primary research articles that were then cited in the review. conflict of interest: none declared. the initial entry of sars-cov- (an enveloped virus) into host cells depends on ace and tmprss . the s protein of sars-cov- binds to the functional receptor ace and employs tmprss for its priming. s protein is cleaved by tmprss at s ' site which results in virus/membrane fusion. both ace and tmprss facilitate the virus transport into the target cell through the early and late endosomes where eventually the viral genome will be released into the cell cytoplasm. sars-cov- infection could influence the balance of ras, which leads to ang ii accumulation through the ace/angii/at r axis and eventually causes acute lung injury. cq/hcq may block sars-cov- fusion with the host cell and entry into the target cell through elevating the ph in the endolysosomal system and/or by interfering with the glycosylation of the ace receptor and the s protein. figure the effects of chloroquine and hydroxychloroquine on the cardiovascular system. cq/hcq could protect against accelerated atherosclerosis targeting tlr signaling, cytokine production, t-cell and monocyte activation, oxidative stress, and endothelial dysfunction. however, cq/hcq interferes with the glycosylation of ace and this leads to dysregulation of the ras, which eventually causes imbalance of the ace/angii/at axis and the ace /ang - /masr axis. meanwhile, cq/hcq can cause cardiotoxicity which may increase the risk of cv disease. therefore, cq/hcq may have a paradoxical effect on the cv system. china novel coronavirus i and research t. a novel coronavirus from patients with pneumonia in china a new coronavirus associated with human respiratory disease in china coronaviridae study group of the international committee on taxonomy of v. the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- clinical features of patients infected with novel coronavirus in wuhan zhong ns and china medical treatment expert group for c. clinical 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macrophage-expressed ifnbeta contributes to apoptotic alveolar epithelial cell injury in severe influenza virus pneumonia cellular inhibitor of apoptosis protein ciap protects against pulmonary tissue necrosis during influenza virus infection to promote host survival calcium uptake via endocytosis with rapid release from acidifying endosomes chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion mechanism of uncoating of influenza b virus in mdck cells: action of chloroquine binding and entry characteristics of porcine circovirus in cells of the porcine monocytic line d / . the journal of general virology antiviral therapies against ebola and other emerging viral diseases using existing medicines that block virus entry fluorescence probe measurement of the intralysosomal ph in living cells and the perturbation of ph by various agents hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro in vitro 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canadian journal of cardiology effects of quinine, quinidine, and chloroquine on alpha alpha nicotinic cholinergic receptors risk of qt interval prolongation associated with use of hydroxychloroquine with or without concomitant azithromycin among hospitalized patients testing positive for coronavirus disease potential role of oral rinses targeting the viral lipid envelope in sars-cov- infection key: cord- -r g w b authors: ayele mega, teshale; feyissa, temesgen mulugeta; dessalegn bosho, dula; kumela goro, kabaye; zeleke negera, getandale title: the outcome of hydroxychloroquine in patients treated for covid- : systematic review and meta-analysis date: - - journal: can respir j doi: . / / sha: doc_id: cord_uid: r g w b background: the pandemic of coronavirus disease (covid‐ ) caused by severe acute respiratory syndrome coronavirus (sars-cov- ) resulted in an unprecedented public health challenge worldwide. despite urgent and extensive global efforts, the existing evidence is inconclusive regarding the medications used for the treatment of covid- . purpose: to generate an up-to-date evidence for the clinical safety and efficacy of hydroxychloroquine (hcq) with or without azithromycin (az) among patients treated for covid- . data source. pubmed, cochrane central, litcovid, web of science, scopus, biorxiv, embase, medrxiv, and wiley online library were searched from / / to / / . study selection. three investigators assessed the quality of the studies. data extraction. data about study characteristics, effect estimates, and the quality of the studies were extracted by two independent reviewers and cross-checked by the third reviewer. data synthesis. the data of , (hcq group, ; hcq + az group, , ; control group, ) participants were included. hcq was compared with standard care for virologic efficacy, disease progression, mortality, and adverse effects. hcq was also compared with hcq + az for qtc prolongation, admission to the intensive care unit, and mortality. the study found hcq did not alter the rate of virologic cure (or = . ; % ci: . – . ) and the risk of mortality (or = . ; % ci: . – . ). the pooled prevalence for mortality was . % ( % ci: . %– . %). moreover, hcq did not impact disease progression (or = . ; % ci: . – . ) but resulted in a higher risk of adverse effects (or = . ; % ci: . – . ). hcq was also compared against hcq + az, and no difference was observed in qtc prolongation above ms (or = . ; % ci: . – . ), admission to the intensive care unit (or = . ; % ci: . – . ), and mortality (or = . ; % ci: . – . ). however, in the analysis of single-arm studies, about . % ( % ci: . %– . %) of patients have developed an absolute increase of qtc greater than ms, and . % ( % ci: . %– . %) of patients discontinued their medication. conclusion: this meta-analysis and systematic review, which included a limited number of poorly designed studies of patients with covid- , revealed hcq is intolerable, unsafe, and not efficacious. similarly, hcq + az combination was not different from hcq alone in curbing mortality and icu admission. e pandemic of coronavirus disease (covid- ) caused by severe acute respiratory syndrome coronavirus (sars-cov- ) resulted in an unprecedented public health challenge worldwide [ ] . as of may , , there were more than . million documented cases, and over , patients have succumbed to this disease globally [ ] . e morbidity and mortality due to covid- have found to increase with age and the presence of comorbid conditions such as diabetes, hypertension, coronary heart disease, or chronic obstructive lung disease [ ] . with the rising death toll and a vaccine unlikely very soon, extensive global efforts are underway to develop safe and effective therapeutics against covid- [ ] . among the efforts undergoing to treat the disease, repurposing of old medications is a compelling strategy for which their safety profile, pharmacokinetics, and potential drug interactions are well studied [ ] . initially, a combination of lopinavir and ritonavir was utilized as the first-line agent in wuhan, china, the epicenter of the disease. however, a previous study [ ] failed to show the beneficial clinical effects of this combination. indeed, it has received considerable criticism from the scientific community [ ] . meanwhile, the aminoquinolines, chloroquine (cq), and hydroxychloroquine (hcq) have emerged as a potent inhibitor of sars-cov- in vitro, and some studies also demonstrated their clinical benefit among hospitalized covid- patients [ ] [ ] [ ] . on march , , the united states food and drug administration (fda) granted emergency authorization that allowed the use of these drugs in hospitalized covid- -pneumonia [ ] . to date, regardless of limited evidence, hcq with or without azithromycin (az) is widely utilized in clinical settings to treat thousands of covid- patients around the world [ ] . e studies supporting the use of hcq had suffered from methodological flaws including small sample size and ill quality of design creating difficulty in measuring the true clinical effects. e first study from france showed hcq and az combination as an effective therapy for covid- . in this open-label non-randomized clinical trial, a total of patients were treated with hcq at a dose of mg three times daily for days, and the data showed a significant reduction in viral carriage at day post-inclusion compared to controls ( . % clearance by day vs. . % clearance by day in control groups). interestingly, the addition of az to hcq (n � ) resulted in a % virological cure on day postinclusion, compared with . % virological cure in the hcq alone arm (n � ) and . % virological cure in the control arm (n � ) [ ] . similarly, million et al. [ ] showed hcq/az combination to be safe with a lower death rate. on the contrary, a more recent study conducted by molina and his colleagues [ ] failed to show evidence of a strong antiviral activity or clinical benefit of hcq in combination with az for the treatment of hospitalized patients with severe covid- . notably, the patients included in this study belonged to the severe covid- category and had significant comorbidities including solid and hematological cancers, hiv, and obesity. a large observational study conducted in the usa reported that hcq use among patients hospitalized with covid- was not associated with either a greatly lowered or increased risk of intubation or death [ ] . a very recent study by mahévas et al. [ ] and rosenberg et al. [ ] also did not show significant differences in terms of in-hospital mortality among patients receiving hcq with/without az compared with standard care. besides, a systematic review by sarma and his colleagues [ ] concluded that treatment with hcq had benefits in terms of fewer cases showing radiological progression, time to body temperature normalization, and the number of cough days compared to standard treatment. however, no difference was seen in terms of virological cure, death, or clinical worsening of the disease. e safety of hcq with/without az in covid- patients, including cardiac arrest and qtc prolongation, was also investigated by several studies [ , ] . interestingly, both drugs can potentially cause qtc prolongation, leading to life-threatening ventricular arrhythmias and torsade de pointes [ ] . critically ill admitted covid- patients with multiorgan failure and metabolic derangements and those having other drugs that can increase the risk of qtc prolongation are at greater risk [ ] . a study conducted in new york recorded higher rates of cardiac arrest among patients receiving a combination of hcq and az [ ] . similarly, ramireddy et al. [ ] reported a significant number of patients with qtc-interval prolongation, and the highest qtc values were recorded in those treated with a combination of hcq and az. despite numerous studies with small sample size, the efficacy and safety of hcq in covid- patients remained unclear. given the inconclusiveness of the existing evidence and awaiting findings from large randomized controlled clinical trials to clear the controversy, we conducted a systematic review and meta-analysis in the interim to investigate the safety and effectiveness of hcq in the clinical setup. e objective of this review is to synthesize an evidence for the safety, efficacy, and tolerability of hcq with or without az among patients treated for covid- . is review was described by the preferred reporting items for systematic reviews and meta-analysis (prisma) framework. e studies were identified from pubmed, cochrane central, litcovid, web of science, sco-pus, biorxiv, embase, medrxiv, and wiley online library. e search was conducted to include human studies published in english language from / / to / / . e search terms included -ncov, novel coronavirus, covid- , coronavirus disease- , hydroxychloroquine, plaquenil, and hydroxychloroquine sulphate. details of the search strategy for some databases are annexed (appendix a). study designs with single-group prospective/retrospective observational studies and controlled clinical trials were pooled using meta-proportion, while prospective/retrospective observational studies and controlled clinical trials comparing hcq with or without az versus usual care or hcq with hcq plus az were pooled using revman version . . controlled clinical trials with serious risk of bias were not included in the pooled analysis, and their findings were narrated descriptively. after removing all irrelevant articles, tam, tmf, and gzn independently reviewed articles for data quality and methodological validity using standardized critical appraisal instruments obtained from https://www.joannabriggs.org/ assets/docs/jbc/...sr.../jbi-sr-protocol-template.docx. any disagreement was handled by consulting kkg and ddb. data extraction was handled by dd and kk using the standardized data extraction tool available at https://www.joannabriggs.org/ assets/docs/jbc/...sr.../jbi-sr-protocol-template.docx. studies were considered if they included patients who received hcq alone or in combination with other specific treatment modalities for covid- infection. both controlled clinical trials (ccts) and observational studies with and without the comparator group were considered for inclusion. data on at least one of the following outcomes had to be available for inclusion: virologic efficacy, mortality, disease progression, adverse effects, qtc prolongation, and drug discontinuation due to adverse effects (tolerability). studies conducted among pediatric covid- patients, case reports, preclinical studies, and studies that did not report outcomes with hcq in covid- were excluded. e risk of bias for comparative clinical trials [ , [ ] [ ] [ ] was assessed using the cochrane risk of bias tool for randomized controlled studies [ ] . e study by gautret et al. [ ] was a non-randomized clinical trial and hence assessed using the robins-i scale [ ] . e results of the risk of bias for the studies included in the meta-analysis are found in appendix b. e modified newcastle-ottawa quality assessment scale [ ] was used for observational studies, and the full results are presented in appendix b. e virologic efficacy, mortality, disease progression, adverse effects, qtc prolongation, and drug discontinuation due to adverse effects (tolerability) were assessed. virologic efficacy is defined as two negative results of sars-cov- in nasopharyngeal swab using rt-pcr assay with samples obtained hours apart. disease progression is defined as the need for admission to the intensive care unit, the need for mechanical ventilation, and hospital admission of previously mild cases. adverse effect is defined as any adverse effect (side effect) reported in a study except qtc prolongation. qtc prolongation is defined as an increase of greater than ms from baseline, and absolute qtc increases to greater than ms. open meta [analyst] was used to analyse the proportions of mortality, qtc prolongation, and tolerability in single-arm studies [ , , , [ ] [ ] [ ] [ ] . e pooled proportion of the outcomes was reported with its % confidence interval (ci). revman . was used to estimate the risk of virologic efficacy, mortality, disease progression, and adverse effects in studies that compared hcq with usual care or hcq with hcq plus az. e odds ratios (ors) and % ci were calculated to estimate the effect sizes. meta-analysis using the mantel hazel method was conducted, and either the fixed-effect or random-effects model was applied. a fixed-effect model was used when the heterogeneity was low to moderate [ ] ; otherwise, the random-effects model was applied. e databases ( databases) search produced articles. after removing the duplicates and excluding articles with thorough evaluation for inclusion using titles and abstracts, full-text articles were assessed for eligibility. furthermore, full-text articles were subjected to critical appraisal, and articles were dropped with reasons. twenty full-text articles ( controlled clinical trials with patients and observational studies with , patients) were included in the final analysis. of these, [ , , , , - , , - ] were double-arm studies ( figure ). ese studies compared hydroxychloroquine (hcq) either with usual/standard care or hcq with hcq and azithromycin (az). e details of the studies are described in table . two of the controlled clinical trials [ , ] were at the preprint stage. both the double-arm (table ) and single-arm (table ) studies were subjected to meta-analysis to estimate the effects of the interventions. e outcomes assessed with double-arm studies include virologic efficacy [ , , ] , clinical efficacy (mortality [ , , , , , ] and disease progression [ , , , , , , ] ), safety (risk of adverse effects) [ , [ ] [ ] [ ] , and tolerability and qt prolongation [ , , ] . outcomes were assessed for both hcq versus standard care and hcq versus hcq + az. e outcome assessed in single-arm studies include the proportions of mortality, qtc prolongation [ , , , [ ] [ ] [ ] ] , and drug discontinuation [ , [ ] [ ] [ ] . to estimate the risk of virologic cure, the data of patients ( hcq and non-hcq groups) from two controlled clinical trials [ , ] , with moderate risk of bias, were pooled. although gautret et al. [ ] reported improved virologic cure rate among the hcq group as compared to the non-hcq group ( / versus / ), the finding was not included in the pooled data due to its serious risk of bias. hence, the pooled result indicated that the virologic cure rate of the hcq group was not statistically different from the non-hcq/standard care group (or � . ; % ci: . - . ). e test for the overall effect was z � . (p � . ) (tau � . ; chi � . , df � (p � . ); i � %) ( figure ). e finding was generated from five observational studies [ , , , , ] comprising the data of , covid- patients ( , hcq and , non-hcq groups). e overall result indicated that treatment with hcq did not result in improved survival (or � . ; % ci: . - . ) as compared to the routine care. e test for the overall effect, z � . (p � . ). however, the interpretation of this finding might be limited by the substantial heterogeneity; heterogeneity: tau � . ; chi � . , df � (p ≤ . ); and i � % ( figure ) . a controlled clinical trial [ ] also reported one death out of hcq-exposed patients and no death out of in the opposite arm. of note, the study was removed from pooled analysis as it carries a serious risk of bias (appendix a). likewise, the data of , covid- patients from single-arm observational studies [ , , , ] were included to determine the pooled prevalence of mortality among patients treated with hcq with or without az. e pooled prevalence was . % ( % ci: . %- . %) with considerable heterogeneity (i � . %, p < . ) ( figure ). e heterogeneity could be attributed to the age difference of the covid- patients, as the studies with death reports had a median age of greater than years. in the three [ , , ] of the observational studies, the cause of death was respiratory and multiorgan failure. ere was no death due to arrhythmogenic adverse effects. e outcome coded as "disease progression" included the need for admission to the intensive care unit, the need for mechanical ventilation, and hospital admission of previously mild cases. e data of , covid- patients ( , hcq and , non-hcq group) extracted from one controlled clinical trial [ ] and five observational studies [ , , , , ] were pooled. e overall random effect analysis indicated hcq therapy did not appear to halt disease progression (or � . ; % ci: . - . ). e test result for the overall effect, z � . is finding was also replicated by subgroup analysis of observational studies (or � . ; % ci: . - . ). however, in subgroup analysis, the finding from a single controlled clinical trial (or � . ; % ci: . - . ) was in favor of the hcq group ( figure ). ough the study had no risk of bias, the small sample size ( in each arm) used made the interpretation of the finding extremely tricky. several studies also indicated hcq may not improve the rate of disease progression in covid- patients. in a controlled clinical trial [ ] , which was not included in the pooled analysis, patients were progressed to severe disease in the hcq group ( / versus / ). moreover, in three noncomparative studies [ , , , ] , out of , covid- patients treated with hcq with or without az, patients were transferred to icu and intubated. e data from four controlled clinical trials [ , [ ] [ ] [ ] of covid- patients ( hcq and from the non-hcq group) were included to assess overall adverse effects (except qtc prolongation) among hcq-exposed patients. ree controlled clinical trials [ ] [ ] [ ] were pooled, and one controlled clinical trial [ ] was described narratively due to its risk of bias a. in the pooled analysis, the odds of adverse effects among covid- patients treated with hcq patients were increased by . (or � . ; % ci: . - . ). e test for the overall effect case studies (n = ) was statistically significant (p � . ), and the summary effect of the meta-analysis was heterogeneity: chi � . , df � (p � . ); i � % ( figure ). besides, gautret et al. [ ] found more adverse effects among patients randomized to hcq ( / versus / ). qtc prolongation was reported in two ways in most of the studies. e cut-off points, an increase in greater than ms from baseline, and absolute qtc increase to greater than ms were used as a threshold to discontinue medications responsible or suspected to cause qtc prolongation. ese cut-off points were described by recent guidelines and fda [ , ] . an absolute qtc prolongation greater than ms was reported in two observation studies [ , ] , which compared hcq alone with hcq plus az. a study by rosenberg et al. [ ] reported that after ecg screening, patients ( . %) had a qtc prolongation in the combination group, whereas patients ( . %) developed qtc prolongation in the hcq group. in the current review, the data of patients from two observational studies [ , ] were pooled to estimate the risk of qtc prolongation in patients exposed to hcq versus hcq plus az. e result indicated the risk of qtc prolongation above ms due to hcq was statistically not different from those receiving hcq plus az (or � . ; % ci: . - . ) with the test for the overall effect of z � . (p � . ); heterogeneity: chi � . , df � (p � . ); i � % (figure ) . however, the findings from noncomparative studies appeared much concerning. seven single-arm studies reported data on qt prolongation after hcq ± az exposure. six studies ( , patients) were reported a baseline increase in qtc by more than ms [ , , , [ ] [ ] [ ] after hcq ± az exposure. e result of the analysis showed about . % ( % ci: . %- %) of patients had an increase of qtc by more than ms from the baseline. considerable heterogeneity was present between studies (q � . , i � . %, p < . ) (figure ). several studies also raised concerns regarding qtc prolongation following hcq exposure with or without other medications. in four studies [ , [ ] [ ] [ ] , concomitant use of other qt-prolonging medications was reported ( of therapy. similarly, from five single-arm studies [ , [ ] [ ] [ ] ] , which comprised of covid- patients treated with hcq ± az, about . % ( % ci: . %- . %) of patients had developed an absolute increase of qtc greater than ms ( figure ). however, a study by million et al. [ ] reported there were no patients who had an increased qtc greater than ms. a meta-regression was conducted to assess the effect of baseline use of other drugs suspected to cause qtc prolongation to above ms. indeed, the result did not show any significant effect (coefficients (q) � − . ; % ci: − . - . ; p � . ). in turn, the pooled prevalence of drug discontinuation due to the increased qtc prolongation to greater than ms in three studies [ ] [ ] [ ] and to greater than ms from baseline in one study [ ] among covid- patients treated with hcq with or without az was . % ( % ci: . - . ). e heterogeneity among the included studies was also acceptable enough (i � . , p � . ) ( figure ). in one study [ ] , three patients had discontinued the treatment due to adverse events other than qtc prolongation. e data of , ( hcq and hcq + az) covid- patients were included to estimate the independent effect of az on mortality and icu admission [ , ] . accordingly, hcq plus az did not improve the risk of mortality (or � . ; % ci: . - . ) and icu admission (or � . ; % ci: . - . ). e overall pooled result showed a statistically insignificant result for the composite outcome (or � . ; % ci: . - . ). e test for the overall effect was z � . (p � . ), and the heterogeneity was tau � . ; chi � . , df � (p � . ); i � % ( figure ). e global community is in the state of urgency to mitigate the health and economic crisis instigated by covid- . chloroquine (cq) and its derivative hydroxychloroquine (hcq) have traditionally been used for the treatment of malaria and certain autoimmune diseases. e drugs have possible activity against sars-cov- and sars-cov- in vitro and in clinical practice [ ] . however, clinical studies were reporting contradictory results on the efficacy and safety of hcq when used for treating covid- patients. us, systematic review and meta-analysis of the existing studies was performed to explore the efficacy, safety, and tolerability of hcq with or without az in covid- patients. e finding of this meta-analysis suggested the use of hcq did not result in a rapid viral clearance. it also failed to improve survival and rate of disease progression. e pooled prevalence of mortality was higher in patients exposed to hcq with or without az. moreover, hcq exposure carried a significant risk of adverse effects and a sizable proportion of patients ended up with drug discontinuation. on the other hand, a combination of hcq and az did not result in increased risk of qtc prolongation, improved survival, or preventing admission to icu. however, the pooled proportion of observational studies indicated an alarming rate of qtc prolongation among patients receiving hcq with or without az. e finding of this study showed an absence of rapid viral clearance (or � . ; % ci: . - . ) in patients treated with hcq compared to the standard care. however, the review by yang et al. [ ] found better virologic efficacy with statistically insignificant results. e finding of this review was based on the data from a single study [ ] , which had a high risk of bias. more importantly, the former review compared hcq + az with the standard care (control group). is arm of the study had a virologic cure rate of % reported from patients. nevertheless, the current review considered the arm which compared hcq alone versus the control group, which reported a virologic cure rate of % in the hcq arm versus . % in the control arm. e study by shamshirian et al. [ ] had also questioned the virologic potency of hcq + az (rr � . , % ci: . - . ). moreover, the optimal duration for virologic clearance is not well known. gautret et al. [ ] , jun et al. [ ] , and tang et al. [ ] reported a virologic cure after , , and days of treatment, respectively. erefore, drawing a conclusion from such findings irrespective of the incurred heterogeneity may be erroneous. of note, all the included studies were open-labeled, non-randomized, and the authors did not describe the type of treatment given for the control group. similarly, hcq could not demonstrate improved survival (or � . ; % ci: . - . ) among covid- patients. gautret et al. [ ] also reported an episode of death in the hcq group, but not in the control group. previous reviews [ ] [ ] [ ] reported either increased mortality among hcq groups or no statistically significant difference. as compared to our review, earlier reviews included a limited number of studies, while sarma et al. [ ] combined result of mortality and clinical worsening. our finding was not different from former studies [ , ] . is review also found a . % pooled prevalence of mortality from five observational studies [ , , , , ] among covid- patients treated with hcq with or without az. in three [ , , ] of these studies, respiratory and multiorgan failure was the cause of death. ere was no reported death due to adverse effects. in all of the included studies, patients had active comorbidity. ere was significant heterogeneity among the studies (i � . %, p ≤ . ), which could be attributed to the differences in disease severity and age of the participants. yet, meta-regression analysis did not produce any evidence. furthermore, hcq therapy was not significantly associated with slowing the composite end point of disease progression (or � . ; % ci: . - . ), unlike the finding by chen et al. [ ] (or � . ; % ci: . - . ). e finding by chen et al. [ ] may not be dependable as it included limited number of patients ( in each arm). e subgroup analysis result of observational studies also indicated hcq therapy was not significantly affecting disease progression (or � . ; % ci: . - . ). analogous results were reported by the preceding studies [ , , ] . however, one review indicated that hcq may prevent progression to severe disease among covid- patients [ ] . since the previous review included a limited number of studies, its finding must be interpreted cautiously. our finding was in agreement with reviews by sarma et al. [ ] and shamshirian et al. [ ] . nonetheless, these studies reported better outcomes of radiological progression in the hcq arm, a finding generated from two poorly designed controlled trials [ , ] . e safety of hcq with or without az has been questioned by several studies [ , ] . diarrhoea, vomiting, blurred vision, rash, and headache were commonly reported adverse effects. in this review, the risk of adverse effects among covid- patients treated with hcq was . (or � . ; % ci: . - . ). shamshirian et al. [ ] also found an increase in the odds of the adverse effect among patients exposed to hcq (or � . , % ci: . - . ). similar findings were also reported by other studies [ ] [ ] [ ] . however, one study reported a conflicting finding where hcq may be safe and effective, though the authors hinted more data are required for a definitive conclusion [ ] . a study conducted in china reported . % of adrs in a cluster of covid- patients. e predominant adverse effects were drug-induced gastrointestinal disorders and liver system disorders [ ] . cardiac side effects including conduction disturbances (bundle-branch block, incomplete or complete atrioventricular block, qt prolongation, and subsequent torsade de pointes) and cardiomyopathy (hypertrophy and congestive heart failure) were also described [ , ] . in this review, although the risk of qtc prolongation above ms among those exposed to hcq (or � . ; % ci: . - . ) may not appear concerning, hcq/az combination could be more worrisome. a finding generated from five single-arm studies [ , [ ] [ ] [ ] ] , which comprised the data of covid- patients treated with hcq ± az, showed . % ( % ci: . %- . %) in the pooled prevalence of qtc prolongation above ms. in addition, a finding generated from other observational studies [ , , , [ ] [ ] [ ] indicated . % ( % ci: . %- %) of patients had an increase in qtc by more than ms from the baseline. is finding was not similar to the data synthesized from double-arm studies. is is because of the fact that the double-arm section had compared the data of hcq alone with hcq + az. as adverse cardiovascular sequels, such as myocarditis, acute myocardial infarction, and heart failure, have been reported in covid- patients and these off-label therapies are not familiar to cardiovascular clinicians managing these patients [ ] , emergency care physicians should outweigh the risk and the benefits. in a nutshell, numerous studies [ , , , , , ] have raised concerns over the cardiac safety of this combination. drug discontinuation due to adverse effects was also a common finding. e current study showed . % of patients discontinued treatment due to an increase in qtc prolongation. another study also indicated . % of covid- patients treated with hcq with or without az have discontinued treatment due to adverse effects. similarly, one patient out of discontinued hcq in patients receiving hcq with or without az after three days due to qtc prolongation [ ] . ough the combination of hcq + az was effective in some studies [ , ] , the other study [ ] found it carries more hazard of death (rr � . ; % ci: . - . ) as compared to the control group. our findings showed a lack of evidence for curbing mortality and intensive care unit admission of this combination (or � . ; % ci: . - . ). it is irrefutable that our study has several limitations. e inclusion of studies with a high risk of bias and methodological flaws, combining findings from controlled and uncontrolled studies, may limit its generalizability. we also reported more than one outcome, for instance, icu admission and need of hospitalization under a single heading, namely, disease progression. is may not show the true picture of the real-world. e existence of heterogeneity, uniform treatment of all cases (mild to severe), and inclusion of limited studies with a small sample size for outcomes such selection: ( ) representativeness of the exposed cohort: a, consecutive eligible participants were selected, participants were randomly selected, or all participants were invited to participate from the source population; b, not satisfying requirements in part (a) or not stated. ( ) selection of the nonexposed cohort: a, selected from the same source population; * b, selected from a different source population; c, no description. ( ) ascertainment of exposure: a, structured injury data (e.g., record completed by medical staff); * b, structured interview; * c, written self-report; d, no description. ( ) for a demonstration that the outcome of interest was not present at the start of the study: * a, yes; b, no or not explicitly stated. b comparibility: for comparability of cohorts based on the design or analysis: * a, study controls for previous injury; * b, study controls for age. c outcome: ( ) assessment of outcome: a, independent or blind assessment stated or confirmation of the outcome by reference to secure records (e.g., imaging and structured injury data); * b, record linkage (e.g., identified through icd codes on database records); * c, self-report with no reference to original structured injury data or imaging; d, no description. ( ) was follow-up long enough for outcomes to occur? * a, yes (≥ months); b, no (< months). ( ) adequacy of follow-up of cohorts: a * , complete follow-up-all participants accounted for; * b, subjects lost to follow-up unlikely to introduce bias (< % lost to follow-up, or description provided of those lost * ); c, follow-up rate < % and no description of those lost provided; d, no statement. d total is out of stars. note: ≥ , high-quality study; - , moderate quality study; < , lowquality study. as virologic efficacy may result in biased findings. we could not retrieve some of the important findings such as disease severity scale for each study and the treatments used in the standard care setting. is systematic review, which included a limited number of poorly designed controlled clinical trials and several real-world studies of patients with covid- requiring hospitalization, found that the use of a regimen containing hcq with or without az did not offer clinical benefit. hcq with or without az did not improve the rate of virologic cure, disease progression, and mortality. ese 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pharmacotherapy in covid- ; a narrative review for emergency providers hydroxychloroquine versus covid- : a periodic systematic review and meta-analysis hydroxychloroquine versus covid- : a periodic systematic review and meta-analysis hydroxychloroquine in patients with covid- : a systematic review systematic and statistical review of coronavirus disease treatment trials efficacy and safety of current therapeutic options for covid- -lessons to be learnt from sars and mers epidemic: a systematic review and meta-analysis hydroxychloroquine in covid- : a systematic review and meta assessment of hydroxychloroquine and chloroquine safety profiles-a systematic review and meta-analysis incidence of adverse drug reactions in covid- patients in china: an active monitoring study by hospital pharmacovigilance system qt prolongation, torsades de pointes and sudden death with short courses of chloroquine or hydroxychloroquine as used in covid- : a systematic review safety considerations of chloroquine and hydroxychloroquine in treatment of covid- an algorithm for managing qt prolongation in coronavirus disease (covid- ) patients treated with either chloroquine or hydroxychloroquine in conjunction with azithromycin : possible benefits of intravenous lidocaine inpatient use of ambulatory telemetry monitors for covid- patients treated with hydroxychloroquine and/or azithromycin covid- : therapeutics and their toxicities e authors declare that they have no conflicts of interest. all the authors contributed to the conceptualization and design of the research. tam, tmf, and gzn conducted the database search, screening, quality assessment, and analysis. tam, tmf, kkg, ddb, and gzn carried out data extraction and wrote the manuscript. kmg and ddb handled the discrepancies that arise during data extraction. all the authors read and approved the final manuscript. e search strategy was created with the assistance of the librarians at the jimma university drug information center.e medley desktop was used as a reference manager. randomized controlled trials and the modified newcastle-ottawa quality assessment scale for the included observational studies (figures and and table additional data not included in the article can be obtained from the corresponding author upon request. canadian respiratory journal key: cord- -q d rvnj authors: sun, jingkang; chen, yuting; fan, xiude; wang, xiaoyun; han, qunying; liu, zhengwen title: advances in the use of chloroquine and hydroxychloroquine for the treatment of covid- date: - - journal: postgraduate medicine doi: . / . . sha: doc_id: cord_uid: q d rvnj coronavirus disease (covid- ), caused by severe acute respiratory syndrome coronavirus (sars-cov- ), is spreading worldwide. antiviral therapy is the most important treatment for covid- . among the drugs under investigation, anti-malarials, chloroquine (cq) and hydroxychloroquine (hcq), are being repurposed as treatment for covid- . cq/hcq were shown to prevent receptor recognition by coronaviruses, inhibit endosome acidification, which interferes with membrane fusion, and exhibit immunomodulatory activity. these multiple mechanisms may work together to exert a therapeutic effect on covid- . a number of in vitro studies revealed inhibitory effects of cq/hcq on various coronaviruses, including sars-cov- although conflicting results exist. several clinical studies showed that cq/hcq alone or in combination with a macrolide may alleviate the clinical symptoms of covid- , promote viral conversion, and delay disease progression, with less serious adverse effects. however, recent studies indicated that the use of cq/hcq, alone or in combination with a macrolide, did not show any favorable effect on patients with covid- . adverse effects, including prolonged qt interval after taking cq/hcq, may develop in covid- patients. therefore, current data are not sufficient enough to support the use of cq/hcq as therapies for covid- and increasing caution should be taken about the application of cq/hcq in covid- before conclusive findings are obtained by well-designed, multi-center, randomized, controlled studies. coronavirus disease- (covid- ) caused by severe acute respiratory syndrome coronavirus (sars-cov- ) is rapidly spreading worldwide, resulting in the third outbreak of coronaviruses in the st century. the pandemic of covid- constitutes a serious threat to the whole world [ ] . to control the pandemic of covid- , effective and easily accessible antiviral drugs and vaccines are urgently needed, in addition to the implementation of epidemiological measures such as strict quarantine. however, until now, no drugs have been demonstrated to be effective against covid- . among the various drugs under investigation are repurposed antimalarial drugs chloroquine (cq) and its analog hydroxychloroquine (hcq), which are among the most used drugs because they are easy to obtain and have a proven favorable safety record at relatively low cost. cq/hcq are derivatives of -aminoquinoline. they are lipophilic weak bases that quickly pass across cell membranes and accumulate in acidic organelles, such as lysosomes, endoplasmic reticulum and golgi [ ] . cq/hcq are used to treat and prevent malaria attacks due to their anti-plasmodium activity and to treat autoimmune diseases such as systemic lupus erythematosus (sle) and rheumatoid arthritis (ra) owing to their immunomodulatory activity [ ] . in addition, cq/hcq display antibacterial, antifungal and antiviral activities [ ] . in vitro studies have shown that cq/hcq possess antiviral activity against rna viruses, such as hiv [ ] , rabies virus [ ] and polio virus [ ] and various dna viruses as diverse as hepatitis b virus [ ] and herpes simplex virus [ ] . this article reviews the current status of cq/hcq against sars-cov- and their use in the treatment of covid- . cov activity than hcq. in addition, cq has both a prophylactic and a therapeutic advantage. vincent et al. tested various concentrations of cq ( . - µm) added - h prior to sars-cov infection and found that . , , and µm cq reduced infectivity by %, %, and %, respectively; when cq was added immediately after virus adsorption, . - µm and - µm reduced the infection by % up to - %; addition of cq and h after virus adsorption was still significantly effective, yet to achieve equivalent antiviral effect, a higher concentration of cq was needed [ ] . in hrt- cells, the antiviral activity of cq against hcov-oc had an ec of . (± . ) μm, cc of . (± . ) μm, and si of . [ ] . an in vivo study found that cq could exert anti-hcov-oc activity transplacentally or via maternal milk. the data from mouse models showed that . % of the pups survived when pregnant mice were treated with mg/kg of cq, and survival rates decreased in a dose-dependent manner, with % and % survival when treated with mg/kg and mg/kg cq, respectively [ ] . the survival rate of newborn mice via maternal milk was . % with mg/kg of cq [ ] . in another mouse study, cq strongly attenuated hcov-oc replication in the brain and prevented the infection from spreading to the spinal cord [ ] . the above studies confirmed that cq/hcq have a broad-spectrum anti-hcov activity in vitro and in vivo. in veroe cells, the ec , cc and si of cq against sars-cov- were . μm, > and > . , respectively. cq functioned at the entry, and post-entry stages of sars-cov- infected cells [ ] . in the same cell line, at different multiplicities of infection (mois, . , . , . , and . ) of sars-cov- , the ec for cq ( . , . , . , and . μm) was slightly lower than that of hcq ( . , . , . , and . μm). consequently, the si of cq ( . , . , . , and . ) was slightly higher than that of hcq ( . , . , . , . ) [ ] . these results indicate that the anti-sars-cov- activity of cq seems to be more potent than hcq in vitro. however, another in vitro cell experiment showed that after sars-cov- infection of veroe cells, the ec values for cq were . μm and . μm, and ec values for hcq were . μm and . μm, at and h, respectively; when administered prior to sars-cov- infection of veroe cells, ec values for cq were > μm and . μm, and the ec values for hcq were . μm and . μm, at and h, respectively [ ] . these results showed that the anti-sars-cov- activity of cq was worse than hcq in vitro. the conflicting results of these two studies may be related to different cell culture methods and experimental conditions. in short, these in vitro studies show that cq/hcq have strong anti-sars-cov- activity. the s protein of sars-cov- is cleaved by host proteases into two subunits, s and s [ ] . the s subunit binds to the host cell surface receptor angiotensin-converting enzyme (ace ) for virus attachment, and the s subunit fuses the virus and the host cell membrane [ ] . the investigation of the effect of cq on ace in veroe cells showed that effective anti-sars-cov- concentrations of cq had no significant effect on the synthesis and glycosylation of s protein on the surface of sars-cov, and although it had no significant effect on the cell surface expression of ace , cq could destroy the glycosylation at the terminal glycosylation site of ace [ ] . therefore, the mechanism of anti-cov activity of cq/hcq may be at least partly related to the impairment of terminal glycosylation of ace , which may result in reduced binding affinities between ace and sars cov s protein, thereby blocking receptor recognition ( figure ). in addition to protein membrane receptors, infection of host cells by hcovs also relies on sialic acid-containing glycoproteins and gangliosides, which are used by a broad range of viruses as receptors, such as influenza [ ] and hcovs including sars-cov [ ] and hcov-oc [ , , ] . a recent molecular structure analysis showed that sars-cov- not only uses ace as a receptor, but also recognizes highly conserved gangliosides on the host cell surface through sialic acid [ , ] . cq/hcq binds sialic acids and gangliosides with high affinity, which can prevent the attachment of sarscov- s protein to gangliosides [ ] . cq had inhibitory effect on quinone reductase (qr ) involved in the biosynthesis of sialic acids [ , ] . hence, the mechanism of anti-cov activity of cq/hcq may also be related to hindering the recognition process of sialic acid and ganglioside ( figure ). covs are enveloped rna viruses, and their cell entry processes involve a principal route of receptor-mediated endocytosis [ ] . membrane fusion takes place in the endosomal compartment after endocytosis, which needs additional triggers such as ph acidification or proteolytic activation [ ] . multiple cellular proteases, such as trypsin, furin, proprotein convertase (pc) family, cathepsins, transmembrane protease/serine (tmprss) proteases and elastase, are involved in s protein activation, which can induce membrane fusion [ ] . among them, cathepsin l, with anoptimal ph of . to . , is most commonly associated with activation of a variety of cov s proteins [ ] , such as sars-cov [ ] , mers-cov [ ] , hcov- e [ ] , and mouse hepatitis virus (mhv- ) [ ] . a recent study found that sars-cov- enters /hace cells mainly through endocytosis, in which cathepsin l is critical for priming of sars-cov- s protein [ ] . a study investigated the detailed mechanism of action of cq/hcq in inhibiting sars-cov- entry, and co-localization of sars-cov- with early endosomes (ees) or endolysosomes (els) in veroe cells, and the results showed that cq/hcq hampered the transport of sars-cov- from ees to els, indicating that cq/hcq might inhibit endosomal maturation [ ] . these studies revealed that the mechanism of anti-cov activity of cq/hcq may involve the inhibition of the endosome acidification process, which might inactivate lysosomal proteases, thus interfering with the fusion of virus and host membranes [ , ] ( figure ). the mitogen-activated protein kinase (mapk) pathway transmits signals from the cell surface to the nucleus involved in the infection of covs such as mhv [ ] and sars-cov [ ] . cq could inhibit hcov- e replication in human embryonic lung epithelial cells (l ) through suppressing the activation of p mapk [ ] . moreover, hcq could markedly induce the production of cellular reactive oxygen species (ros), which play an important role in the activation of innate immunity [ ] . hcq also could trigger the host defense mechanism through the mitochondrial antiviral signaling (mavs) pathway, resulting in anti-dengue virus activity [ ] . therefore, cq/hcq may also exert their antiviral activity by suppressing the activation of p mapk pathway and affecting the host defense machinery (figure ). cq/hcq regulate the release of various pro-inflammatory factors, which are important immunomodulators. intracellular alkalinization by cq/hcq inhibits lysosomal activity, preventing antigen processing, major histocompatibility complex (mhc) class ii expression and immune activation [ ] . this process can inhibit t cell activation and block expression of cd on the surface of cd + t cells [ ] . cq also reduces cytokines such as interleukin (il)- , il- and tumor necrosis factor-α (tnf-α) produced by t cells and b cells [ ] . at the same time, changes of endosomal ph can interfere with tolllike receptor (tlr) signaling, such as tlr and tlr processing, inhibiting the activation and production of cytokines [ ] . cq/hcq also weaken the cyclic gmp-amp (cgamp) synthase (cgas) activity by inhibiting cytosolic dna, thereby reducing type i interferon production [ ] . in vitro, cq/hcq can also inhibit phospholipase a , altering the metabolism of arachidonic acid, and reducing the production of prostaglandins [ ] . some clinical studies have found that high concentrations of cytokines and pro-inflammatory factors such as il- and il- are elevated in the plasma of critically ill patients infected with sars-cov- [ , ] , suggesting that cytokine release syndrome (crs) is associated with disease severity. in the aspect of immune response, hcq/cq therefore are likely to inhibit crs, delaying the progression of covid- ( figure ). only two published clinical reports have studied the efficacy of cq in covid- patients (table ) . one study used cq to treat more than patients with covid- and claimed that cq was superior to the control group in suppressing the deterioration of pneumonia, improving lung imaging, promoting viral conversion and shortening the course of disease. serious adverse effects were not observed in these patients figure . schematic representation of the possible mechanisms of cq/hcq against covs replication and modulating immune response. cq/hcq may synergistically exert antiviral and immunomodulatory effects on covid- through multiple mechanisms including hindering the receptor recognition process by influencing the affinity of ace and s protein, and the affinity for sialic acid and ganglioside; inhibiting the membrane fusion process by suppressing endolysosome acidification; suppressing the p activation and affecting host defense machinery, and preventing mhc class ii expression (block expression of cd on the surface of cd + t cell) and tlr signaling and reducing the production of cytokines through inhibiting the activation of t cells and b cells. ace , angiotensin-converting enzyme ; covid- , coronavirus disease ; cq, chloroquine; hcq, hydroxychloroquine; covs, coronaviruses; mapk, mitogen-activated protein kinase; mhc-ii, major histocompatibility complex class ii; tlr, toll-like receptor; cgas, cyclic gmp-amp synthase; ifn, interferon; il, interleukin; tnf-α, tumor necrosis factor-α. a macrolide, on in-hospital outcomes for covid- was not observed. large multinational real-world data and large number of study populations. there may be potential confounders; it did not measure qt intervals and stratify the arrhythmia pattern; it did not determine whether the increased risk of death inhospital and use of drug treatment regimens were directly related to cardiovascular risk; it did not observe the risk of the drug dose-response analysis. [ ] . however, this report did not provide any details about the study design and patient data, thus it is difficult to evaluate the validity. recently, a parallel, double-blind, randomized, phase iib clinical trial was performed in brazil [ ] . in this study, severe covid- patients were randomly divided into two groups: patients received high-dose cq ( mg/ times/day for day) and patients received lowdose cq ( mg/ times on day and then mg/ time/ day for days). the -day mortality rate in the high-dose group was more than double that in low-dose group ( . % vs. . %). the high-dosage group exhibited more instance of corrected qt (qtc) interval prolongation (> milliseconds (ms); of [ . %]) than the low-dosage group ( of [ . %] ). these findings suggest that the higher cq dosage should not be recommended for critically ill patients with covid- [ ] . several trials evaluated the efficacy of hcq for the treatment of covid- (table ). in a randomized clinical trial from wuhan about hcq treatment of mild covid- [ ] , out of patients received hcq ( mg/ times/day for days). the results showed that the temperature recovery time in the hcq group was improved compared with the control group (average days, . vs. . ); the cough relief time was shorter in the hcq group than the control group (average days, . vs. . ); and the improvement rate of pneumonia in the hcq group was higher than the control group ( . % vs. . %). however, only % of patients ( / ) in hcq group and % of patients ( / ) in control group had cough at baseline and the duration of cough was not described. improvement of symptoms were small and the trial was terminated prematurely. these factors and the low sample size compromise the reliability of the results of this study. importantly, evaluation of hcq in the covid- pandemic areas have shown that hcq can help patients with mild symptoms, and may potentially reduce transmission in areas lacking isolation facilities [ , ] . however, in areas with strict isolation standards, the use of hcq to reduce transmission or for treatment of mild covid- cases may not be beneficial in risk-benefit analysis [ , ] . nevertheless, there is an urgent need for drugs and therapeutics in severe cases, which require randomized controlled trials. a study from four french tertiary care centers included patients hospitalized for covid- and requiring oxygen ( l/min): patients received hcq ( mg/day) within hours of admission (hcq group) and did not (no-hcq group) [ ] . the results showed that the patients transferred to the icu or died within days and developed ards within days had no significant differences between the hcq group and no-hcq group. eight patients in the hcq group ( . %) discontinued hcq due to electrocardiogram alterations. these results do not support the use of hcq for treating hospitalized covid- -related hypoxic pneumonia patients. it is worth noting that in the study's propensity score model, four possible important prognostic variables were unbalanced and a center effect was not considered, which all can cause bias for study results. in a multicenter, randomized, parallel trial about hcq in patients with mainly mild to moderate covid- [ ] , patients received 'standard care' and patients received hcq ( mg daily for days, and then mg daily for weeks [mild to moderate disease] or weeks [severe disease]). the results showed that the days negative conversion probability in 'standard care'＋hcq group was . %, similar to the 'standard care' group ( . %). hcq did not show additional benefits of viral elimination in patients with mild to moderate covid- . however, the study could not evaluate the antiviral effect of hcq at early stages of disease, which is a critical period of antiviral treatment. in addition, viral rna specimens were mostly from the upper respiratory tract rather than bronchoalveolar lavage fluid, which may cause false negative results. due to the small number of severe patients, this study could not provide evidence regarding the effect of hcq on the disease progression or regression. in another large observational study involving cases of covid- from new york, patients received hcq ( mg/ times on the first day, then mg once a day for days) within or hours of admission and did not [ ] . this study found no correlation between hcq use and significantly higher or lower risk of intubation or death. however, in this study, even after the propensity scorematching, the diseases in patients receiving hcq were more severe at baseline than those in the patients not receiving. notably, according to another recent study [ ] , low dose of hcq reduced fatality of critically ill patients with covid- without apparent toxicity. this retrospective study included patients who need mechanical ventilation, of which received hcq treatment ( mg/ times/day for to days) and did not. the fatalities of the hcq group was significantly lower than no-hcq group ( . % vs . %, p < . ), and the inflammatory cytokine il- in the hcq group decreased significantly from . ( . to . pg/ml) at the beginning of treatment to . ( . to . pg/ml) at the end of treatment. the authors deemed that the anti-inflammatory effect of low-dose hcq and the activity of inhibiting viral replication may have important significance in critically ill patients with covid- . yet, this study is flawed due to its retrospective nature and the small number of hcq treated patients included. in short, some initial studies have shown that hcq appears to have a curative effect on patients with mild covid- , but subsequent studies indicate that hcq had no significant benefit in covid- patients with viral conversion and the risk of intubation or death. although some recent studies show that low-dose hcq could potentially reduce the mortality of severe covid- patients, there are other studies showing that the hcq use had no effect on risk of intubation or death. there are also several reports that investigated the efficacy of cq or hcq in combination with a macrolide in the treatment of covid- (table ) . in an open nonrandom clinical trial conducted in france [ ] , of the participants, patients were given hcq ( mg/ times) with receiving added azithromycin, and controls. the results showed that compared with the control group, hcq alone or in combination with azithromycin could effectively eliminate nasopharyngeal virus in - days. on the th day after treatment, the virus clearance rates of hcq combined with azithromycin, hcq alone and controls were %, . % and . %, respectively (p < . ). this study indicated that the combined application of azithromycin and hcq appears to have a synergistic effect. however, the trial design and the results were unreliable, as six patients in the hcq group discontinued treatment early due to critical illness or intolerance to the drugs and were excluded from the analysis. the assessment of efficacy was based on viral load rather than a clinical endpoint. an observational study in covid- patients evaluated the efficacy of hcq ( mg/ times/day for days) in combination with azithromycin ( mg on the first day, mg/day afterward for days) and showed that all patients' clinical symptoms were improved, except for one patient aged over years who died due to critical illness [ ] . the nasopharynx viral load in most patients decreased rapidly, and the negative rates of viral nucleic acid conversion on days and were about % and %, respectively. about . % of patients had negative virus culture in respiratory specimens on the fifth day. however, this study had no control group, thus the results were difficult to interpret [ ] . some recent studies have yielded different results about the efficacy of hcq combined with azithromycin. a retrospective study including patients ( patients received hcq, patients received hcq + azithromycin and patients received no hcq) from usa [ ] showed that the rates of ventilation in the hcq, hcq+azithromycin and no hcq groups had no significant differences. unfortunately, thehcq group (but not in the hcq+azithromycin group) had a higher risk of death from any case than the no hcq group. this study showed no evidence that the use of hcq, either with or without azithromycin, reduced the risk of mechanical ventilation in patients hospitalized with covid- . noticeably, in patients treated with hcq alone, an association with increased overall mortality was observed [ ] . in this study, the subjects included were only men and most of them were black, which may affect the generality of the results. in addition, the patients who received hcq or azithromycin were more severe, which may also affect the results. in a retrospective multicenter cohort study of a random sample of covid- patients from hospitals in new york [ ] , totaling patients, received hcq and azithromycin, received hcq alone, received azithromycin alone and received neither drug (hcq or azithromycin). the results showed that the hospital mortality rate of patients receiving hcq＋azithromycin was . %, hcq alone was . %, azithromycin alone was . % and neither drug was . %. in adjusted cox proportional hazards models, compared with patients receiving neither drug, there were no significant differences in hospital mortality rate for patients receiving hc＋azithromycin, hcq alone, or azithromycin alone. in this study, the sample size is large and includes patients with long-term, complex and ongoing hospitalization. however, the mortality rate of this study was limited to inhospital deaths, and patients discharged during the study period were considered alive, which may underestimate the morality rate. recently, a multinational registry analysis about hcq or cq with or without second-generation macrolides (especial azithromycin and clarithromycin) for treatment of covid- was reported [ ] . a total of , patients were included in this study. of these, received cq, received cq with a macrolide, received hcq and received hcq with a macrolide and patients as control group. after controlling various confounding factors related to disease, when compared with the mortality in the control group ( . %), cq group was . %, cq with a macrolide group was . %, hcq group was . % and hcq group with a macrolide was . %; each group was associated with an increased risk of hospital mortality independently. apart from this, compared with the control group ( . %), cq group ( . %), cq with a macrolide group ( . %), hcq group ( . %) and hcq with a macrolide group ( . %) were independently associated with a risk for ventricular arrhythmia during hospitalization [ ] . this study showed that cq or hcq (used alone or combination with a macrolide) was associated with an increased hazard for in-hospital death and an increased risk of ventricular arrhythmias. this study included a large number of patients, but it is not a randomized clinical trial. in short, some small studies have shown that hcq combined with azithromycin could quickly and effectively eliminate viruses, but the design of these studies was flawed in many aspects, making the results unconvincing. several subsequent studies have shown that the combination of hcq or cq and macrolides (azithromycin or clarithromycin) has no obvious correlation with a reduced risk for mechanical ventilation, and may even increase the risk of arrhythmia and in-hospital mortality. in summary, although cq/hcq appeared to exhibit a favorable effect on covid- patients in some initial studies of small numbers of patients, the most recent studies with larger sample sizes revealed that cq/hcq exhibited no significant improvement of disease but even an increased overall mortality in covid- patients. the studies on the combination of hcq or cq and macrolides (azithromycin or clarithromycin) also showed conflicting findings. therefore, caution should be taken regarding the use of cq/hcq treatment in covid- due to the uncertainty of efficacy, the potential adverse effects and the various defects in the studies. according to the chinese clinical trial registry (chictr) (http://www.chictr.org.cn/index.aspx) and the international clinical trials registry platform (ictrp) (https://www.who.int/ ictrp/en/), currently, there are more than ongoing clinical trials for cq/hcq. current findings suggest that cq/hcq alone or in combination with macrolides should not be recommended for widespread use in covid- (except in clinical trials). results from these ongoing prospective, randomized, controlled studies are required before these drugs are recommended for the treatment of covid- . cq/hcq are basic medications for malaria with a long history of reliable safety records [ , ] . however, the therapeutic window of cq is narrow, and the toxic dose is times higher than the therapeutic dose [ , ] . the most common adverse effects of taking cq are gastrointestinal discomforts, such as nausea, vomiting, diarrhea, and anorexia. these symptoms are mild and can be controlled by reducing the dose [ ] . however, long-term and high-dose cq intake can cause irreversible damage to the ear, cardiovascular system, and blood system, such as neurological deafness, conduction disorder cardiomyopathy, and leukopenia, though these adverse effects are very rare [ ] . compared to cq, hcq has fewer adverse effects, which may be related to its lower toxicity. in animal models, hcq was about % less toxic than cq [ ] . reportedly, only overdoses (average daily dose > . mg/kg) and long-time (more than years) ingestion of hcq can cause retinopathy [ ] . cq/hcq have similar pharmacokinetic characteristics, fast absorption in the gastrointestinal tract, fast excretion in liver and kidney, and a long half-life ( - days) [ , ] . therefore, liver and kidney dysfunction may aggravate adverse effects. high-dose cq ( mg/ times/day for day) is associated with increased qtc interval prolongation in critically ill patients with covid- [ ] and should not be recommended. in the initial trial from france, of the patients receiving hcq treatment, patients discontinued hcq owing to ecg modifications within days. among them, patients had a prolonged qtc interval more than ms, and one patient developed a first-degree atrioventricular block within days [ ] . in a randomized clinical trial from wuhan, out of patients receiving hcq treatment had minor adverse effects (headache and rash) [ ] . prolonged qt interval after taking hcq may also develop in icu covid- patients [ ] . a recent observation showed that patients who received hcq for the treatment of pneumonia associated with covid- were at high risk of qtc prolongation ( %), and concurrent use of azithromycin was associated with greater changes in qtc ( %) [ ] . another observation in covid- patients admitted to icu showed that qtc intervals increased in % of patients receiving hcq with or without azithromycin, prolonged qtc was observed in % of patients after a duration of the treatment for to days, and of ( %) patients treated with hcq and azithromycin and of ( %) of those treated with hcq alone developed an increase in qtc of ms or greater [ ] . the use of cq, cq with a macrolide, hcq and hcq with a macrolide were all found to be independently associated with increased risk for ventricular arrhythmia in hospitalized covid- patients [ ] . therefore, clinicians should carefully weigh risks and benefits if considering cq/ hcq with or without a macrolide. when cq/hcq are used, electrocardiogram examination should be routinely performed before taking the medicine, with close monitoring of qtc and concomitant medication usage. cq/hcq should be more cautiously used in patients with existing heart disease, and the use of qt interval prolonging drugs, such as antiarrhythmic drugs, antihistamines, and moxifloxacin should be avoided. in addition, close attention should be paid to symptoms after taking drugs and the drugs should be stopped in time if there are intolerable adverse reactions. in addition, cq is extremely dangerous for patients with glucose -phosphate dehydrogenase (g pd) deficiency because of the possible induction of hemolytic anemia [ ] . therefore, more caution should be given for patients with g pd deficiency when cq is considered for the treatment and the best way should be to detect g pd deficiency before the use of cq. at present, the covid- pandemic is continuing worldwide. it is still an urgent need to find effective therapies and vaccines for treatment and prevention. cq/hcq have diverse biological activities, and their mechanisms against covs including sars-cov- are not yet fully clarified. current studies show that cq/ hcq can prevent receptor recognition by covs, inhibit endosome acidification, which interferes membrane fusion, and exhibit immunomodulatory activity. these multiple mechanisms may work together to exert a therapeutic effect on covid- . a number of in vitro studies have revealed that cq/hcq have inhibitory effects on various covs, including sars-cov [ , ] , mers-cov [ ] and sars-cov- [ ] [ ] [ ] . however, conflicting results also exist on the in vitro activity of cq/hcq against sars-cov- [ , ] . several clinical studies have shown that cq/hcq may alleviate the clinical symptoms of covid- , promote viral conversion, and delay the progression of the disease, with less serious adverse effects [ , , , ,] . however, previous studies showed that cq had anti-ebola virus activity in cell culture, but it had conflicting results in animal models [ , ] . in addition, cq has shown beneficial results against chikungunya virus in vitro, but in animal models it aggravates the infection and lacks therapeutic effect [ ] . more importantly, in recent studies the use of hcq did not show any favorable effect on patients with covid- and high-dose cq treatment of severe covid- patients may even increase the risks of mortality and qtc interval prolongation [ , ] . in addition, the optimal daily dose and duration of treatment course are not yet clear. one study suggested that the dose of hcq should be mg/ times for day, mg/ times/day for days based on the physiological pharmacokinetic model [ ] . a prospective study of hcq on covid- patients ( cases) admitted to the icu in france showed that the first daily dose of mg/ time for day, and mg/ times/day for days was recommended to maintain the hcq treatment level ( - mg/l) based on physiologically pharmacokinetic (pbpk) models for covid- patients in icu [ ] . whether the dosage of cq or hcq should be varied according disease severity is also unclear. a rodent study showed that cq could exert anti-hcov-oc activity transplacentally or by way of maternal milk [ ] . however, in humans, the efficacy of cq in the prevention and treatment of sars-cov- infection to both the mother and the child remains to be investigated. clinical trials in france showed that hcq combined with azithromycin could enhance the virus clearance [ ] , but the subsequent reports did not support this combination [ , ] . furthermore, cq/hcq alone or in combination with a macrolide induced high rate of adverse effects, especially prolonged qtc, in the use for covid- treatment [ , , ] . therefore, current data are not sufficient enough to support the routine use of cq/hcq as therapies for covid- and increasing caution should be taken for the application of cq/hcq, alone or in combination with other drugs, in covid- before the conclusive findings are obtained by well-designed, multicenter, randomized, controlled studies. novel coronavirus ( -ncov): update on rd coronavirus outbreak of st century antimalarials -are they effective and safe in rheumatic diseases? reumatologia chloroquine analogues in drug discovery: new directions of uses, mechanisms of actions and toxic manifestations from malaria to multifarious diseases recycling of chloroquine and its hydroxyl analogue to face bacterial, fungal and viral infections in the st century the potential place of chloroquine in the treatment of hiv- -infected patients ammonium chloride and chloroquine inhibit rabies virus infection in neuroblastoma cells chloroquine 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fda-approved drugs for inhibitors of biological threat agents chloroquine inhibited ebola virus replication in vitro but failed to protect against infection and disease in the in vivo guinea pig model paradoxical effect of chloroquine treatment in enhancing chikungunya virus infection we are very grateful to dr. adam kim from cleveland clinic for the english language editing of our manuscript. this manuscript received no funding. the authors have no financial or other relationships to disclose.peer reviewers on this manuscript have no relevant financial or other relationships to disclose. jks and zwl wrote the article, and contributed to the concept of this article, definition of intellectual content, and data acquisition; ytc, xdf, xyw contributed to data acquisition; qyh reviewed the manuscript for its intellectual content. no potential conflict of interest was reported by the authors. http://orcid.org/ - - - yuting chen http://orcid.org/ - - - key: cord- -i p p o authors: ruamviboonsuk, paisan; lai, timothy y. y.; chang, andrew; lai, chi-chun; mieler, william f; lam, dennis s. c. title: chloroquine and hydroxychloroquine retinal toxicity consideration in the treatment of covid- date: - - journal: asia pac j ophthalmol (phila) doi: . /apo. sha: doc_id: cord_uid: i p p o the proposed doses of chloroquine (cq) and hydroxychloroquine (hcq) for treatment of covid- ( mg/day for days, cq; mg first day then mg/day for days, hcq) in many guidelines worldwide, are considerably higher than the maximum recommended daily safe doses of both agents (≤ . mg/kg/day, cq; ≤ . mg/kg/day, hcq) for development of retinal toxicity. irreversible retinal damage can occur if the exposure to the safe doses is > years. it is not known whether exposure to high doses over a short period of time can also cause the damage. we recommend that before prescribing cq or hcq, history of ocular disease should be obtained to avoid the prescription if appropriate. if either agent is to be used, routine baseline ocular examination is not absolutely necessary. patients who do not have ocular disease should also be informed about the potential risk of retinal toxicity. both agents, however, have not yet been proven to be beneficial to covid- . w ith the occurrence of pandemic of the coronavirus disease (covid- ) announced by the world health organization in early march and the number of cases still on the rise in all continents in late march, many therapeutic options have been proposed for this novel and potentially fatal disease. apart from antiviral agents, chloroquine (cq) and hydroxychloroquine (hcq) have been examined for their roles in treatment of covid- . this may be because both cq and hcq have been postulated to reduce viral replication in other coronavirus infections. , according to a recent systematic review, there are almost ongoing randomized controlled clinical trials on both medications for treatment of covid- and all of them are in china. the details including dosing regimens of cq and hcq in these trials have been summarized in the review. although there has not yet been a completed clinical trial and the world is waiting eagerly for the results of these trials and other trials on other treatment options, many authorities have chosen to adopt cq and hcq in the guidelines for treatment of covid- based on in vitro studies, , nonrandomized trial, and anecdotal evidence. [ ] [ ] [ ] [ ] [ ] [ ] as the therapeutic doses of cq and hcq recommended in the trials and guidelines are relatively high compared with the maximum daily safe dose that is related to cq and hcq retinal toxicity, this issue of retinal toxicity should be taken into consideration when employing these medications for treatment of covid- worldwide. according to the recommendation by the american academy of ophthalmology, the most significant major risk factors for cq and hcq retinal toxicity are high dose and long duration of use. other risk factors include concomitant renal disease and use of tamoxifen. the maximum daily dose from this recommendation is . mg/kg real body weight for hcq, and . mg/kg real body weight for cq. as shown in table , the doses of cq or hcq for treatment of covid- in various treatment guidelines worldwide are well beyond these recommended dosing regimens. the royal college of ophthalmologists (rco) in the uk also addressed the importance of safe dose and duration of prescription of cq and hcq for the development of retinal toxicity. although no absolute safe dose was identified, the rco recommends the daily dose of hcq to be < mg/kg/day for < years as relatively safe for retinal toxicity. however, no safe dose of cq was recommended and the rco identified those who receive cq for > year as having risk of retinal toxicity. despite the fact that the daily doses of both cq and hcq for the treatment of covid- exceed the daily safe doses of both agents, the treatment may still be considered relatively safe for retinal toxicity. the toxicity, which causes irreversible retinal damage and visual loss despite ceasing prescription, requires exposure to the safe dose for a long period of time, generally in excess of years. in general, the recommendation for screening for retinal toxicity from cq and hcq is within the first year of use as baseline and then annual screening after a year of use for cq and years of use for hcq. both american academy of ophthalmology and rco recommended the screening should be conducted sooner if the major risk factors are present. , in the case of treatment of covid- using cq and hcq, the major risk factor is the use of higher than generally recommended dosage, although over a relatively short period of time, that is for about a week. there has not yet been a report on retinal toxicity associated with this kind of treatment. nonetheless, it has been reported that retinal toxicity can develop even after < year of high dose of hcq use ( mg daily) in an oncology trial. in this report, of patients who received the high dose of hcq showed abnormalities of the macula on retinal imaging modalities and multifocal electroretinogram without visual symptoms. these patients did not have any known risk factors, such as renal disease, concomitant retinotoxic agents, or co-existing retinal disease. it is not known whether the retinal toxicity from high-dose cq and hcq is underreported in the literature due to suboptimal and nonuniform ocular screening methods. both cq and hcq are known for their binding affinity with melanin in retinal pigment epithelium which can be a mechanism of the toxic effects. both agents have also been shown to cause damage to the photoreceptor layer and outer nuclear layer of the retina, whereas cq can cause damage to the inner retina as well. light absorption and metabolism of cone cells may also play roles for the damages. these mechanisms may lead to clinically characteristic "bull's eye" maculopathy after chronic exposure to both agents even in the safe dose. it is not known whether exposure to the high dose over a short period may also cause similar cellular damages as with the chronic exposure. given that patients with covid- who may require treatment are commonly older patients, it is possible that some may already have coexistent age-related macular degeneration. it is still not known whether the diseased macula would be more vulnerable to damage with exposure to the high dose of either cq or hcq even over a short duration. routine baseline ocular examination is not absolutely necessary for patients with covid- who are undergoing treatment with cq and hcq but should be considered if manpower and expertise are available and extreme precautions should be taken during the examination. it is relevant, however, to take a history of ocular disease, particularly macular disease, in patients with covid- who are older than years before prescribing cq or hcq as treatment, to rule out age-related macular degeneration or other macular abnormalities. coexistent retinal pathology is listed as a contraindication of using cq and hcq in patients with covid- in the treatment guideline of belgium. as treatment with cq or hcq is not yet proven to be beneficial, but instead can be harmful, in covid- , choosing other options of treatment in this group of patients with the preexisting disease may be more appropriate. for patients in whom cq or hcq is still considered as a treatment option, the potential benefits and risks of retinal toxicity and other systemic complications shall be thoroughly discussed with patients and well documented on written consent form before the treatment or trial of cq or hcq. following recovery from covid- with the treatment using cq or hcq, the patients should also be informed to visit ophthalmologists if they encounter any abnormal visual symptoms. in summary, the bottom line at the present time is that neither cq nor hcq has been proven to be effective in the treatment of covid- , although there is certainly a vast interest in its possible benefit. further controlled clinical trial data will be necessary to help better address this issue. despite the current situation of covid- pandemic, many adverse effects of either cq or hcq should still be weighed against its potential benefit. for retinal toxicity, the risk of having irreversible retinal damage and visual loss may outweigh the unproven benefit of both agents in some patients. detecting the risk is easy. it can be done by simply taking a history of previous or co-existing ocular disease from the patients, then other options of treatment should be considered if appropriate. covid- : a recommendation to examine the effect of hydroxychloroquine in preventing infection and progression in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro a systematic review on the efficacy and safety of chloroquine for the treatment of covid- hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial thai treatment guideline for covid- medicamenteuze behandelopties bij patiënten met covid- (infecties met sars-cov- ) multicenter collaboration group of department of science and technology of guangdong province and health commission of guangdong province for chloroquine in the treatment of novel coronavirus pneumonia interim clinical guidance for patients suspected of/ confirmed with covid- in belgium therapeutic options for covid- patients j cdc national institute for the infectious diseases "l. spallanzani" irccs. recommendations for covid- clinical management recommendations on screening for chloroquine and hydroxychloroquine retinopathy ( revision) the royal college of ophthalmologists recommendations on screening for hydroxychloroquine and chloroquine users in the united kingdom: executive summary consensus-statement rapid onset of retinal toxicity from high-dose hydroxychloroquine given for cancer therapy hydroxychloroquine retinopathy. eye (lond) could chloroquine /hydroxychloroquine be harmful in coronavirus disease (covid- ) treatment? the weight of nations: an estimation of adult human biomass the authors acknowledge varis ruamviboonsuk, md, in assisting manuscript preparation. key: cord- -fcdwitxy authors: ayerbe, luis; risco-risco, carlos; ayis, salma title: the association of treatment with hydroxychloroquine and hospital mortality in covid- patients date: - - journal: intern emerg med doi: . /s - - -x sha: doc_id: cord_uid: fcdwitxy this study investigates the association between the treatment with hydroxychloroquine and mortality in patients admitted with covid- . routinely recorded, clinical data, up to the th of april , from the patients with covid- , admitted in hospitals in spain between the st of march and the th of april were used. the following variables were extracted for this study: age, gender, temperature, and saturation of oxygen on admission, treatment with hydroxychloroquine, azithromycin, heparin, steroids, tocilizumab, a combination of lopinavir with ritonavir, and oseltamivir, together with data on mortality. multivariable logistic regression models were used to investigate the associations. at the time of collecting the data, patients had died, had been discharged home from the hospitals, were still admitted, and had been transferred to hospitals not included in the study. median follow-up time was (iqr – ) days. hydroxychloroquine had been used in patients. hydroxychloroquine was associated with lower mortality when the model was adjusted for age and gender, with or ( % ci): . ( . – . ). this association remained significant when saturation of oxygen < % and temperature > °c were added to de model with or . ( . – . ) p < . , and also when all the other drugs, and time of admission, were included as covariates. the association between hydroxychloroquine and lower mortality observed in this study can be acknowledged by clinicians in hospitals and in the community. randomized-controlled trials to assess the causal effects of hydroxychloroquine in different therapeutic regimes are required. in december , an outbreak of covid- , a novel disease caused by the virus sars-cov- , was declared in china. in the first months of , covid- spread throughout the world [ , ] . the number of new cases in some areas of the world is currently declining; however, it continues to rise worldwide with many countries having second outbreaks [ ] . it has been estimated that % of affected patients have mild symptoms, and in the rest, hospital care can be necessary, and mortality is less than % [ , , ] . the large number of simultaneous cases of covid- has overloaded hospitals in many countries, making it very difficult to provide an adequate care. many governments have used costly and disruptive policies to confine and distance the population to reduce the transmission of the disease and prevent a second wave [ ] [ ] [ ] . these measures will need to be maintained in some manner until vaccines or effective treatments become available to avoid the risk of later epidemics [ , ] . covid- is negatively affecting all areas of healthcare, and is also having an adverse impact on the entire society and the international economy. using limited evidence and clinical experience, doctors have treated covid- patients with different drugs to eliminate or reduce the presence of the virus, including hydroxychloroquine (hcq) [ ] [ ] [ ] [ ] . the use of this drug is based on its anti-inflammatory and antiviral effect [ , ] . there are some in vitro data supporting the ability of hcq to inhibit sars-cov- activity [ , ] . hcq may lead to the clinical improvement of the patients, it is safe, economical, and easy to use, and it can be given both to admitted and outpatients. if the treatment with hcq is confirmed to be effective, it could be used in the early stages of the disease, and decrease the need for admissions, the mortality, the transmission of the infection, and its impact on other areas of health care. however, the use of hcq is supported by limited evidence, and it may not lead to any clinical benefit. studies on the clinical outcomes associated with hcq are required. research policies are currently shifting towards the investigation of more expensive treatments [ ] [ ] [ ] [ ] . studies on hcq could inform evidence-based and affordable management for covid- , which may be particularly relevant when the world is facing an economic crisis, more so in areas where health care is based on limited resources. this study investigates the association between treatment with hcq and mortality, in hospitalized patients with covid- . the clinical records up to the th of april , of all the patients with covid- (n = ), admitted in all spanish hospitals, of the private healthcare provider hm hospitales, were reviewed. these hospitals are based in the provinces of barcelona, coruña, león, madrid, pontevedra, and toledo [ ] . patients had been diagnosed with polymerase chain reaction test of respiratory samples for sars-cov- , between the st of march and the th of april . seven patients had been admitted before the st of march . data on age and gender, together with temperature and saturation of oxygen at the time of admission, were collected. data on treatments at any time during admission with hcq were collected. once it was approved for clinical use in covid- patients, hcq was started immediately after admission, and it was stopped if any abnormalities were identified in the ecgs that were done on a daily basis to patients who were taking it. hcq was dosed as mg twice daily the first day, followed by mg twice daily for - days. data on treatments with azithromycin, steroids, heparin, tocilizumab, a combination of lopinavir with ritonavir, and oseltamivir, were collected, as well. the limited evidence on which these treatments were based, and the rapidly evolving clinical protocols, resulted in these drugs being given in many different specific preparations, doses, and frequency. no information on specific preparations of these drugs, dosage, duration of treatment, or route of administration, were collected. finally, data on death were recorded. the age of patients treated and not treated with hcq was compared with t tests. the proportion of men and women for those treated and not treated with hcq was compared with chi-squared tests. the association between treatment with hcq and mortality was examined with four different logistic regression models: model one was adjusted for age and gender; model two included age and gender, together with temperature > °c, and saturation of oxygen < % on admission, which were both associated with mortality in an exploratory analysis; model three had all the variables previously mentioned together with treatment with azithromycin, steroids, heparin, tocilizumab, a combination of lopinavir with ritonavir, and oseltamivir; finally, to account for the change in clinical management during the study period, model four was adjusted for all the previously mentioned demographic, clinical severity measures, and drugs, together with a categorical variable for date of admission (before the th of march, - th of march, - st march, st- th of april, and - th of april). all covariates included in the four models were considered potential confounders. no further variables were included to avoid the complex interpretation of results and collinearity. the software stata . was used for the analysis [ ] . missing data were treated as a separate category for temperature and oxygen saturation. sensitivity analyses were made to compare estimates based on using missing data as categories with, ( ) estimates based on complete data dropping variables with missing observations, and ( ) complete case analysis; dropping cases with missing data. among the patients whose records were reviewed, were men, were women, and the mean age was . . at the time of collecting the data, had died, patients had been discharged home from hospitals, were still admitted, and had been transferred to hospitals not included in the study. median (iqr) follow-up time was ( - ) days. data on treatment with hcq were available for patients. there was a younger age (p < . ), and a higher proportion of men (p = . ) for those who received hcq. among the patients who had been treated with hcq, had died. (table ) . hcq was associated with lower mortality when the model was adjusted for age and gender with or: . ( . - . ), p value < . . when the model was adjusted for age, gender, together with temperature > °c and saturation of oxygen < % on admission, and also when the model included all the previous variables plus treatment with all drugs, the association between use of hcq and lower mortality remained significant. the analysis of interaction suggested that hcq was associated with lower mortality, and there was no difference for those taking (n = ), or not (n = ), azithromycin, as well. in the final model where all the previous demographic, clinical variables, and drugs were introduced, together with time of admission, the association between treatment with hcq and lower mortality was also significant. (table ). treatment with hcq was associated with lower mortality in patients admitted with covid- . this study has strengths and limitations. patients were not randomized and the differences in mortality may be explained by factors other than the use of hcq. in an effort to control for the severity of disease, available markers were considered, and adjusted for, in all the models. the observation of a large number of unselected patients admitted in hospitals, and the analyses run with some variations to test the consistency of the results are also strengths of this study. however, residual confounding is always present in all observational research. the association, that was consistent across a set of models that adjusted for different potential confounders, provides support to an independent positive effect of hcq. for all models, the sensitivity analyses described in methods were used to assess the impact of missing data on the two markers of disease severity, and results were consistent with those presented. hcq has been used in all phases of covid- since two studies, with no control arm and small sample size, published in march , reported it to be associated with a reduction of the viral carriage and clinical improvement [ , , ] . this has allowed for its effects to be observed since then in a large number of different patients in many countries. our results, showing that hcq is associated with positive outcomes, are consistent with the ones first reported in march . a number of observational studies later conducted in china, france, spain (in a hospital not included in our project), and the usa, have also reported the association between hcq and lower mortality [ ] [ ] [ ] [ ] [ ] [ ] . three of these studies included over participants [ , , ] . in one of these studies, only patients having mechanical ventilation were included [ ] . in another one, hcq was also associated with lower probability of admission to intensive care, shorter hospital admissions, and shorter duration of viral shedding [ ] . in two of these studies, favourable results of hcq combined with azithromycin were reported [ , ] , and in another one, the lower mortality was observed for those on hcq in combination with azithromycin and zinc [ ] . the later study was based on primary care, and reported an association between treatment and lower rates of hospital admissions, as well. another observational study conducted in primary care, were mortality was not the outcome, showed significantly shorter time to clinical recovery for those treated with hcq or hcq plus azithromycin [ ] . in our study, the addition of azithromycin to hcq does not seem to add any clear benefit. a large multicentre observational study reported no association between hcq, or hcq with azithromycin, and lower mortality but significantly higher rates of discharge home were observed for patients treated by hcq [ ] . in a recent meta-analysis, studies were classified as big data, when electronic medical records had been used, or clinical studies, when details of treatments were reported and the study had been conducted by the same physicians who cared for the patients. it reported, among clinical studies, an association of chloroquine derivates with clinical improvement, lower mortality, and viral carriage [ ] . finally, a small rct reported the association of hcq with shorter time both to clinical recovery and to reach viral rna negativity [ ] . the absence of any positive effects of the treatment with hcq has also been reported. a number of observational studies have reported that hcq, either alone or in combination with azithromycin, was not associated with lower mortality [ ] [ ] [ ] [ ] . three of these studies included over participants [ , , ] . one of these studies included patients who needed oxygen [ ] and another one used mortality or need for intubation as an outcome [ ] two rcts have also reported no association between hcq and survival [ , ] . one of them also reported no benefit in need for admissions or clinical improvement [ ] . the lack of clinical improvement was reported in another trial [ ] . finally, two more rcts have reported no association between hcq and virological clearance [ ] or prevention of disease in individuals exposed to it [ ] . a number of factors could explain the difference between our results and the ones observed in these studies [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] including the following: the clinical-epidemiological design of our work; [ ] the involvement of all patients admitted with covid- , regardless their past medical history, the time between onset of symptoms and the start of treatment, the duration of admission, and the need for oxygen; the different statistical approach; and the observation in our work of patients from private hospitals, who are likely to have a high socioeconomic status [ ] . the safety of the hcq has been questioned, as it could negatively impact the immune response to the virus, or cause abnormalities in the ecg [ , , ] . however, none of the studies that we have reviewed, reporting no benefit on hcq, show an increased mortality associated with it [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . further rcts, observational studies, and summaries of both types of evidence to assess the associations between hcq and survival are necessary. future studies could also address at what dosage, and in what phase of the disease, does hcq lead to the best possible outcome, for patients with different past medical histories [ ] . the interventional evidence on the management of covid- is still limited. therefore, clinicians could acknowledge the results presented in this study. the positive effect of hcq seems consistent and its use could be considered in clinical settings. hcq is easy to administer, and its use in ambulatory patients, to reduce symptoms, prevent admissions, decrease mortality, and the transmission of the disease, could also be considered by clinicians and future researchers. the author(s) declare that they have no conflict of interest. the ethics committee of hm hospitales approved this study. data were anonymized before the authors could access it. no results that may make patients identifiable are reported. the study was conducted in accordance with the ethical standards as laid down in the declaration of helsinki and its later amendments. informed consent this study was conducted on already available clincal data and informed consent was not required. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. namin g-thecoron aviru s-disea se-(covid - )-and-the-virus -that-cause s-it who ( ) coronavirus disease (covid- ) pandemic covid- coronavirus pandemic estimating clinical severity of covid- from the transmission dynamics in wuhan covid- : learning from experience covid- : risk of second wave is very real, say researchers first-wave covid- transmissibility and severity in china outside hubei after control measures, and second-wave scenario planning: a modelling impact assessment the global impact of covid- and strategies for mitigation and suppression clinical and microbiological effect of a combination of hydroxychloroquine and 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critically ill patients with covid- outcomes of covid- patients treated with hydroxychloroquine/azithromycin and other regimens in marseille, france: a retrospective analysis treatment with hydroxychloroquine, azithromycin, and combination in patients hospitalized with covid- early hydroxychloroquine is associated with an increase of survival in covid- patients: an observational study covid- outpatients-early risk-stratified treatment with zinc plus low dose hydroxychloroquine and azithromycin: a retrospective case series study azithromycin and hydroxychloroquine accelerate recovery of outpatients with mild/ moderate covid- hydroxychloroquine with or without azithromycin and in-hospital mortality or discharge in patients hospitalized for covid- infection: a cohort study of in-patients in france clinical efficacy of chloroquine derivatives in covid- infection: comparative meta-analysis between the big data and the real world efficacy and safety of chloroquine or hydroxychloroquine in moderate type of covid- : a prospective open-label randomized controlled study hydroxychloroquine and tocilizumab therapy in covid- patientsan observational study clinical efficacy of hydroxychloroquine in patients with covid- pneumonia who require oxygen: observational comparative study using routine care data observational study of hydroxychloroquine in hospitalized patients with covid- association of treatment with hydroxychloroquine or azithromycin with in-hospital mortality in patients with covid- in new york state hydroxychloroquine in nonhospitalized adults with early covid- : a randomized trial hydroxychloroquine with or without azithromycin in mild-to-moderate covid- hydroxychloroquine in patients with mainly mild to moderate coronavirus disease : open label, randomised controlled trial ) a randomized trial of hydroxychloroquine as postexposure prophylaxis for covid- race, socioeconomic deprivation, and hospitalization for covid- in english participants of a national biobank could chloroquine /hydroxychloroquine be harmful in coronavirus disease (covid- ) treatment? qt interval prolongation and torsade de pointes in patients with covid- treated with hydroxychloroquine/azithromycin optimizing hydroxychloroquine dosing for patients with covid- : an integrative modeling approach for effective drug repurposing acknowledgements thank you to all clinicians and administrators of hospitales hm (spain) on which data this study is based.funding salma ayis was funded by the national institute for health research (nihr) biomedical research centre based at guy's and st thomas' nhs foundation trust and king's college london. the views expressed are those of the authors and not necessarily those of the nhs, the nihr, or the department of health. key: cord- -p foay k authors: ahmad, i.; alam, m.; saadi, r.; mahmud, s.; saadi, e. title: doxycycline and hydroxychloroquine as treatment for high-risk covid- patients: experience from case series of patients in long-term care facilities date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: p foay k importance: patients in long-term care facilities (ltcf) are at a high-risk of contracting covid- due to advanced age and multiple comorbidities. without effective treatments, outbreaks in such facilities will become commonplace and will result in severe morbidity and mortality. the effectiveness of doxycycline (doxy) and hydroxychloroquine (hcq) combination therapy in high risk covid- patients in long-term care facilities is not yet understood. objective: the goal of this analysis is to describe outcomes after use of doxy-hcq combination in high-risk covid- patients in ltcf. design: case-series analysis. setting: three ( ) ltcfs in new york. participants: from march to march , , fifty-four ( ) patients, residents of three ( ) ltcfs in new york and diagnosed (confirmed or presumed) with covid- , were included in this analysis. exposure: all patients who were diagnosed (confirmed or presumed) with covid- received doxy-hcq combination therapy along with standard of care. main outcomes and measures: patients characteristics, clinical recovery, radiological improvements, medication side-effects, hospital transfer, and death were assessed as outcome measures. results: a series of fifty-four ( ) high-risk patients, who developed a sudden onset of fever, cough, and shortness of breath (sob) and were diagnosed or presumed to have covid- , were started with a combination of doxy-hcq and % (n= ) patients showed clinical recovery defined as: resolution of fever and sob, or a return to baseline setting if patients are ventilator-dependent. a total of % (n= ) patients were transferred to acute care hospitals due to clinical deterioration and % (n= ) patients died in the facilities. naive indirect comparison suggests these data were significantly better outcomes than the data reported in mmwr (reported on march , ) from a long-term care facility in king county, washington where % patients were hospitalized, and % patients died. conclusion: the clinical experience of this case series indicates doxy-hcq treatment in high-risk covid- patients is associated with a reduction in clinical recovery, decreased transfer to hospital and decreased mortality were observed after treatment with doxy-hcq. the covid- pandemic is straining the u.s. healthcare system due to increased hospitalization, icu admissions, and mortality. as of may , , a total of , patients and , , patients were afflicted with covid- in new york state and the united states, respectively . in addition, , patients were hospitalized, and , patients died in new york alone . thus, new york has become the epicenter of the world for the covid- pandemic at the time of this publication . the covid- virus affects the upper and lower respiratory tract, leading to acute respiratory failure. consequently, pro-inflammatory cytokines are released such as tumor necrosis factor (tnf), interferonalpha (ifna), il- β, and il- , all of which are mediators of lung inflammation and fever . therefore, symptoms of a covid- infection include fever, cough, and shortness of breath (sob). in addition, the virus is associated with acute cardiac injury in . % of hospitalized patients, manifesting as an ejection fraction decline and an elevation in troponin i levels . recent small studies in france and china demonstrated the limited efficacy of hydroxychloroquine (hcq) alone or in combination with azithromycin (az) , , . however, concerns have been raised about the use of the az-hcq combination due to higher arrhythmogenic potential in high-risk patients with pre-existing cardiac comorbidities and/or acute cardiac injury due to covid- . according to the food and drug administration (fda) labeling, side-effects of az include "prolonged cardiac repolarization and qt interval, imparting a risk of developing cardiac arrhythmia and torsades de pointes" . additionally, qtc prolongation was found in a dose-dependent manner when chloroquine, an hcq analogue, was used alone or in combination with az . doxycycline (doxy) is considered to be an anti-inflammatory, immunomodulatory, and cardioprotective drug . both in-vitro and in-vivo studies demonstrated that doxycycline directly modulates the expression . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may , . . covid- pathophysiology (il- , il- β , tnf , mmp , and il- ). in addition, kalish et al showed a novel immunosuppressive mechanism for minocycline (a doxy analogue), as well as its possible additive anti-inflammatory effect when combined with chloroquine or hcq . no cardiac side-effects are noted per the fda labeling of doxy . in addition, during reperfusion after myocardial injury, an acute release of matrix metalloproteinases- (mmp- ) is noted , and mmp inhibition after myocardial infarction yields preservation of left ventricular function . doxy reduces the adverse lv remodeling in patients with acute st-segment elevation myocardial infarct (stemi) and lv dysfunction, possibly via mmp inhibition pathway . these potential cardioprotective effects may have an additional implication in the covid- patients with acute myocardial injury. due to these scientific findings, we have undertaken a thorough review of this case series of high-risk covid- patients in nursing homes, who were treated with a combination of doxy-hcq. from march to march , , we analyzed the clinical outcomes of fifty-four ( ) high-risk patients who developed a sudden onset of fever, cough, and sob, were diagnosed or presumed to have covid- , and were treated with doxy ( mg po bid for days) and hcq (two regimens: i) mg po tid for days or ii) mg po bid one day, then mg daily for days). the patients were residents of three ( ) long term care facilities (ltcf) in new york. patients' characteristics, clinical recovery, radiological improvements, medication side-effects, hospital transfer, and death were assessed as outcome measures in this population. consent was obtained from all patients and/or families before starting doxycycline and hydroxychloroquine to treat presumed and confirmed covid- infection and approved by the oversight medical boards. the retrospective review was approved by corporate clinical services. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . high-risk patients were defined as patients who had at least one comorbidity, such as hypertension, diabetes, coronary artery disease (cad), congestive heart failure (chf), chronic obstructive pulmonary disorder (copd), and cerebrovascular accident (cva). clinical recovery was defined as a resolution of fever and sob, or a return to baseline settings for ventilator-dependent patients. a return to baseline ventilator settings was defined as a return of oxygen saturation and mode of ventilator to a baseline level. chest x-rays were ordered at the onset of symptoms. follow-up chest r-rays were ordered if clinically indicated. in this series of fifty-four ( ) patients, the median age was yrs. (range - yrs.). table summarizes the characteristics and outcomes of all covid- patients treated with doxy-hcq combination in ltcfs in new york. % (n= ) tested positive for covid- and % (n= ) were presumed to have the viral infection due to clinical features. % (n= ) of patients showed clinical recovery, defined by resolution of fever and sob, or a return to baseline ventilator setting. % (n= ) of patients died and % (n= ) of patients were transferred to acute care hospitals due to clinical deterioration. all patients became and remained afebrile within - days and sob resolved or returned to baseline with - days of doxy-hcq initiation. % of patients (n= ) showed an improvement in chest x-rays. % of patients (n= ) did not show chest x-ray improvement. % (n= ) did not display any side-effects of doxy-hcq. % (n= ) had a seizure and hcq was immediately terminated. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint there were patients who did not complete the -day course of doxy-hcq due to hospital transfers, death, or side-effects (table ) . excluding these patients, a total of patients completed the doxy-hcq combination therapy; all patients clinically recovered (table ). the covid- pandemic created a huge burden on the u.s. healthcare system and increased the risk of patients. compared to this study, our case series patients were much older and had significant multiple comorbidities. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint so far, no study was published from the united states using hcq alone or in combination with az or doxy. as a matter of fact, this study is the first case series conducted globally using a combination of doxy-hcq in covid- patients in nursing home settings. we treated our moderate to severe patients in out-of-hospital settings and prevented hospital transfer in the majority of patients. naive indirect comparison showed a better outcome than the data reported in mmwr (reported on march , ) from a long-term care facility in king county, washington where % of patients were hospitalized and % patients died. this effect can possibly be explained by the anti-inflammatory mechanism of action of doxy-hcq. however, data from the ltcf from king county, washington was found to be similar to our population. these data also showed a reduction of hospitalization by % among ltcf residents compared to previously reported data by similar populations in mmwr. a limitation of our case-series analysis is that there was no control population available. doxy and hcq combination therapy is known to be anti-inflammatory, and immunomodulatory in both in-vitro and in-vivo studies. in addition, hcq has anti-viral properties. although this sample size is small (n= ), the results suggest that early intervention of doxy-hcq may improve the clinical outcome of high-risk covid- patients suffering from moderate-severe symptoms in ltcf. these data is also associated with a reduction of hospitalization by % among moderate to high severity covid- ltcf residents compared with previously reported data by similar populations from king county, given the massive pandemic created by covid- virus, there is an emergent therapeutic need to manage this disease with effective and safe drugs in an out-of-hospital setting for high-risk patients to . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint reduce hospitalization and icu admission and manage acute ventilator shortage. a well-designed clinical study is urgently needed to identify the appropriate patient population, optimize dosing regimen, and assess side-effects of doxy-hcq combination therapy. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . unknown**** % * chest x-ray either not ordered, or not required or pending ** recovery defined by resolution of fever and shortness of breath, or return to baseline vent setting *** % (n = ) patients recovered and vent setting is returned to baseline **** not available as patients were transferred to hospital ! dropouts means patients who did not complete a full course of doxy-hcq therapy due to transfer to hospital, death or side-effects. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . * this table includes all patients who completed a full course ( day) of doxy-hcq therapy and excludes any patients who were transferred to hospital, death or developed side-effects due to doxy-hcq and did not complete the -day course. ** recovery defined by resolution of fever, shortness of breath or return to baseline vent setting . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint *** chest x-ray either not ordered, or not required or pending . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint induction of pro-inflammatory cytokines (il- and il- ) and lung inflammation by coronavirus- (covi- or sars-cov- ): anti-inflammatory strategies association of cardiac injury with mortality in hospitalized patients with covid- in wuhan, china. jama cardiol hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial a pilot study of hydroxychloroquine in treatment of patients with common coronavirus disease- (covid- ) doxycycline reduces lipopolysaccharide-induced inflammatory mediator secretion in macrophage and ex vivo human whole blood models modulation of cytokine and cytokine receptor/antagonist by treatment with doxycycline and tetracycline in patients with dengue fever an alternative therapy for idiopathic pulmonary fibrosis by doxycycline through matrix metalloproteinase inhibition anti-inflammatory properties of low and high doxycycline doses: anin vitrostudy early short-term treatment with doxycycline modulates postinfarction left ventricular remodeling early short-term doxycycline therapy in patients with acute myocardial infarction and left ventricular dysfunction to prevent the ominous progression to adverse remodeling: the tiptop trial clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in covid- patients with at least a six-day follow up: an observational study covid- in a long-term care facility -king county minocycline promotes the generation of dendritic cells with regulatory properties minocycline inhibits antigen processing for presentation to human t cells: additive inhibition with chloroquine at therapeutic concentrations key: cord- -hpgrbhkl authors: cairoli, ernesto; espinosa, gerard title: hydroxychloroquine in the treatment of covid- : how to use it waiting for conclusive scientific evidence() date: - - journal: med clin (engl ed) doi: . /j.medcle. . . sha: doc_id: cord_uid: hpgrbhkl nan letter to the editor hidroxicloroquina en el tratamiento del covid- : como utilizarla a la espera de evidencia científica concluyente. hydroxychloroquine in the treatment of covid- : how to use it waiting for conclusive scientific evidence autores: ernesto cairoli md, phd a, b , gerard espinosa md, phd c . to date, there is no effective treatment against the novel severe acute respiratory syndrome coronavirus (sars-cov- ), responsible for the covid- disease. numerous clinical studies are evaluating the utility of antiviral and immunomodulatory drugs, where antimalarials such as chloroquine and hydroxychloroquine (hcq) are one of the alternatives studied . so far, clinical experience in the use of hcq arises mainly from treatment in patients with systemic lupus erythematosus (sle), whose long-term effects show multiple benefits. however, high cumulative doses have been associated with serious adverse effects, especially in the retina and myocardium . many healthcare protocols propose the use of hcq in the treatment of covid- . however, it is important to consider adverse myocardial effects, such as the development of severe arrhythmias , . in the covid- patient, possible cardiac involvement is mainly related to factors: ) underlying heart disease (often silent in older patients); ) myocardial involvement caused by the infection and the inflammatory response itself, which leads to myocarditis with elevated troponins; ) acute toxicity probably associated with the use of antimalarials in high doses, more evident in chloroquine treatments and ) concomitant use of other treatments that, together with hcq, prolong the corrected qt interval (qtc), with the risk of serious ventricular arrhythmias - . in the absence (pending) of conclusive scientific evidence, what considerations should be taken into account when using hcq in the treatment of covid- ? it is necessary to change the way in which hcq is usually used in patients with sle, adapting its prescription and control of potential adverse effects to this new therapeutic scenario. the following considerations aim to optimize the hcq treatment of covid- : -when the doctor considers that hcq can be useful, it should be initiated as early as possible after diagnosing the infection, due to the decrease in viral replication and dissemination demonstrated in vitro and in vivo . -using hcq in an acute treatment ( days), with loading dose ( ░mg/every ░h) the first day and days of maintenance ( ░mg/every ░h), after requesting an informed medical consent (with covid- being an indication not contemplated in the smpc). -minimize the risk of prolonged qtc. for this, a baseline electrocardiogram (ecg) must be performed prior to the start of treatment. if the qtc is greater than or equal to ░ms, hcq should not be started. if the qtc is less than ░ms in men or less than ░ms in women, treatment can be initiated, repeating the ecg in ░h. if the qtc is greater than or equal to ░ms or an increase greater than or equal to ░ms is observed, treatment should be discontinued . -avoid or discontinue the simultaneous use of drugs that prolong the qtc, particularly azithromycin, clarithromycin, levofloxacin, moxifloxacin, ciprofloxacin, haloperidol, quetiapine, risperidone, domperidone and ondansetron, among others , . -keep a close watch on potassium, calcium and magnesium levels due to their arrhythmogenic potential, as well as glycemia in patients with diabetes due to the risk of hypoglycemia . -consider not administering or discontinuing hcq in advanced stages of infection due to the possibility of a covid- -induced myocarditis , . -it is not necessary to adjust the dose based on renal or hepatic function, nor does it require ophthalmological control before or after treatment . -pending the result of several active studies, hcq should not be indicated prophylactically as there is no evidence to support its preventive use or postexposure to avoid covid- infection. in short, when prescribing hcq in covid- , different precautions should be taken from those currently considered for sle patients. it should be indicated in its window of opportunity and consider the existing multifactorial myocardial involvement, seeking to avoid cardiovascular adverse effects. the results of different controlled and j o u r n a l p r e -p r o o f randomized studies that confirm or refute the usefulness of hcq in the treatment of covid- will contribute to define its role in this clinical setting. the authors declare that they have not received funding to carry out this study. uso de antipalúdicos en el tratamiento del covid- : una ventana de oportunidad? antimalarialinduced cardiomyopathy: a systematic review of the literature cardiovascular complications in patients with covid- : consequences of viral toxicities and host immune response guidance of minimizing risk of druginduced ventricular arrhytmia during treatment of covid- : a statement from the canadian heart rhythm society description and proposed management of the acute covid- cardiovascular syndrome covid- and chloroquine/hydroxychloroquine: is there ophthalmological concern? key: cord- -mnj zmkn authors: hussain, nowair; chung, emily; heyl, jonathan j; hussain, bisma; oh, michael c; pinon, candis; boral, soumya; chun, david; babu, benson title: a meta-analysis on the effects of hydroxychloroquine on covid- date: - - journal: cureus doi: . /cureus. sha: doc_id: cord_uid: mnj zmkn introduction since december , severe acute respiratory syndrome coronavirus (sars-cov- ) has rapidly spread throughout the world with a large medical and economic impact. on march , , the world health organization (who) classified sars-cov- as a pandemic. as a result of this worldwide public health crisis, politicians, elected officials, and healthcare professionals emergently began trialing hydroxychloroquine (hcq) in efforts to treat and prevent the transmission of the virus. this meta-analysis was performed to assess the effects of hcq on patients with covid- . methods this meta-analysis adheres to the preferred reporting items for systematic reviews and meta-analyses (prima) guidelines. selected articles published between december and july were found utilizing the following search engines: pubmed, google scholar, cochrane library, disasterlit, clinicaltrials.gov, medrxiv, and embase. two independent physician reviewers screened eligible articles that met the inclusion and exclusion criteria of the analysis. the outcome measures analyzed were mortality rate, rate of disease progression/improvement, rate of disease severity, and adverse effects of treatment. six out of studies that met the study’s eligibility criteria were selected and further analyzed, with a total of participants (n= ). conclusion from the studies analyzed, it was found that groups treated with hcq had an overall mortality rate that was . times greater than that of the control group. hcq treated patients had higher rates of adverse clinical outcomes and side effects compared with the control populations. lastly, there was a . times higher rate of improvement in the group of hcq treated patients with mild to moderate symptoms as compared to the control group. coronavirus disease (covid- ) originated in wuhan, hubei province of china on december , [ ] [ ] [ ] [ ] . the rapid spread of the virus led the world health organization (who) to announce covid- as a pandemic on march , [ ] . the spread of sars-cov- resulted in an enormous public health crisis with high patient mortality and significant economic consequences [ , ] . furthermore, covid- is a complex, multifaceted, multi-system disease process that spares no one [ ] . the covid- acute respiratory distress syndrome consists of a period of cytokine storm, which is noted particularly in the later stages of advanced severe respiratory failure [ ] . covid- patients have increased levels of plasma pro-inflammatory cytokines and chemokines [ ] [ ] . these cytokines and chemokines are il b, il ra, il , il , il , il , basic fgf , gcsf, gmcsf, ifng, ip , mcp , mip a, mip b, pdgfb, tnfa, and vegfa [ ] [ ] . high patient mortality is caused by the disarray of these host cytokines, causing damage to the lungs and leading to multi-system organ failure [ ] [ ] . the sars-cov- virus has an affinity for the ciliated cells of the respiratory conducting airway, with increased viral replication as it progresses further along the respiratory tract and gastrointestinal mucosa [ ] . the sars cov- infection occurs in three distinct stages: an asymptomatic stage, an upper airway stage, and, finally, the conducting airway response stage, which leads to the classically seen ground-glass infiltrates on chest x-ray and clinical hypoxia with progression to acute respiratory distress syndrome (ards) and multi-system dysfunction [ ] . in stage , the virus binds to the angiotensin-converting enzyme (ace ) receptor, a transmembrane protease, serine (tmprss ). tmprss is ubiquitous in the human body; it is found in the nasal cavity and lung and is also expressed throughout the intestine and prostate [ ] . ace receptors can also be found in the heart, esophagus, kidneys, stomach, bladder, and ileum [ , ] . as sars-cov- progresses down the respiratory tract, the virus begins to activate a more potent immune response and certain patients may manifest clinically with respiratory failure and ards. most patients will have a mild disease, with the disease restricted to the upper respiratory tract [ ] . about one out of five sar-cov- infected patients will progress to more severe respiratory disease and further to ards [ ] . the proposed mechanism is the destruction of type ii pneumocytes once the virus reaches the alveoli [ ] . the virus would then begin the replication process within these cells and the cell would undergo apoptosis, releasing viral particles. this cellular apoptosis results in diffuse alveolar damage with the formation of hyaline membranes, which decrease gas exchange and lead to clinical hypoxia. furthermore, the healing of the affected areas may worsen the patient's condition through more severe parenchymal scarring and fibrosis. because the cytokines mentioned above have binding sites within the lungs, they may serve as therapeutic targets. -aminoquinolones such as hydroxychloroquine (hcq) and chloroquine have gained a lot of steam in the medical field and media for their possible efficacy against covid- . hcq has immunomodulatory properties and was originally developed as an antimalarial drug with further applications in patients with rheumatoid arthritis and systemic lupus erythematosus [ ] . in vitro studies of hcq have additionally shown antiviral properties; it supposedly prevents covid- related ards [ , [ ] [ ] . the treatment of covid- positive patients with hcq has been met with controversy, as there have been no large multicenter randomized control trials to support its use. up to this point, there is a lack of statistically significant reduction in morbidity or mortality in covid- patients who have undergone hcq trials. the treatment of covid- with a combination of hydroxychloroquine and azithromycin was first proposed in a controversial, small non-randomized trial from the south of france that concluded that the drug combination was effective for the treatment of covid- [ ] . criticism was brought on immediately when it was presented for peer review due to many methodological flaws, with the biggest being the lack of a randomized control group [ ] . this led to various expert researchers criticizing the efficacy of hydroxychloroquine, with the majority concluding no statistically significant difference between treatment groups. the emergent approval of hcq at the height of the covid- pandemic was considered controversial but necessary given the overwhelming lack of effective treatment options at that time. the controversy was limited not only to the unknown efficacy and side-effect profile of hcq but also to the limited supply of the drug [ ] . this systematic review and meta-analysis adheres to the preferred reporting items for systematic reviews and meta-analyses (prisma) guidelines ( figure ) [ ] . the search terms used were hydroxychloroquine, chloroquine, azithromycin, covid- , coronavirus, and sars-cov- . using these terms, the systematic search strategies used were boolean and fuzzy logic, truncated terms, and wild cards. selected articles published between december and july were found utilizing the following search engines: pubmed, google scholar, cochrane library, disasterlit, clinicaltrials.gov, medrxiv, and embase. two independent physician reviewers screened eligible articles that met the analysis' inclusion and exclusion criteria. the inclusion criteria were ( ) age range - , ( ) prospective control trial, and ( ) use of hydroxychloroquine, chloroquine, lopinavir-ritonavir, or azithromycin. the exclusion criteria were ( ) presence of a co-morbid medical condition, i.e., advanced heart, liver, or renal disease or diabetes mellitus, ( ) treatment with remdesivir, convalescent plasma, corticosteroids, vaccines, il- inhibitors, t-cell therapy, α-ketoamide inhibitors, resiniferatoxin, teicoplanin, favipiravir, extracorporeal therapy, or hcq prophylaxis. the outcome measures analyzed were hcq's effect on covid- mortality rate, rate of disease progression/rate of improvement, rate of disease severity, and adverse effects of treatment. six out of studies that met the study's eligibility criteria were selected and further analyzed, with a total of participants (n= ). data collected from six different studies looked at the effects of hcq on patients with clinically proven covid- infection. these six studies have been labeled s , s , s , s , s , and s for better data visualization. each study varies in sample sizes and the distribution of treatment and control groups. the defined outcomes studied in this meta-analysis are: ) the mortality rate of patients after applying hcq on patients with covid- ) the rate of progression/improvement of covid- disease ) the rate of covid- disease severity, for example, after applying the hcq treatment, the rate of which patients went on to develop severe conditions such as acute hypoxic respiratory failure and adult respiratory distress syndrome. the random-effects model was used on the assumption that the study effect estimates show more variance when drawn from a single population [ ] . therefore, this follows the so-called assumption of exchangeability [ ] . this means that in a random-effects model fit, not only do assumptions of the effects of individual studies deviate from the true intervention effect of all studies due to sampling error but that there is another source of variance introduced by the fact that the studies do not stem from one single population [ ] . the studies are sampled from a "universe" of populations [ ] . in this study, the random effect model is a suitable choice because it is a risky assumption to state all the studies along with their respective effect sizes stem from a single homogeneous population. among the six studies considered for meta-analysis, information on mortality rates was available in two of them, details of which are provided below (tables - ) . these studies are perfectly homogeneous as tau^ is (equivalently, h^ is ). see figures - for more information. study has more uncertainty in its results as evident due to the wide spread of the horizontal line. studies and both do not cross the effect line at , indicating that they are not in agreement with the mortality rate of hcq treated covid- positive patients. there is a marginal asymmetry between the studies, however, as the number of studies is small, this result can be attributed purely to chance rather than any actual publication bias. among the six studies considered for meta-analysis, information on disease progression rates are available in four of them, details of which are provided below (tables - ). these studies are a bit heterogeneous, though by a very small amount. see figures - for more information. interpretation study has more uncertainty in its results as evident by the width of the horizontal line [ ] . all studies, except study , are in agreement with the results of a disease progression rate of hcq treatment in patients with covid [ ] . the studies are symmetric except study , which falls outside the triangle. this is in line with the conclusion drawn from the funnel plot. however, as evidenced by the funnel plot, study has very low power and thus its effect can be ignored. among the six studies considered, information on disease severity rates are available in four, the details of which are provided below (tables - ). here, the estimated average log relative risk is equal to ˆμ=- . ( % ci: - . to . ) [ ] . for easier interpretation, these values are transformed back to the relative risk scale through exponentiation (i.e., exp(ˆμ) = . with % ci: . to . ). the interpretation of these results suggests that the disease severity rate in hcq treated individuals is on average . that of the non-hcq individuals. the null hypothesis h : μ= can be rejected (p < . ). these studies exhibit heterogeneity by a moderate amount. see figures - for more the side effects of -aminoquinolones are known to be dose-dependent increased risks for retinopathy, methemoglobinemia, and gastrointestinal (gi), renal, and cardiac toxicity [ ] . hcq co-administered with medications such as azt further increases the risk of toxicity, particularly prolongation of the qt interval on electrocardiogram. the borba et al. study revealed that males aged with severe covid symptoms and heart disease are at high risk for developing hcq-related cardiac complications such as qt prolongation at higher doses of hcq [ ] . this toxicity is especially noted when combined with azt, which is known to prolong the qt interval in populations with cardiac disease [ ] . the studies by tang et al. [ ] and chen j et al. [ ] showed greater hcq-related gi side effects as well. in a post-marketing study by the food and drug administration (fda), it was also shown that the use of -aminoquinolones increased rates of cardiac arrhythmias, ventricular tachycardia, fibrillation, and torsades de pointes. their analysis also noted adverse cardiac events in combination with the use of other qt-prolonging medications such as azithromycin [ ] . as a result, the fda has cautioned the use of hcq in covid- patients, especially outside of the inpatient hospital setting [ ] . similarly, this meta-analysis supports that hcq treated patients are more likely to have adverse side effects. it also appears that treatment with hcq has a fatality rate of approximately . higher than with the control group. the non-randomized study performed by gautret et al. in the south of france included a total of young patients with positive pcr test results and milder covid- disease with no advanced comorbid medical conditions. a % reduction in viral load was noted at one week with a low dose of hcq with azt [ ] . this study was not powered to detect mortality outcomes. similarly, yang et al. [ ] , mingxing et al. [ ] , and chen j et al. [ ] studied females with a median age of and mild covid- related upper respiratory/pneumonia symptoms, without co-existing comorbid medical disease. patients were stated to have improved time to clinical resolution in the hcq treatment arm [ , [ ] [ ] . these results seem to be in line with the meta-analysis' of a slight disease improvement in covid- patients treated with hcq as compared with the controls. furthermore, recent studies show a gender disparity, in that females show better outcomes as compared to similar male cohorts [ ] . this gender disparity is seen in a recent study that noted that male patients with advanced age or multiple comorbid medical conditions are at higher risk for mortality [ , ] . the studies in this meta-analysis did not include these high-risk patients with underlying complex co-morbid medical conditions, severe cases of covid- , ards, or critical care patient populations. of note, the studies included in this meta-analysis have various definitions of control groups, which might affect the conclusion. however, with respect to the disease progression and severity meta-analysis, it appears that most of the studies are in agreement with the results, with slight exceptions, which might be attributed to chance. to get a more robust conclusion, the metaanalysis can be performed on more studies rather than six prospective control trials. currently, there are hcq clinical trials in the active recruitment phase [ ]; as the pandemic continues to unfold, these future large multicenter randomized controlled clinical trials may be included in the meta-analysis to conclude the size effect of hcq on covid- . covid- ) pandemic and economic impact preferred reporting items for systematic reviews and meta-analyses: the prisma statement early transmission dynamics in wuhan, china, of novel coronavirusinfected pneumonia who announces covid- outbreak a pandemic coronavirus timeline: tracking the critical moments of covid- coronavirus disease case surveillance -united states in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) the epidemiology and pathogenesis of coronavirus disease (covid- ) outbreak covid- : pathogenesis, cytokine storm and therapeutic potential of interferons extrapulmonary manifestations of covid- virology, epidemiology, pathogenesis, and control of covid- pathogenesis of covid- from a cell biology perspective sex differences in sars-cov- infection rates and the potential link to prostate cancer mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology in vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial current and resolved drug shortages and discontinuations reported to fda statistical methods for immunogenicity assessment a pilot study of hydroxychloroquine in treatment of patients with common coronavirus disease- (covid- ) effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus (sars-cov- ) infection. a randomized clinical trial efficacy of hydroxychloroquine in patients with covid- : results of a randomized clinical trial treating covid- with chloroquine hydroxychloroquine in patients mainly with mild to moderate covid- : an open-label, randomized, controlled trial hydroxychloroquine: small effects in mild disease center for drug evaluation and research office of surveillance and epidemiology. pharmacovigilance memorandum search of: hydroxychloroquine. recruiting studies: covid -list results our study looks at three disease outcome measures of treatment with hcq in patients with covid- : mortality rates, progression rates, and severity rates. in terms of mortality rates, it appears treatment with hcq has a fatality rate that is . times greater than that of the control group. similarly, hcq treated patients are more likely to have an adverse clinical outcome and side effects. lastly, there was a . -times higher rate of clinical improvement in the group of hcq treated patients, with mild to moderate symptoms as compared to the control group. human subjects: all authors have confirmed that this study did not involve human participants or tissue. animal subjects: all authors have confirmed that this study did not involve animal subjects or tissue. conflicts of interest: in compliance with the icmje uniform disclosure form, all authors declare the following: payment/services info: all authors have declared that no financial support was received from any organization for the submitted work. financial relationships: all authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. other relationships: all authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work. key: cord- -oxxle authors: samuel, sharmeen; friedman, richard a.; sharma, chetan; ganigara, madhusudan; mitchell, elizabeth; schleien, charles; blaufox, andrew d. title: incidence of arrhythmias and electrocardiographic abnormalities in symptomatic pediatric patients with pcr positive sars-cov- infection including drug induced changes in the corrected qt interval (qtc). date: - - journal: heart rhythm doi: . /j.hrthm. . . sha: doc_id: cord_uid: oxxle background: there is limited data regarding the electrophysiological abnormalities and arrhythmias in children with covid- , including those associated with treatment using potentially pro-arrhythmic hydroxychloroquine (hcq) and azithromycin (azn). objectives: to describe the electrophysiologic findings and arrhythmias associated with pediatric covid- and its treatment. methods: a single center retrospective chart review was undertaken and included all patients with ) symptoms of covid- , and ) pcr (+) nasopharyngeal swabs for sars-cov- who were placed on continuous telemetry for the duration of their hospitalization during march through may, . results: thirty-six patients were included in the study. significant arrhythmias were found in (non-sustained (ns) ventricular tachycardia in and sustained atrial tachycardia in ). all were self-resolving and half prompted prophylactic anti-arrhythmic therapy. patients with significant arrhythmias were likely to have non-cardiac co-morbidities ( / ), but these were not more common than in patients without arrhythmias ( / , p= ). the use of hcq with or without azn was associated with statistically significant qtc prolongation ( + msec vs + msec, p< . ). qtc was not statistically different in patients with and without arrhythmias ( + msec vs + msec, p= ). conclusions: in pediatric patients with pcr positive active covid- infection, significant arrhythmias are infrequent, but more common than expected in a general pediatric population. comorbidities are not more common in patients with arrhythmias than in patients without arrhythmias. covid- treatment using hcq is associated with qtc prolongation, but was not associated with arrhythmias in pediatric patients. the coronavirus disease caused by the severe acute respiratory syndrome coronavirus- (sars-cov- ) has been declared a global pandemic by the world health organization (who) and has affected millions of people worldwide . while typically the virus known to affect the respiratory system, there is adult literature of cardiovascular involvement, including electrophysiologic abnormalities and anecdotal reports of sudden cardiac death [ ] [ ] [ ] [ ] . currently, literature regarding electrophysiological abnormalities in children with covid- infection is lacking. additionally, some of the medications that have been used for treatment of covid- infection, such as hydroxychloroquine (hcq) and azithromycin (azn), are known to cause corrected qt (qtc) interval prolongation, therefore potentially predisposing patients to malignant ventricular arrhythmia.s , however, there is little current data on the electrophysiologic consequences of these drugs in the setting of active covid- in pediatric patients. this is a single centered retrospective observational study conducted at cohen children's medical center (ccmc), located in the covid- epicenter in new york city. the study was conducted in accordance with the declaration of helsinki and was approved by the northwell institutional review board (irb) through its covid- research consortium. all pediatric patients between the ages of - years who were sufficiently medically ill to require hospital admission with confirmed covid- infection by positive result on polymerase chain reaction testing of nasopharyngeal specimens between march , and april , were included in the study if they had been placed on continuous telemetry for the duration of their hospitalization. patients with ) igg antibodies for sars-cov- , ) any known history of known arrhythmias, long qt syndrome or other channelopathies, or ) hemodynamically significant congenital heart disease, were excluded from analysis. some patients are included in other ccmc covid reports, but the analyses presented here are original. as per hospital protocol, covid- specific medications including hcq with or without azn were initiated at the discretion of the infectious disease team for patients needing supplemental oxygen for hypoxia in the setting of positive sars-cov- , if the baseline qtc was less than milliseconds (msec) measured on lead ii via lead ecg or telemetry. all patients were treated with days of hcq ± azn, with the standard dosing of hcq being mg/kg/dose (maximum dose mg/dose) on day and . mg/kg/dose (maximum dose mg/dose) on the subsequent days, and azn dose being mg/kg/dose (maximum dose mg) on day and mg/kg/dose (maximum dose mg) on subsequent days. other covid- specific medications such as ivig, steroids, remdesivir, anakinra and tocilizumab were reserved for patients with more severe manifestations and multiple comorbidities. arrhythmia treatment was at the discretion of the attending electrophysiologist (ab). as part of the treatment protocol, patients underwent baseline -or -lead electrocardiograms (ecg), when possible, prior to initiation of treatment and were placed on continuous telemetry rhythm monitoring while on treatment. to reduce contact between health care personnel and covid- patients, ecgs were performed only when there was an absolute clinical indication. ecg interval, axis, and voltage criteria were evaluated via davignon et al. otherwise, all rhythm monitoring and qtc calculations were performed using lead ii obtained via telemetry. rhythm tracings were collected daily and independently interpreted by both a pediatric cardiology fellow (ss) and a pediatric electrophysiologist (ab) in a retrospective fashion for the purposes of research, each blinded to other variables. when there was initial lack of agreement in qtc measurement, tracings were reviewed together and consensus was achieved. all qtc measurements were made from telemetry recordings. baseline measurements were made shortly prior to therapy with hdq or azn. subsequent manual measurements were made randomly once per day in the morning, with few exceptions. these daily measurements were recorded and the longest of them is referred to as the longest daily measured qtc. the qtc interval was measured using the bazett's formula (qtc = qt /√ rr). in patients with conduction abnormalities the qtc was calculated using an adjusted qt (adjusted qt: aqt=qt-qrs in excess of ms). significant arrhythmias included type ii second degree av block, complete heart block, non-sustained (ns) or sustained supraventricular/atrial and ventricular tachycardias. sustained arrhythmias are defined as those lasting more than seconds and/or causing hemodynamic collapse. single ectopic complexes were recorded but not considered significant arrhythmias. rates, duration, and factors associated with arrhythmias were also recorded. qtc values of > and > msec were considered abnormal intervals in males and females, respectively as per aha/accf/hrs recommendations for the standardization and interpretation of the electrocardiogram . in addition to descriptive analyses, several sub-analyses were performed including: ) comparisons of qtc prolongation were made between patients who received hcq with or without azn, hcq alone, hcq with azn, and those who received neither drug. and ) comparisons were made between patients who developed significant arrhythmias and those who did not, information including demographics, pre-existing conditions, laboratory data including electrocardiographic and echocardiographic data, cardiac enzymes, respiratory support, need for vasopressors and covid- specific medications used were collected and entered in project's secure descriptive data is presented as average +/-standard deviation. continuous parametric analyses were performed using the student's t-test. unpaired tests were used in comparing data from different patients while paired tests were used when comparing data points from the same patient at different times. the fisher exact test was used for comparing categorical variables between small groups while the chi square test was used for categorical comparisons of larger groups. clinical correlations were performed using pearson's correlation. thirty-six patients met criteria for inclusion in the study. two patients with large vsd and significant shunts were excluded. no patients were excluded for a channelopathy diagnosis. the demographics of all included patients are presented in table . the majority of patients were adolescents and there were more boys than girls. minority children were disproportionately represented. interestingly, / of patients had a significant co-morbidity with over a quarter having a hematological or oncologic diagnosis. patients were sick for an average of + days prior to hospitalization and manifested the following symptoms: fever ( %), cough ( %), shortness of breath ( %), chest pain ( %), myalgia ( %), head ache ( %) and gastro-intestinal symptoms ( %). elevated troponin t was noted in / ( %) with an average maximum value of + ng/l, with one outlier patient having a maximum level of ng/l. c-reactive protein (crp) was elevated in / ( %) patients with an average of + mg/l. pro-brain natriuretic peptide (pro-bnp) was elevated / ( . %) with an average of + pg/ml. echocardiograms were performed in patients, of whom had ventricular dysfunction (left ventricular ejection fraction < %) while the average lvef was + %. hcq alone was administered to ( %) patients, a combination of hcq+azn to ( %) while ( %) patients received neither medication. other covid- medications used included ivig ( ), steroids ( ), remdesivir ( ), tocilizumab ( ), and anakinra ( ). patients were admitted for an average of + days with ( %) patients requiring picu admission, where ( . %) patients required vasoactive support during their hospital admission. one patient with walker-warburg syndrome and multiple brain anomalies died from complications related to severe respiratory insufficiency. twenty-eight patients had at least one -lead ecg and all patients were on continuous telemetry with reviewable strips. ten patients had significant findings on the -lead ecg including low voltage qrs complexes ( %), left ventricular hypertrophy ( %), right ventricular hypertrophy ( %), left axis deviation ( %), right axis deviation ( %), significant st segment changes ( %). no patients had second degree or complete heart block. three patients had infrequent isolated premature atrial ectopic beats (pac), patient had frequent isolated pacs, patient had infrequent isolated late cycle unifocal premature ventricular ectopic beats (pvc), and patient had frequent isolated late cycle unifocal pvcs. six patients ( %) had significant arrhythmias (tables and ). while all arrhythmias were self-limited and hemodynamically tolerated, prophylactic anti-arrhythmic therapy was started in . two patients were started on antiarrhythmics because of recurrence while another was started on labetalol for coexisting hypertension. there was no mortality among patients with significant arrhythmias. while there was no association between significant arrhythmias and the presence of comorbid noncardiac medical conditions (p= ), non-cardiac co-morbidities in children with arrhythmias was common ( / ) with patients having co-morbidities and/or associated circumstances known to place the patients at independent risk for arrhythmias including: mediastinal mass and pericardial effusion (n= ) and electrolyte disturbances (n= ). two others were diagnosed with myocarditis secondary to covid- . the maximum troponin level was higher in patients with significant arrhythmias compared to those without ( ± ng/l vs ± ng/l, p= . ). however, there was no difference in the proportion of patients with significant arrhythmias who had abnormally elevated troponins compared to the proportion of those without arrhythmias having abnormally elevated troponins ( / vs / , p = . ). this discrepancy can be explained by a single outlier in the arrhythmia group with very high troponin levels. other relevant variables such as demographics (age, weight, gender, and race/ethnicity), prehospitalization symptoms and duration of symptoms, laboratory variables (pro-bnp; maximum crp, and electrolytes), echocardiographic findings (%ef and presence of lv dysfunction), and significant ecg findings (longest daily measured qtc and baseline ecg abnormalities) were not significantly associated with arrhythmias. there was a trend for arrhythmias to be more common in patients with baseline ecg abnormalities compared to those without these abnormalities ( / vs / , p = . ). covid- treatment including the use of hcq with or without azn was not associated with significant arrhythmias (p= . ).eighty-six percent interobserver agreement was achieved for qtc measurement. in discrepant cases, tracings were reviewed by both interpreters together and consensus was achieved in all remaining tracings. among the entire study cohort, the average baseline qtc was ± msec and the longest daily measured qtc during the hospital course was ± msec. no patients had an abnormal baseline or longest daily measured qtc. adolescents aged - years had a statistically significantly longer (but still clinically normal) baseline qtc compared to younger children ( + msec vs + msec, p = . ). baseline qtc was not different in males vs females. both baseline and longest daily measured qtc was not correlated with ef and was not significantly different in those with lv dysfunction compared to those with normal lv function on echocardiographic evaluation. there was an unexpected trend for the qtc to be shorter in patients with abnormal troponin levels vs those with normal troponin levels ( + msec vs + msec, p = . ) baseline qtc was not statistically different for patients who received hcn alone ( + msec) vs those who also received azn ( + msec), and vs those who received neither drug ( + msec). longest daily measured qtc after starting therapy was not different in patients who received hcn with or without azn compared to those who received neither drug ( + msec vs + msec, p = . ). however, on comparing baseline to longest daily measured qtc intervals after starting hcq +/-azn showed that the qtc statistically significantly prolonged (but still clinically normal) for those patients treated with hcq alone ( + msec to + msec, p = . ), but not for those treated with both drugs ( + msec to + msec, p = . ). this is illustrated in figure . importantly, the longest daily measured qtc after starting treatment with hcq with or without azn occurred after day of treatment in , after days in , after days in , after days in , and after days in . however, of these, the baseline qtc was longer than the longest daily measured qtc after starting treatment in . in patients with post treatment rhythm strips available, the post treatment qtc was shorter than the baseline qtc in , the same in , and longer in . to our knowledge, this is the largest study reporting the electrophysiologic abnormalities in children with covid- . there are several main findings of this paper including the following: ) significant arrhythmias in pediatric patients with covid- infection are infrequent but occur at incidence higher than expected in a general pediatric population, ) comorbidities are not more common in covid- patients with arrhythmias. and ) hcq, is associated with qtc prolongation is not associated with arrhythmias despite previous publications. , arrythmias arrhythmias in the present study occurred more frequently than expected in a general pediatric population. in the current study, % of patients had significant arrhythmias of whom ( % of the study population) had ns vt. in a population of children with structurally normal hearts, less than % would be expected to have multiform pvcs, couplets, or ns vt on ambulatory monitoring. while one could argue that the patients in the present study were monitored for a longer period ( days) than the average child is monitored in the ambulatory setting, the incidence of ns vt in the present study still seems high. although no episode required acute intervention for hemodynamic embarrassment, / patients with ns vt received prophylactic therapy and thus may have been prevented from experiencing subsequent episodes with different durations and/or hemodynamic implications. as more insights are acquired about the novel sars-cov- virus and the multi-system involvement that occurs with it, the effects of the virus on the cardiovascular system are being revealed. currently data among adult patients on the incidence of new arrhythmias in patients with covid- infection are limited. patients with covid- infection may develop arrhythmia due to acute cardiac injury related to direct myocardial injury from myocarditis, hypoxia-mediated injury, worsening of preexisting coronary artery disease, as a result of systemic inflammatory response syndrome or due to the effects of medications used in the management of covid- . in a study from wuhan by wang et al., of adult patients who were hospitalized with covid- related pneumonia, arrhythmias were reported in . % of the general cohort and in % patients admitted to the intensive care unit. of the patients, hypertension, cardiovascular disease and diabetes were present in , and % of patients respectively. as often found in other clinical circumstances, results obtained from adult studies cannot be directly extrapolated to the pediatric patient population. in our present study, no patient had preexisting cardiovascular morbidity including ischemic heart disease or hypertension. while troponin levels were higher in pediatric patients with arrhythmias, there was no increased risk of significant arrhythmias in patients with elevated troponin, suggesting that the association is related to the small number of patients in the study and one outlier patient with a severely elevated troponin. this lack of association between abnormal troponin levels and arrhythmias is also different from what has been reported in adults and may be related to the pathophysiologic differences in the etiologies of troponin elevation in adults compared to children. while the pediatric population presented here did not have preexisting cardiovascular morbidity, a high proportion of these patients did have other co-morbidities. whether there was a selection bias in that pediatric patients with co-morbidities were more likely to be admitted or whether these patients were actually sicker and required hospitalization rather than home management is not clear from our data. although there was no statistically significant increased risk of arrhythmias in patients with co-morbid conditions, / patients with significant arrhythmias had co-morbidities that may have independently placed patients at risk for arrhythmias including mediastinal masses and pericardial effusions in two, electrolyte derangements in one, and myocarditis in two others. this finding illustrates the point that providers need to be focused on the common associations between arrhythmias while being vigilant for new associations with covid- . another distinction between adults and children was that in the present study, there was no significant association between arrhythmias with, duration of illness or hospitalization, icu admission, use of vasoactive medications, or mortality. this is in contrast to adult studies that have shown increased morbidity and mortality in patients with covid- infection and cardiovascular complications including arrhythmias. , this may in part be related to the self-limited nature of the arrhythmias seen in the present study. among the various therapies that have been advocated for the management of covid- , hcq and azn have been used in the adult and pediatric patient population. however, it has a serious side effect of prolonging qt interval and pre-disposing patients to ventricular arrhythmias such as torsades de pointes. ,, while the present pediatric study also found a statistically significantly longer longest daily measured qtc in those patients receiving hcq, treatment was not terminated in any of the patients in this study as none of them developed clinically significantly prolonged qtc intervals. in fact, none had qtc intervals that were abnormal and the development of arrhythmias was not associated with qtc prolongation. while the degree of qtc lengthening found in the present study for those receiving hcq with or without azn ( msec) was shorter than that reported in adult covid patients receiving hcq and azn, the timeline of lengthening ( - days in / patients in whom there was lengthening) was similar to that reported in adult studies. in a study by chorin et al., of adult patients receiving hcq and azn for covid- infection, there was prolongation of the qtc from a baseline average of ± ms (mean ± s.d.) to a maximal average value of ± ms (p < . ), which occurred on day . ± . of therapy, including a subset of patients developed severe qtc prolongation of > msec; however no torsades de pointes events recorded for any patients. it must be acknowledged that it is unclear if the change in qtc seen in the present study was necessarily related to treatment in all patients. while the longest daily measured qtc was occurred during treatment in % patients, it was found that the baseline qtc was longer in % of patients treated with hcq +/-azn and the longest daily measured qtc after treatment was started was not statistically different for those treated with hcq +/-azn compared to those who received neither drug. furthermore, there was no difference between baseline qtc and the longest daily measured qtc after treatment was started for those who received both drugs. thus, routine variation in qtc duration may account for changes in qtc in some patients and may, in part, account for the lack of association with arrhythmias. while evidence for the efficacy of hcq and azn in treating covid- is lacking, it is important to realize that the effects of any treatment must be studied in children as well as adults and not to assume that the risks for children are the same, less, or more than that seen in adults. , limitations: as a single-centered retrospective chart review, several inherent limitations exist, including a small study cohort which limited the ability to make statistical comparisons. being retrospective in nature, certain variables were not obtained in all patients or collected at the same time points in all patients. management protocols that were instituted during the pandemic were revised frequently for efficacy as well as patient and staff safety, creating variability in data set completeness at various times during the pandemic. in addition, igg testing was not available at the beginning of the pandemic. one patient with possible kawasaki syndrome, who did not have arrhythmias or qtc prolongation, did not have igg tested upon admission. however, unlike our subsequent experience and what has been reported with mis-c patients, this patient's pcr was positive on the first test, suggesting she had active covid at the time of admission. in pediatric patients with pcr positive active covid- infection, significant arrhythmias are infrequent, but occur at an incidence higher than expected in a general pediatric population. comorbidities are not more common in patients with arrhythmias than in patients without arrhythmias. however, providers still need to be vigilant for comorbidities that may independently place patients at risk for arrhythmias. covid- treatment using hcq leads to significant qtc prolongation, but was not associated with arrhythmias in pediatric patients. the long term sequelae of arrhythmia development in this population and their impact on outcome needs to be studied. who declares covid- a pandemic an outbreak of severe kawasaki-like disease at the italian epicentre of the sars-cov- epidemic: an observational cohort study. the lancet cardiac involvement in covid- patients: risk factors, predictors, and complications: a review cardiac arrhythmias in covid- infection guidance for cardiac electrophysiology during the coronavirus (covid- ) pandemic from the heart rhythm society covid- task force; electrophysiology section of the american college of cardiology; and the electrocardiography and arrhythmias committee of the council on clinical cardiology drug-induced ventricular tachycardia association of treatment with hydroxychloroquine or azithromycin with in-hospital mortality in patients with covid- in aha/accf/hrs recommendations for the standardization and interpretation of the electrocardiogram: part iv: the st segment, t and u waves, and the qt interval: a scientific statement from the american heart association electrocardiography and arrhythmias committee, council on clinical cardiology; the american college of cardiology foundation; and the heart rhythm society. endorsed by the international society for computerized electrocardiology assessment of qt intervals in a case series of patients with coronavirus disease (covid- ) infection treated with hydroxychloroquine alone or in combination with azithromycin in an intensive care unit race to find covid- treatments accelerates paces/hrs expert consensus statement on the evaluation and management of ventricular arrhythmias in the child with a structurally normal heart cardiac manifestations of coronavirus (covid- ) clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china cardiovascular implications of fatal outcomes of patients with coronavirus disease (covid- ) jama cardiol clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study efficacy of hydroxychloroquine in patients with covid- : results of a randomized clinical trial effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus (sars-cov- ) infection: a randomized clinical trial the qt interval in patients with covid- treated with hydroxychloroquine and azithromycin urgent guidance for navigating and circumventing the qtc-prolonging and torsadogenic potential of possible pharmacotherapies for coronavirus disease (covid- ) baseline qtc (solid bars) is compared to the longest daily measured qtc after initiation of therapy (hashed bars) seen during treatment for each treatment strategy. p values for paired t-test comparing baseline and longest daily measured qtc after initiation of therapy values for each group are displayed • patients > years, number (%) ( %) gender • males, number (%) ( . %) • females, number (%) key: cord- -zn f lk authors: liu, jia; cao, ruiyuan; xu, mingyue; wang, xi; zhang, huanyu; hu, hengrui; li, yufeng; hu, zhihong; zhong, wu; wang, manli title: hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro date: - - journal: cell discov doi: . /s - - - sha: doc_id: cord_uid: zn f lk nan hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro jia liu , ruiyuan cao , mingyue xu , , xi wang , huanyu zhang , , hengrui hu , , yufeng li , , zhihong hu , wu zhong and manli wang dear editor, the outbreak of coronavirus disease (covid- ) caused by the severe acute respiratory syndrome coronavirus (sars-cov- / -ncov) poses a serious threat to global public health and local economies. as of march , , over , cases have been confirmed in china, including deaths as well as over , confirmed cases in other countries. such huge numbers of infected and dead people call for an urgent demand of effective, available, and affordable drugs to control and diminish the epidemic. we have recently reported that two drugs, remdesivir (gs- ) and chloroquine (cq) phosphate, efficiently inhibited sars-cov- infection in vitro . remdesivir is a nucleoside analog prodrug developed by gilead sciences (usa). a recent case report showed that treatment with remdesivir improved the clinical condition of the first patient infected by sars-cov- in the united states , and a phase iii clinical trial of remdesivir against sars-cov- was launched in wuhan on february , . however, as an experimental drug, remdesivir is not expected to be largely available for treating a very large number of patients in a timely manner. therefore, of the two potential drugs, cq appears to be the drug of choice for large-scale use due to its availability, proven safety record, and a relatively low cost. in light of the preliminary clinical data, cq has been added to the list of trial drugs in the guidelines for the diagnosis and treatment of covid- (sixth edition) published by national health commission of the people's republic of china. cq (n -( -chloro- -quinolinyl)-n ,n -diethyl- , pentanediamine) has long been used to treat malaria and amebiasis. however, plasmodium falciparum developed widespread resistance to it, and with the development of new antimalarials, it has become a choice for the prophylaxis of malaria. in addition, an overdose of cq can cause acute poisoning and death . in the past years, due to infrequent utilization of cq in clinical practice, its production and market supply was greatly reduced, at least in china. hydroxychloroquine (hcq) sulfate, a derivative of cq, was first synthesized in by introducing a hydroxyl group into cq and was demonstrated to be much less (~ %) toxic than cq in animals . more importantly, hcq is still widely available to treat autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis. since cq and hcq share similar chemical structures and mechanisms of acting as a weak base and immunomodulator, it is easy to conjure up the idea that hcq may be a potent candidate to treat infection by sars-cov- . actually, as of february , , seven clinical trial registries were found in chinese clinical trial registry (http://www.chictr.org.cn) for using hcq to treat covid- . whether hcq is as efficacious as cq in treating sars-cov- infection still lacks the experimental evidence. to this end, we evaluated the antiviral effect of hcq against sars-cov- infection in comparison to cq in vitro. first, the cytotoxicity of hcq and cq in african green monkey kidney veroe cells (atcc- ) was measured by standard cck assay, and the result showed © the author(s) open access this article is licensed under a creative commons 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(fig. a) . to better compare the antiviral activity of cq versus hcq, the dose-response curves of the two compounds against sars-cov- were determined at four different multiplicities of infection (mois) by quantification of viral rna copy numbers in the cell supernatant at h post infection (p.i.). the data summarized in fig. a and supplementary table s show that, at all mois ( . , . , . , and . ), the % maximal effective concentration (ec ) for cq ( . , . , . , and . μm) was lower than that of hcq ( . , . , . , and . μm). the differences in ec values were statistically significant at an moi of . (p < . ) and moi of . (p < . ) (supplementary table s ). it is worth noting that the ec values of cq seemed to be a little higher than that in our previous report ( . μm at an moi of . ) , which is likely due to the adaptation of the virus in cell culture that significantly increased viral infectivity upon continuous passaging. consequently, the selectivity index (si = cc /ec ) of cq ( . , . , . , and . ) was higher than that of hcq ( . , . , . , . ) at mois of . , . , . , and . , respectively. these results were corroborated by immunofluorescence microscopy as evidenced by different expression levels of virus nucleoprotein (np) at the indicated drug concentrations at h p.i. (supplementary fig. s ). taken together, the data suggest that the anti-sars-cov- activity of hcq seems to be less potent compared to cq, at least at certain mois. both cq and hcq are weak bases that are known to elevate the ph of acidic intracellular organelles, such as endosomes/lysosomes, essential for membrane fusion . in addition, cq could inhibit sars-cov entry through changing the glycosylation of ace receptor and spike protein . time-of-addition experiment confirmed that hcq effectively inhibited the entry step, as well as the post-entry stages of sars-cov- , which was also found upon cq treatment (supplementary fig. s ). to further explore the detailed mechanism of action of cq and hcq in inhibiting virus entry, co-localization of virions with early endosomes (ees) or endolysosomes (els) was analyzed by immunofluorescence analysis (ifa) and confocal microscopy. quantification analysis showed that, at min p.i. in untreated cells, . % of internalized virions (anti-np, red) were observed in early endosome antigen (eea )-positive ees (green), while more virions ( . %) were transported into the late endosomal-lysosomal protein lamp + els (green) (n > cells for each group). by contrast, in the presence of cq or hcq, significantly more virions ( . % for cq and . % for hcq; p < . ) were detected in the ees, while only very few virions ( . % for cq and . % for hcq; p < . ) were found to be co-localized with lamp + els (n > cells) (fig. b, c) . this suggested that both cq and hcq blocked the transport of sars-cov- from ees to els, which appears to be a requirement to release the viral genome as in the case of sars-cov . interestingly, we found that cq and hcq treatment caused noticeable changes in the number and size/morphology of ees and els (fig. c) . in the untreated cells, most ees were much smaller than els (fig. c) . in cqand hcq-treated cells, abnormally enlarged ee vesicles were observed (fig. c , arrows in the upper panels), many of which are even larger than els in the untreated cells. this is in agreement with previous report that treatment with cq induced the formation of expanded cytoplasmic vesicles . within the ee vesicles, virions (red) were localized around the membrane (green) of the vesicle. cq treatment did not cause obvious changes in the number and size of els; however, the regular vesicle structure seemed to be disrupted, at least partially. by contrast, in hcq-treated cells, the size and number of els increased significantly (fig. c , arrows in the lower panels). since acidification is crucial for endosome maturation and function, we surmise that endosome maturation might be blocked at intermediate stages of endocytosis, resulting in failure of further transport of virions to the ultimate releasing site. cq was reported to elevate the ph (see figure on previous page) fig. comparative antiviral efficacy and mechanism of action of cq and hcq against sars-cov- infection in vitro. a cytotoxicity and antiviral activities of cq and hcq. the cytotoxicity of the two drugs in vero e cells was determined by cck- assays. vero e cells were treated with different doses of either compound or with pbs in the controls for h and then infected with sars-cov- at mois of . , . , . , and . . the virus yield in the cell supernatant was quantified by qrt-pcr at h p.i. y-axis represents the mean of percent inhibition normalized to the pbs group. the experiments were repeated twice. b, c mechanism of cq and hcq in inhibiting virus entry. vero e cells were treated with cq or hcq ( μm) for h, followed by virus binding (moi = ) at °c for h. then the unbound virions were removed, and the cells were further supplemented with fresh drug-containing medium at °c for min before being fixed and stained with ifa using anti-np antibody for virions (red) and antibodies against eea for ees (green) or lamp for els (green). the nuclei (blue) were stained with hoechst dye. the portion of virions that co-localized with ees or els in each group (n > cells) was quantified and is shown in b. representative confocal microscopic images of viral particles (red), eea + ees (green), or lamp + els (green) in each group are displayed in c. the enlarged images in the boxes indicate a single vesicle-containing virion. the arrows indicated the abnormally enlarged vesicles. bars, μm. statistical analysis was performed using a one-way analysis of variance (anova) with graphpad prism (f = . , df = , , ***p < . ). of lysosome from about . to . at μm . to our knowledge, there is a lack of studies on the impact of hcq on the morphology and ph values of endosomes/ lysosomes. our observations suggested that the mode of actions of cq and hcq appear to be distinct in certain aspects. it has been reported that oral absorption of cq and hcq in humans is very efficient. in animals, both drugs share similar tissue distribution patterns, with high concentrations in the liver, spleen, kidney, and lung reaching levels of - times higher than those in the plasma . it was reported that safe dosage ( - . mg/kg per day) of hcq sulfate could generate serum levels of . - . μm in humans . therefore, with a safe dosage, hcq concentration in the above tissues is likely to be achieved to inhibit sars-cov- infection. clinical investigation found that high concentration of cytokines were detected in the plasma of critically ill patients infected with sars-cov- , suggesting that cytokine storm was associated with disease severity . other than its direct antiviral activity, hcq is a safe and successful anti-inflammatory agent that has been used extensively in autoimmune diseases and can significantly decrease the production of cytokines and, in particular, pro-inflammatory factors. therefore, in covid- patients, hcq may also contribute to attenuating the inflammatory response. in conclusion, our results show that hcq can efficiently inhibit sars-cov- infection in vitro. in combination with its anti-inflammatory function, we predict that the drug has a good potential to combat the disease. this possibility awaits confirmation by clinical trials. we need to point out, although hcq is less toxic than cq, prolonged and overdose usage can still cause poisoning. and the relatively low si of hcq requires careful designing and conducting of clinical trials to achieve efficient and safe control of the sars-cov- infection. remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro first case of novel coronavirus in the united states review of side effects and toxicity of chloroquine animal toxicity and pharmacokinetics of hydroxychloroquine sulfate chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion new insights into the antiviral effects of chloroquine ebola virus and severe acute respiratory syndrome coronavirus display late cell entry kinetics: evidence that transport to npc + endolysosomes is a rate-defining step effect of phospholipidosis on the cellular pharmacokinetics of chloroquine fluorescence probe measurement of the intralysosomal ph in living cells and the perturbation of ph by various agents choloroquine: a review dosage of antimalarial drugs for children with juvenile rheumatoid arthritis and systemic lupus erythematosus. a clinical study with determination of serum concentrations of chloroquine and hydroxychloroquine clinical features of patients infected with novel coronavirus in wuhan we thank professor zhengli shi and dr. xinglou yang from wuhan institute of virology and professor fei deng from national virus resource center for providing sars-cov- strain (ncov- betacov/wuhan/wiv / ); professor xiulian sun for kind help in statistical analysis; professor zhenhua zheng for kindly providing the anti-lamp rabbit polyclonal antibody; prof. zhengli shi for kindly providing the anti-np polyclonal antibody; beijing savant biotechnology co., ltd for kindly providing the anti-np monoclonal antibody; min zhou and xijia liu for their assistance with this study; jia wu, jun liu, hao tang, and tao the authors declare that they have no conflict of interest.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.supplementary information accompanies the paper at (https://doi.org/ . /s - - - ). key: cord- - kg nf authors: chivese, t.; musa, o. a. h.; hindy, g.; wattary, n.; badran, s.; soliman, n.; aboughalia, a. t.; matizanadzo, j. t.; emara, m. m.; thalib, l.; doi, s. title: a meta-review of systematic reviews and an updated meta-analysis on the efficacy of chloroquine and hydroxychloroquine in treating covid infection date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: kg nf abstract background there is an urgent need for an efficacious and safe treatment for covid . several trials testing a variety of therapeutics are on-going. some in-vitro studies found the anti-malarial drug chloroquine (cq), and its derivative, hydroxychloroquine (hcq), are effective against covid . however, systematic reviews and meta-analyses of clinical trials in humans have produced conflicting findings on the efficacy and safety of these drugs. guidelines vary considerably and are hotly debated at political and scientific levels. therefore, it has become necessary to provide a summary of the effectiveness and safety of these drugs in treating covid infection, using an overview of the existing systematic reviews and meta-analyses. objective to synthesize the findings presented in systematic reviews and meta-analyses as well as to update the evidence using a meta-analysis in evaluating the efficacy and safety of cq and hcq with or without azithromycin for the treatment of covid infection. methods the design of this meta-review followed the preferred reporting items for overviews of systematic reviews including harms checklist (prio-harms). a comprehensive search included several electronic databases in identifying all systematic reviews and meta-analyses as well as experimental studies which investigated the efficacy and safety of cq, hcq with or without antibiotics as covid treatment. manual searches of the reference list of all included studies and a citation search of the top papers supplemented the search. findings from the systematic reviews and meta-analyses were reported using a structured summary including tables and forest plots. the updated meta-analysis of experimental studies was carried out using the distributional-assumption-free quality effects model. risk of bias was assessed using the assessing the methodological quality of systematic reviews (amstar) tool for reviews and the methodological standard for epidemiological research (master) scale for the experimental studies. the main outcomes for both the meta-review and the updated meta-analysis were; mortality, transfer to the intensive care unit (icu), intubation or the need for mechanical ventilation, worsening of illness, viral clearance and the occurrence of adverse events. results a total of reviews with primary studies comprising , participants were included. most of the primary studies were observational (n= ) and the rest were experimental studies. two meta-analyses reported a high risk of mortality with similar ors of . for hcq with azithromycin. however, four other meta-analyses reported contradictory results with two reporting a high risk of mortality (or ~ . to . ) and the other two reporting no significant association between hcq with mortality. most reviews reported that hcq with or without azithromycin had no significant effect on virological cure, disease exacerbation or the risk of transfer to the icu, need for intubation or mechanical ventilation. after exclusion of studies that did not meet the eligibility criteria, the updated meta-analysis contained eight experimental studies ( rcts and quasi-experimental trial), with a total of participants of whom were on either cq/hcq or combined with azithromycin. cq/hcq with or without azithromycin was significantly associated with a higher risk of adverse events (rr . , %ci . - . , i = %, n = studies). hcq was not effective in reducing mortality (rr . , %ci . - . , i = %, n= studies), transfer to the icu, intubation or need for mechanical ventilation (rr . , %ci . - . , i = %, n= studies) virological cure (rr . , %ci . - . , i = %, n= studies) nor disease exacerbation (rr . , %ci . - . , i = %, n= studies). conclusion there is conclusive evidence that cq and hcq, with or without azithromycin are not effective in treating covid- or its exacerbation. there is an urgent need for an efficacious and safe treatment for covid . several trials testing a variety of therapeutics are on-going. some in-vitro studies found the antimalarial drug chloroquine (cq), and its derivative, hydroxychloroquine (hcq), are effective against covid . however, systematic reviews and meta-analyses of clinical trials in humans have produced conflicting findings on the efficacy and safety of these drugs. guidelines vary considerably and are hotly debated at political and scientific levels. therefore, it has become necessary to provide a summary of the effectiveness and safety of these drugs in treating covid infection, using an overview of the existing systematic reviews and meta-analyses. to synthesize the findings presented in systematic reviews and meta-analyses as well as to update the evidence using a meta-analysis in evaluating the efficacy and safety of cq and hcq with or without azithromycin for the treatment of covid infection. the design of this meta-review followed the preferred reporting items for overviews of systematic reviews including harms checklist (prio-harms). a comprehensive search included several electronic databases in identifying all systematic reviews and metaanalyses as well as experimental studies which investigated the efficacy and safety of cq, hcq with or without antibiotics as covid treatment. manual searches of the reference list of all included studies and a citation search of the top papers supplemented the search. findings from the systematic reviews and meta-analyses were reported using a structured summary including tables and forest plots. the updated meta-analysis of experimental studies was carried out using the distributionalassumption-free quality effects model. risk of bias was assessed using the assessing the methodological quality of systematic reviews (amstar) tool for reviews and the methodological standard for epidemiological research (master) scale for the experimental studies. the main outcomes for both the meta-review and the updated meta-analysis were; mortality, transfer to the intensive care unit (icu), intubation or the . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the severe acute respiratory syndrome coronavirus (sars-cov ) is currently known to be among one of the most contagious viruses in the history of pathogens ( ) . this novel human coronavirus emerged in wuhan in december and has since spread from person to person in an efficient and sustained way to cause a global pandemic, with the disease named coronavirus disease- (covid ) ( ) . to date, . million people have been infected with covid and more than , have died as of th of july ( ) . although the disease is largely controlled in china, where it originated, and italy, which became the epicentre of the disease in europe, subsequently, the cases and deaths continue to increase globally, with the highest increases in the usa, brazil and india. concerns for world economies has forced the relaxation of lockdowns and restrictive measures even in some of the worst affected countries ( ) ( ) ( ) . however, the fatalities from covid are expected to rise further as lockdowns are eased and people begin to interact more ( , ) . this continued increase in deaths from covid infection has warranted the need to investigate effective vaccines and therapies. of several therapeutic drugs that have been suggested, chloroquine (cq), an antimalarial drug in the class of -aminoquinolones with anti-inflammatory, antiviral and anti-thrombolytic properties, and its derivative, hydroxychloroquine (hcq) have been repurposed and are being used widely for the treatment of covid ( , ) . cq and hcq are antimalarials as well as used as disease-modifying antirheumatic drugs (dmards) ( ) . there are several mechanisms which have been suggested for the expected effect of the two drugs against viruses in general and sars-cov- in particular. one of these mechanisms is based on inhibiting the ability of the virus to enter the cell. sars-cov- harbours a spike (s) protein, which is considered a crucial element in the virus replication cycle as it binds to angiotensin-converting enzyme (ace ) expressed in the lungs of the host cell receptor ( , ) . the antiviral effect of both cq and hcq is thought to be through their ability to interfere with the glycosylation of ace and thus prevent the proper binding of the s protein. ( ) . moreover, virus entry occurs through receptor-mediated endocytosis, which needs an acidic ph to complete the fusion and deliver the viral genome into the cell. both cq and hcq are weak bases and thought to . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint inhibit this process that the sars cov- virus needs for replication ( ) . apart from their direct antiviral properties, cq and hcq also seem to have immune-modulatory effects which help to reduce over-activation of the immune system from covid ( ) . hcq has been used more frequently than cq in treating covid as it has been reported to be more potent in vitro ( ) , can be used in higher doses for a longer time with a lower risk of adverse events compared to cq and is more widely available ( , ) . further, both drugs are relatively cheap, and their use in the treatment of covid is based on promising results from in vitro studies ( , ) and some observational studies ( , ) . repurposing the drugs for use in treating covid has been easy as the drugs are already in use for the treatment of malaria, are cheap and have been thought to have a relatively safe profile ( , ) . due to the above reasons and the unprecedented situation and the urgency to curb the covid pandemic, cq and hcq have been approved, on a fast track basis, by the united states food and drug administration and other regulatory bodies, despite the lack of good quality evidence of their efficacy and safety ( , ) . consequently, more than trials of the two drugs are either ongoing or completed and it is necessary to evaluate the evidence so clinicians and regulatory bodies can make informed decisions on the use of these drugs for the treatment of covid infection. initial reports suggested that hcq was effective in the treatment of covid associated pneumonia ( ) and that hcq in combination with the second-generation macrolide antibiotic, azithromycin, resulted in a lower fatality ( ) , and % viral clearance of covid , by day ( ) . however, subsequent findings from both observational studies ( , ) and clinical trials have been contradictory ( ) ( ) ( ) . further, although the two drugs are relatively safe in the treatment of malaria, concerns have been raised about the risk of adverse events associated with their use when treating covid ( ) . the recovery trial ( ), one of the largest trials to-date investigating the optimal treatment for covid , issued a statement that they found no differences in mortality between participants on hcq and those on usual care, and subsequently stopped the hcq arm of the trial. in a similar move, the world health organization stopped the hcq arm of the solidarity trial ( ), citing the lack of . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint efficacy from their interim analysis of the u.k. recovery trial, the french discovery trial ( ) and an unnamed cochrane review ( ). before the stopping of the recovery and solidarity trials, the largest observational study to date, a multinational cohort study ( ) of , participants, had previously reported a to fold increase in the mortality associated with cq or hcq, with or without macrolides. however, the study was subsequently retracted due to concerns over the veracity of the data and its analysis ( ) leaving some degree of uncertainty in its wake. several systematic reviews and meta-analyses investigating the efficacy and the safety of cq and hcq have been published or are in the process of being published ( , , , , ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . similar to the primary studies, systematic reviews and meta-analyses have presented contradictory findings. a key issue that has made it difficult to have conclusive results on the efficacy of cq and hcq with or without the macrolide antibiotics is that clinical trials were few, small and poorly designed. most of the existing systematic reviews have therefore carried out meta-analyses combining data from observational and experimental studies without accounting for the quality of included studies. several clinical trials, with acceptable quality, are now available. therefore, it has become necessary to synthesize all available evidence to provide the best evidence-based assessment on the efficacy and safety of both cq and hcq, with or without macrolide antibiotics, in the treatment of covid infection. in this respect, we conducted this umbrella review with two broad aims; ( ) to assess the efficacy and safety of each of hcq and cq, with or without azithromycin in the treatment of covid by assessing the evidence from existing systematic reviews and metaanalyses, and ( ) to carry out an updated meta-analysis of the existing experimental studies to assess the efficacy of these drugs. this study protocol is registered on prospero (crd ). this study had two components; an overview of all existing systematic reviews and meta-analyses and an updated meta-analysis of all eligible experimental studies that investigated the . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint efficacy of either cq or hcq with or without macrolide antibiotics in the treatment of covid infection. the design of this overview followed the preferred reporting items for overviews of systematic reviews including harms checklist guidelines (prio-harms) ( ) . the updated meta-analysis was carried out according to the preferred reporting items for systematic review and meta-analysis (prisma) ( ) . we conducted electronic searches for all experimental studies, systematic reviews and meta-analyses on the efficacy and safety of cq and hcq for the treatment of covid we used the following search terms for chloroquine and hydroxychloroquine; "chloroquine" or "hydroxychloroquine" or "cq" or "hcq". we used the following search terms for covid infection; "covid " or "coronavirus" or "novel coronavirus" or "sars-cov- " or "covid" or "covid- ". the following search terms were used for the study design; "clinical study", "clinical trial", "trial", "rct", "controlled trial", "randomized controlled trial", "meta-analysis", "rapid review", "review", "systematic reviews". all searches had no limitations on language or location but were limited to studies published during the year . keywords and medical subject headings (mesh) were used for searches for covid , sars-cov , chloroquine and . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint hydroxychloroquine. the full electronic search strategy is given in appendix (supplementary table s ). systematic reviews and/or meta-analyses were included if they compared the efficacy and/or safety of hcq or cq. such reviews should either have a minimum of two clinical studies comparing hcq or cq with or without azithromycin to any other standard treatments, including placebo. participants in the included index primary studies should have had confirmed covid , regardless of age or the severity of illness. reviews were excluded if they were literature reviews, did not include at least two eligible primary studies and if their main scope was on prophylaxis. the updated meta-analysis only included experimental studies which investigated the efficacy of either cq, hcq or either with azithromycin added in the treatment of covid . studies were participants were diagnosed using symptoms were excluded as symptoms have been shown to have poor diagnostic accuracy ( ) . observational studies and any studies without control groups were excluded. studies on animals and in-vitro studies were also excluded. in the case that studies were duplicates, either the study with the most data was used and the other excluded or both studies were combined. studies were included if the intervention included either cq or hcq alone or with azithromycin in any dose combinations and any length of administration. due to the heterogeneous nature of covid treatments in different countries and different disease severity categories, any studies that had control groups that did not include either cq or hcq were acceptable for inclusion in this overview and meta-analysis. studies were also included irrespective of either the severity of disease of included participants or the setting (i.e. either hospital-based or community or both). search results were uploaded on to the rayyan platform ( ) where authors blindly screened titles and abstracts. conflicts were discussed and resolved by consensus between authors. the full text of potentially relevant articles were screened against eligibility criteria for the final inclusion. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint for each study, two authors independently extracted data and assessed quality. the data extracted from the reviews included; type of review (systematic review or metaanalysis), date of publication or submission to the preprint servers, countries with data included in the review, scope of the review, the number and type of index studies included, tools used to assess the risk of bias, risk of bias summary, main comparisons, total participants, mean age, outcomes measured and pooled measures of effect, statistics for heterogeneity, the review conclusion and limitations. additional data included the number of and citations of all included primary studies in the review. the primary studies were further categorised as either experimental or observational, first based on their classification in the parent review and later after an independent assessment by two authors. all experimental studies identified from the assessment were considered for inclusion in the updated meta-analysis. data extracted from experimental studies included; study design (rct or quasi-rct), intervention (whether cq or hcq with or without azithromycin), dosage, route of administration, control treatment description, any co-interventions, setting (community setting or hospital), the country where study carried out, proportions with severe sickness, proportions with comorbidities, mean age, gender distribution, and total with each out outcome in the intervention and control groups. due to the heterogeneous nature of the outcomes assessed in different systematic reviews, meta-analyses and primary studies, we grouped the outcomes into four main groups. the primary outcome was mortality assessed as all-cause mortality. secondary outcomes were disease exacerbation, virological cure, adverse events and included a composite outcome that combined the events of any transfer to the intensive care unit (icu), any need for intubation or any need for mechanical ventilation. disease worsening was defined as any form of symptom worsening such as the need for oxygen, dyspnoea, hospitalization in the case of community-based studies. the virological cure was defined as a negative pcr any time after commencement of treatment. safety was assessed as the occurrence of the known adverse events of cq . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . or hcq or azithromycin. these adverse events included gastric side effects such as diarrhoea and vomiting, ventricular tachycardia possibly as a result of qtc prolongation and headache, blurred vision and rash. two authors independently assessed the quality of each included review using the assessing the methodological quality of systematic reviews (amstar) tool ( ) . each review had a maximum score of if the methodological quality is good and zero if the methodological quality is poor. any disagreements were resolved by discussion between the authors. for the experimental studies, the methodological standard for epidemiological research (master) scale ( ) was used to assess the methodological quality of studies across safeguards listed within standards. the standards assessed were; equal recruitment, equal retention, equal ascertainment, equal implementation, equal prognosis, sufficient analysis and temporal precedence ( ) . a kappa interrater agreement was calculated for each study and the quality safeguard counts averaged if the kappa was at least . . where the kappa was below . , the authors resolved disagreements by discussion or by referring to a third assessor if they did not resolve. the quality counts were used to rank studies for inclusion in a bias adjusted metaanalysis. the characteristics of the included reviews and experimental studies are summarised in table and table respectively. the quality assessment of the included reviews and experimental studies are shown in supplementary tables and , respectively. a map showing the distribution of all the index studies included in the included reviews was created using tableau software ( ). synthesis of findings from different reviews was done using a combination of a structured summary of findings from the reviews and presentation in forest plots ( ) . a table with the findings of each review for each outcome was presented. for outcomes were several meta-analyses were available, forest plots were used to show the . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) magnitude of the effect, the % confidence intervals ( %ci) and the number of included studies. where there were no available meta-analyses, findings from systematic reviews were compared narratively. the overall score from the amstar quality assessment and the i heterogeneity score were incorporated in the interpretation of the findings of each review. the forest plots were created in r statistical software ( ) . for the updated meta-analysis, the quality effects model ( ) was employed to pool the estimates (relative risks), as it is more robust and performs better than the randomeffects model ( ) . in some studies where outcome assessed cases were zero, a post hoc continuity correction adding ( . ) to all cells was employed for valid estimation of the relative risk and its variance. forest plots were used to depict the results of the pooled analysis. the quality effects method uses a relative quality rank from each of the studies to modify the study's variance weight, thereby incorporating the quality of the study quantitatively into the results. the results of random effects analyses were reported in the supplementary material for comparison only if there was heterogeneity as without this it defaults to a fixed effect model similar to the quality effects model. the stata software program ( ) was utilized for meta-analysis. forest plots were used to present the pooled risk ratios and their confidence intervals (cis). the cochran q test p-value was used to test for and the i statistic to quantify heterogeneity ( ) . the i statistic measures consistency and is an indication of the variability in the estimates of the effects that are caused by heterogeneity instead of sampling error, and ranges from a % (no heterogeneity) to % (high heterogeneity). i values above % indicate substantial heterogeneity and above % high heterogeneity. doi plots and lfk index were used for assessing publication bias as they are more reliable ( ) than funnel plots and in this case funnel plots could not be used as they are not recommended when there are less than studies in a meta-analysis ( ) . ethics approval was not required as the study used published data. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint *index publication is the first occurrence of a primary publication in the included reviews. **additional eligible primary studies that had not been initially identified by the search of the relevant reviews or obtained by updating the search of the included reviews. ***study compared high dose chloroquine against a low dose chloroquine. **** study design not clear as both groups received hcq, ***** most participants in study diagnosed using symptoms and not pcr additional records identified through other sources (n = ) records identified through database searching (n = ) duplicates removed (n = ) full-text review articles assessed for eligibility (n = ) included screening of the experimental studies that are included in meta-review • total included in updated meta-analysis = ( rct (n= ) and one qrct) • excluded -no data presented (n= ), cq vs cq*** (n= ), abstract only (n= ), covid not pcr confirmed (n= ), duplicate (n= ), study design unclear **** (n= ) articles with efficacy outcomes included in the meta-review reviews (n= ) additional primary studies (n= ) articles focused on harms outcomes included in the meta-review reviews (n= ) additional primary studies (n= ) articles with efficacy and harms outcomes included in the metareview reviews (n= ) additional primary studies (n= ) no. of index publications* included in the reviews = • experimental studies (n= ) **additional screening for eligible rcts that are not included in the meta-review (included, n= , excluded, covid not pcr confirmed for most participants*****, n= ) . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint the search identified reviews and were excluded by screening the title and abstract (fig ) . most reviews were excluded because they were either literature reviews or review which did not include completed clinical studies (fig and supplementary table s ). the remaining reviews were screened for eligibility through reading the full text and were provisionally included. one additional review was identified through manual searching of references and the citations search. the final included reviews were , of which ( . %) were systematic reviews and metaanalyses and the remainder systematic reviews only (table ) . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . all the included reviews were conducted between april and the st of june . at the time that this meta-review was carried out, of the included reviews were published ( , , , , , , ) and the remaining were preprints ( , , , ( ) ( ) ( ) . all the included reviews assessed the efficacy and the safety of the cq and hcq in the treatment of covid- , except jankelson et al. ( ) which only assessed adverse events associated with cq and hcq. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint most of the included reviews had above safeguards and only reviews ( , ) had or lower safeguard counts on the amstar scale ( supplementary fig. & supplementary table s ). the same reviews did not report that they assessed for risk of bias in their included index studies. one ( ) of the two reviews which did not report assessing the risk of bias in included studies was not published at the time of this umbrella review. the reported quality of included index studies in the reviews was generally poor, with reviews ( , - , , , , ) reporting a low count of safeguards in general. the most common issues were lack of safeguards against selection bias, lack of randomization, lack of allocation concealment bias, absence of blinding and performance and reporting biases. most of the included reviews had the limitations of a small number of included index studies, lack of proper control groups or improper randomization among the included primary studies. an additional limitation of the reviews was the mixing of results from observational and experimental studies in the meta-analyses of efficacy. n -sample size of the index study . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint the total number of index studies in the included reviews, after removing duplicate studies was . most of the included primary studies were conducted in the usa ( studies, participants), followed by china ( studies, participants) and france ( studies, participants) (fig ) . the total number of participants in all the reviews after removing duplicate studies was . all the included reviews included at least experimental studies and the number of included observational studies ranged from zero in reviews ( , , ) to in one review ( ) . the observational index studies were excluded from the updated meta-analysis (supplementary table s ). after screening the studies described as experimental studies in the included reviews, only experimental studies ( , , ( ) ( ) ( ) satisfied the criteria for inclusion in the updated meta-analysis (fig. ) . the reasons for exclusion of the experimental studies were as follows; duplicate study (n= ) ( ) which used data from gautret et al. ( ) , one study had no full text available ( ), one study compared high dose chloroquine to low dose chloroquine ( ), and one study included participants who did not have covid confirmed by pcr ( ). another experimental study was excluded because it was a letter to the editor describing trials with a total of patients but with no clear details about the trials, the participants or any outcomes ( ) . finally one study ( ) was described by the authors as quasi experimental as two institutions treated patients confirmed as positive with hcq but study subjects in both institutions received hcq. one institution received hcq shortly after admission, and in the other institution did not receive hcq until days later when pcr results came through so there was no real control arm and was not published with a submission version of the pdf circulated on the internet. five trials were identified from the additional search, with three included ( , , ) and two excluded ( , ) as they included participants who were diagnosed using symptoms rather than pcr (fig. ) . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint the eight experimental studies included in the updated meta-analysis ( rcts ( ) and taiwan ( ) . only one study used cq ( ) as the experimental drug while the remaining seven used hcq. all seven ( , , - , , ) included rcts had safeguard counts (quality assessment) of at least out of . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint . the quasi-experimental study ( ) had the lowest safeguard counts of . all the included studies, except chen z et al. ( ) , either did not blind or did not report sufficient information about blinding of either participants and research performers. other notable deficiencies included; insufficient reporting of randomisation procedure and insufficient statistical analyses ( supplementary fig. b & supplementary table s ). findings from included reviews a total of meta-analyses, all with amstar quality safeguard counts of at least , reported pooled effect sizes for mortality ( , , , ) (fig. ) . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint of the reviews without meta-analysis, concluded that there was evidence for higher risk of mortality in the hcq group ( , , , ) and one review ( ) reported evidence of lower mortality in the hcq group. two reviews reported evidence of higher mortality in the chloroquine group ( , ) (supplementary table s ). six experimental studies, five rcts ( , , , , ) and one quasi-experimental studies ( ) , assessed mortality, with a total of participants, of which were in . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint the intervention group. there was no significant difference in risk of mortality between participants who received hcq with or without azithromycin and those on standard care (rr . , %ci . - . ; fig ) , which was consistent across studies (i = %). the doi plot (supplementary fig. a) showed minor asymmetry. as horby et al. had the most weight in the meta-analysis, we carried out a sensitivity analysis without this study and the effect of hcq with or without axithromycin on all-cause mortality remained non-significant (rr . , %ci . - . ) ( supplementary fig a) . findings from included reviews a total of reviews investigated the risk of transfer to icu, need for intubation and mechanical ventilation. only one meta-analysis ( ) of studies with a total of participants was done and found a non-significant -fold increase risk of intubation in individuals on hcq (or . , %ci . - . ), with significantly high heterogeneity. the remaining reviews ( , , , , , ) which did a narrative synthesis did not find a difference in the risk of transfer to the icu, intubation or need for mechanical ventilation (supplementary table s ). two rcts ( , ), and the quasi experimental study ( ) , reported data on this outcome with a total of participants, of whom were in the intervention group. there was no significant difference in risk of icu transfer, need for mechanical ventilation or intubation in participants who received hcq with or without azithromycin, compared to those on standard care (rr . , %ci . - . ; fig. a ), consistent across the all the studies (i = %). the doi plot showed major asymmetry which was not resolved after several sensitivity analyses (supplementary fig. b ). . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint findings from included reviews twelve reviews ( , , , , , , , ( ) ( ) ( ) ( ) assessed the outcome of virological cure with of the reviews being meta-analyses. all the meta-analyses ( , , , ) found no significant differences between either hcq alone or hcq+ azithromycin and control, in virological cure (fig. ) . is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint however, the remaining seven reviews ( , , , , , , ) which narratively synthesized this outcome concluded that either hcq alone or hcq+ azithromycin were effective to some extent in the cure of the virus (supplementary table s ). five experimental studies, rcts ( - , ) and the single quasi-experimental study ( ) , assessed virological cure, with a total of participants, of which were in the intervention group. there was no significant difference in viral cure between participants who received hcq with or without azithromycin and those on standard care (rr . , %ci . - . ; fig ) , with some heterogeneity (i = %). there was consistency across studies after removal of the quasi-experimental study, gautret et al., although this did not alter the results of the pooled analysis (rr . %ci . - . , i = %) ( supplementary fig. ) . the doi plot showed major asymmetry which was not resolved after several sensitivity analyses ( supplementary fig. c ). findings from reviews four meta-analyses ( , , , ) , all with amstar score above found no significant effect of either hcq or hcq + azithromycin on disease exacerbation (fig ) . all the reviews without meta-analysis on this outcome concluded that either hcq or hcq+ azithromycin reduced the severity of illness (supplementary table s ). three reviews ( , , ) concluded that pneumonia was improved in the hcq / hcq+ azithromycin arm. singh et al. ( ) and hernandez et al. ( ) concluded that both fever and coughing were improved in the hcq/ hcq+ azithromycin group. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint updated meta-analysis of experimental studies three rcts ( , , ) assessed disease exacerbation, with a total of participants, of which were in the intervention group. there was no significant difference in the risk of disease exacerbation between participants who received hcq and those on standard care (rr . , %ci . - . ; fig. c ), with low heterogeneity across studies (i = . %). the doi plot showed major asymmetry which was not resolved after several sensitivity analyses (supplementary fig. d ). findings from reviews a total of ten reviews investigated the risk of adverse events between hcq or hcq+ azithromycin groups and control. two of these carried out meta-analyses and found pooled odds ratios of . ( %ci . - . , n= studies, participants, i = . %) ( ) and . ( %ci . - . , n= studies, participants, i = %) ( ) . all the remaining reviews ( , , , , , , ) except one ( ) , found an increased risk of adverse events in the hcq/ hcq+ azithromycin group. the most commonly reported adverse events were qtc interval prolongation, ( , - , , ) diarrhea, arrythmia and first-degree av block (supplementary table s ). five rcts ( , ( ) ( ) ( ) ) assessed adverse events, with a total of participants, of which were in the intervention group. the most commonly reported adverse events due to hcq and cq were gastrointestinal (nausea, vomiting, diarrhea and abdominal pain), reported in five trials ( - , , ) , headache ( , , ) and itchiness and rash ( , ) . serious adverse events were very rare and reported in only three is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint did not result in change in either the effect size or the significance (rr . , %ci . - . , i = %) ( supplementary fig. b ). the doi plot showed major asymmetry which was not resolved after several sensitivity analyses ( supplementary fig. e ). . nine of the reviews ( - , , , , , , ) all concluded that there was insufficient evidence to support the use of either cq or hcq with or without azithromycin in the treatment of people with covid disease. two reviews concluded that there was some benefit in using hcq or hcq+ azithromycin. yang et al. concluded that, although they were associated with higher mortality, hcq with or without azithromycin were beneficial, based on their effect on covid viral clearance. sarma et al. concluded that hcq was beneficial based on its efficacy in reducing radiological progression and that the drug was safe. the remaining reviews concluded that either cq or hcq were unsafe based on higher mortality ( ) and a higher risk of adverse events ( , ) . in this meta-review, we summarized the findings of seven systematic reviews and five meta-analyses and carried out an updated meta-analysis to investigate the efficacy and safety of cq and hcq, with or without a second generation macrolide antibiotic (azithromycin) in individuals infected with covid- , limiting our analysis to eight experimental studies which met a strict inclusion criteria. findings on the effect of cq and hcq on mortality were conflicting; two studies suggesting a to -fold increase in mortality associated with hcq, while other two reviews found no significant association with mortality. only two meta-analyses were carried out for hcq combined with azithromycin and both reported a . -fold increase in risk of mortality. the updated meta analyses carried out for this review showed no significant difference in all-cause mortality among those taking either hcq alone or in combination with azithromycin compared to standard care. this meta-review also found the risk of transfer to icu is doubled with either hcq or hcq and azithromycin and two meta-analyses conducted on adverse events showed . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint a -fold increase in the risk associated with hcq or hcq and azithromycin. the reviews also indicated there were no significant effect of hcq or hcq and azithromycin on both disease exacerbation and virological cure. the updated metaanalysis of eight experimental studies, however, showed that individuals on hcq or hcq and azithromycin had a -fold increase in risk of any adverse events. the updated meta-analysis also showed that there was no significant effect of cq/hcq with or without azithromycin on the risk of transfer to the icu, intubation or need for mechanical ventilation, virological cure and disease exacerbation. an important consideration in this meta-review is the impact of methodological limitations on the results of both the primary studies and the systematic reviews and meta-analyses included in this umbrella review. these limitations could primarily have resulted from the urgency of the need to find a cure, at short notice, for a pandemic that seems to be worsening in many countries to date. the limitations include, but are not limited to, small study sample sizes, the scarcity of randomized controlled trials, and the lack of methodological rigueur in the primary studies. all the reviews, except one ( ), included observational studies, which tend to have confounding and may lead to biased estimates of effects. an additional weakness of these observational studies is that patients and clinicians will most likely choose an experimental drug, compared to standard of care which comprises of symptom management during a pandemic with a perceived high risk of death and no cure. faced with a life-threatening illness, patients with severe illness will likely choose the experimental drug in the absence of proven alternatives, while those with mild to moderate disease may not want the experimental drug. the inclusion of observational studies in these meta-analyses seems to have been driven by the lack of good quality experimental studies and the need to use as much of the available information as possible. the observational studies were bigger than the rcts and therefore tended to influence the pooled estimates. the effect of inclusion of the observational studies could have been reduced using quality-adjusted meta-analysis synthesis, which decreases the weight of the observational studies in the overall estimate. however, none of the existing meta-analyses adjusted for the quality of the included studies in their syntheses. an exception is shamshirian et al. ( ) , who . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint carried out a sensitivity analysis with experimental studies only. further, the poor quality of studies included in the reviews is one of the limitations frequently cited by the review authors. some major limitations in the experimental studies include; participants not having confirmed covid disease in one study ( ), controlled clinical trials which had a high risk of selection bias ( , ) , and studies where the veracity of data presented could not be verified ( , ) . an example of the later is a letter about clinical trials in china claiming that cq was effective ( ) but without any data presented. this letter was included in two reviews ( , ) and contributed to the perceived efficacy of cq and hcq, which resulted in regulatory approvals in many countries. the risk of bias associated with a lack of either proper randomization or protection of the allocation sequence in the controlled trials is particularly serious in the case of covid . this is because without an effective cure, and based on the hype about cq/hcq, severely ill patients were more likely to be given the experimental treatment, and consequently have worse outcomes if the treatment was not efficacious. finally, it would be remiss to not mention that the biggest observational study ( ) included in two of the metaanalyses ( , ) was subsequently retracted ( ) . this impacts the findings of these reviews although one of the reviews carried out a sensitivity analysis without this study. the collective impact of these limitations is that even though there are multiple reviews published, there is still uncertainty about the efficacy and safety of cq or hcq with or without second-generation macrolide antibiotics such as azithromycin. the findings of our updated meta-analysis of experimental studies showed no benefit of hcq, with or without azithromycin in reducing the risk of all-cause mortality. our findings are in agreement with findings of two of the included meta-analyses ( , ) but contrast with the two others ( , ) . the two meta-analyses which found a higher risk of mortality had fewer studies and participants than the latter two reviews. singh et al ( ) included three studies with participants from two observational studies ( , ) and the one quasi-experimental study ( ) had a weight of . %, in their meta-analysis. yang et al. ( ) included data from two small trials ( , ) and one observational study, the us veterans study ( ) which had a weight of % in the meta-analysis, and is misclassified as an rct in the meta-analysis. further, all the deaths, except one, occurred in the u.s. veterans' observational study, and therefore the meta-analysis . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . observational studies and only one quasi experimental study which had a weight of . % in the meta-analysis. it is apparent that most of the conclusions from the reviews were driven by data from observational studies. despite the aforesaid, the evidence from our updated meta-analysis and these existing reviews suggests that, at the very least, cq, hcq or cq/hcq+ azithromycin do not have a protective effect against mortality in individuals with covid disease and may be harmful, in the worst-case scenario. from a meta-analysis of one large rct ( ), one small rct ( ) and one quasiexperimental studies ( ) , we found no difference in risk of transfer to the icu, intubation or the need for mechanical ventilation. this finding was in agreement with seven of the included reviews ( , , , , , ) ( ) . in agreement with the included reviews, we did not find any significant effect of hcq, with or without azithromycin, on either virological cure or disease exacerbation but found a -fold increase in risk of adverse events which was expected. it should be noted however, that the occurrence of serious adverse events in the included experimental studies was rare, in agreement with the known safety profile of both cq and hcq. our updated meta-analysis had limitations which include small sample sizes in seven of the eight included trials and high risk of selection bias in the included quasiexperimental study. some of the strengths of this updated meta-analysis include the inclusion of data from experimental studies only and the use of quality effects models to adjust for the weight of the studies in the meta-analysis. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint our findings from the updated meta-analysis suggest that use of hcq (with or without azithromycin) did not result in decreased mortality nor were they any reduction in severe sequela of covid , including transfer to the icu, intubation, mechanical ventilation, virological cure or disease exacerbation. rather use of these drugs are associated with a higher risk of adverse events, mainly gastrointestinal such as vomiting, diarrhea, and nausea. however, serious adverse events are rarely reported. although the available evidence is of heterogeneous quality and, apart from the recovery trial, there are only a few clinical trials with small sample sizes, our findings do not support any further use of either cq or hcq, with or without azithromycin, for the treatment of covid . all the authors declare no conflict of interest . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. 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visually identify publication bias treating covid- with chloroquine hydroxychloroquine in patients with mainly mild to moderate coronavirus disease : open label, randomised controlled trial a pilot study of hydroxychloroquine in treatment of patients with common coronavirus disease- (covid- ) hydroxychloroquine and azithromycin as potential treatments for covid- ; clinical status impacts the outcome effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus (sars-cov- ) infection: a randomized clinical trial empirical treatment with hydroxychloroquine and azithromycin for suspected cases of covid- followed-up by telemedicine clinical outcomes of hydroxychloroquine in hospitalized patients with covid- : a quasi-randomized comparative study open-label, controlled trial to evaluate the efficacy and tolerability of hydroxychloroquine and a retrospective study in adult patients with mild to moderate coronavirus disease hydroxychloroquine for early treatment of adults with mild covid- : a randomized-controlled trial hydroxychloroquine in nonhospitalized adults with early covid- : a randomized trial hydroxychloroquine with or without azithromycin in mild-to-moderate covid- hydroxychloroquine in patients with mainly mild to moderate coronavirus disease : open label, randomised controlled trial treating covid- with chloroquine breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies clinical efficacy of hydroxychloroquine in patients with covid- pneumonia who require oxygen: observational comparative study using routine care data outcomes of hydroxychloroquine usage in united states veterans hospitalized with covid- no funding to report abbreviations covid , sars-cov , hcq, cq, key: cord- -e v ge authors: davoodi, lotfollah; abedi, seyed mohammad; salehifar, ebrahim; alizadeh‐navai, reza; rouhanizadeh, hamed; khorasani, ghasemali; hosseinimehr, seyed jalal title: febuxostat therapy in outpatients with suspected covid‐ : a clinical trial date: - - journal: int j clin pract doi: . /ijcp. sha: doc_id: cord_uid: e v ge background: the aim of this clinical trial was to evaluate the effects of febuxostat (fbx) in comparison with hydroxychloroquine (hcq) on clinical symptoms, laboratory tests and chest ct findings in outpatients with moderate symptoms of covid‐ infection. methods: we conducted a clinical trial involving adult outpatients with the moderate respiratory illness following covid‐ infection. patients were randomly assigned to receive either fbx or hcq for days. the measured variables were needs to hospitalization, clinical and laboratory data including fever, cough, breathing rate, c‐reactive protein level, lymphocytes count at onset of admission and was well as at days of treatments. in addition, ct findings were evaluated on admission and days after initiation of treatment. results: sixty subjects were enrolled in the study with a to ratio in fbx and hcq groups. on admission, fever ( . %), cough ( %), tachypnea ( . %), dyspnea ( %), elevated crp value ( . %) and lung involvement according to chest ct ( %) were documented in enrolled patients with insignificant difference between fbx and hcq groups. fever, cough and tachypnea were significantly mitigated in both groups after five days of treatments without any significant differences between groups. the mean percentages of lung involvement were significantly reduced to . % and % after days of treatment with fbx and hcq, respectively. in adult outpatients with moderate covid‐ infection, the effectiveness of fbx and hcq was not different in terms of resolution of clinical manifestations, laboratory tests and lung ct findings. conclusion: this trial suggests that fbx is as an alternative treatment to hcq for covid‐ infection and may be considered in patients with a contraindication or precaution to hcq. novel non-purine xanthine oxidase (xo) approved for treating hyperuricemia in patients with gout. several studies have already demonstrated the anti-inflammatory [ ] , anti-oxidant [ ] and antiapoptosis effects of fbx [ ] . several preclinical studies showed that fbx inhibits inflammatory responses through reducing the levels of pro-inflammatory mediators such as tumor necrosis factor (tnf)-α, interleukin (il)- β, il- and nf-κb [ ] [ ] [ ] . it protects animal against toxic-induced lung inflammation through downstream inflammatory mediators and oxidative stress [ ] [ ] [ ] . fbx markedly accelerates pulmonary endothelial barrier recovery and improves survival in lipopolysaccharide-induced murine sepsis [ ] . this clinical trial was conducted to assess the effects of fbx and hydroxychloroquine (hcq) on clinical symptoms, laboratory tests and chest ct findings of patients with covid- infection. irct n , the full trial protocol can be accessed at: http://www.irct.ir). the study was performed in accordance with declaration of helsinki. all patients signed the informed consent form. sample size was determined patients in both group based on effect size = . for difference in response rate as a primary endpoint, power = % and alpha = . for this study, as a two-sided superiority trial [ , ] . inclusion criteria were as following ; chest ct finding compatible with covid- infection along with other symptoms of coronavirus infection. bilateral and peripheral ground-glass and consolidative pulmonary opacities were the hallmarks of ct findings. ; any symptoms of respiratory tract accepted article involvement including cough, dyspnea or tachypnea along with a history of contact with a known case of covid- ; creatinine clearance greater than ml/min. the exclusion criteria include: ; suspicious patients for covid- pneumonia who had severe underlying diseases such as cardiovascular, lung and kidney diseases, ; patients with severe pneumonia needing hospitalization, ; patient who were unable to take oral medications and ; concurrent use of azathioprine, didanosine, mercaptopurine or pegloticase (due to drug interaction with fbx). patients were randomized using the balance block method to receive hcq ( patients) or fbx ( patients). hcq were administered one tablet of hcq mg twice daily (amin pharmaceutical company, iran). patients in fbx group took one tablet of fbx mg per day (jalinus pharmaceutical company, iran). all patients were taken acetaminophen mg, as needed, for controlling the fever. the pharmaceutical companies were neither involved in the design nor in the financial support of the study. study drugs were purchased from an official iranian pharmacy. amin and jalinus pharmaceutical companies did not access to the data of the study during trial and prior publication. the treatment duration was five days. both patients and physician did not know the contents of tables. the primary outcome of this study was the rate of hospitalization. secondary outcomes were clinical improvements (e.g., resolution of fever, cough and dyspnea) and improvement of ct findings at days after initiation of the treatment. patients were assessed clinically (e.g., temperature, respiratory rate, cough, and dyspnea) and paraclinically (e.g., cbc-diff and c-reactive protein) at onset of admission and th day of treatment. in addition, the chest ct scans were done at first and days after the onset of treatment. for each patient, the chest ct scan was evaluated for the presence of groundglass opacities and/or consolidation. each five lobe of the lung was assessed and the overall lung involvement was reached by summing the five lobe scores (range of possible scores, - for each lobe and total lung involvement of possible score of - percent). the chest ct was repeated in day and compared with the initial finding. reduced lung ct involvement; not adjusted" values were computed according to this equation: reduced lung ct involvement, not adjusted = day total long involvement -initial total lung involvement reduced lung ct involvement; adjusted values was computed with the following equation that included the initial total lung involvement in the denominator: reduced lung ct involvement; adjusted value = (initial total long involvement -day total long involvement) initial total lung involvement × normality of data was checked with shapiro-wilk test. independent sample t-test and mann-whitney u test (comparison of continuous variables between two groups), wilcoxon matched-pair signed-rank test (comparison of continuous variables before and after treatment), and chi test (comparing the qualitative data) were used for analysis. the method of analysis was intention-to-treat. the spss software version . (spss, inc., chicago, il) was applied for statistical analysis. sixty subjects were enrolled including fbx (n = ) and hcq groups (n = ) ( figure ). six patients ( patient in fbx group and patients in hcq group) were excluded, because patients were not interest to continue the treatment ( figure ). this article is protected by copyright. all rights reserved documented by clinical manifestations and ct findings. there were not between-group significant differences in baseline demographic characteristics, laboratory data (e.g., crp, wbc and lymphocytes counts) and ct scores of lung lesions (table ) . the rate of hospitalization, the primary endpoint of the study, was nor different among groups. six patients ( %, patients in each group) were hospitalized because of developing more severe symptoms. the rate of intensive care unit (icu) care and also mortality rate was not different between patients received fbx or hcq. all hospitalized patients were released from hospitals between to days of hospitalization. patients were re-evaluated at five days after admission and using fbx or hcq. fever, cough and tachypnea significantly mitigated in patients treated with either fbx or hcq after five days of treatment (p< . compared to baseline of each group) ( this article is protected by copyright. had severe disease and % needed critical care [ ] . in italy, approximately % of patients have been hospitalized, whereas nearly % admitted to icu [ ] . there is significant variation in the rate of hospitalization of patients with covid- in the world. however, sample size of our clinical trial was smaller and patients with significant comorbidities such as sever cardiovascular and renal diseases were excluded in our trial. clinical symptoms such as fever, cough and shortness of breath were observed in a large proportion of patients at admission, but these manifestations markedly reduced or resolved (e.g., fever and dyspnea) after days following use of fbx or hcq. it was not observed any statistically difference in mitigating of clinical symptoms between fbx and hcq treatments. low lymphocyte count has been consistently reported in patients with covid- infection (in % of cases) and may indicate the severity of disease and serve as a predictor of prognosis [ ] . more than half of patients show elevated values of crp. patients with severe disease had more prominent laboratory abnormalities than those with non-severe disease [ ] . in our study, lymphopenia was observed in . % of patients at onset of admission and significantly increased after this article is protected by copyright. all rights reserved carriage of sars-cov- in covid- patients were evaluated. the clinical symptoms, laboratory tests and lung ct features were reported to be similar in both groups of patients [ ] . in other study, gautret, et al conducted an uncontrolled non-comparative observational study in a cohort of mildly infected inpatients treated with a combination of hcq and azt over a period of at least three days. a rapid fall of nasopharyngeal viral load tested by qpcr was noted, with % negative at day , and % at day . authors concluded that they have provided evidence of a beneficial effect of coadministration of hcq with azt in the treatment of covid- and its potential effectiveness in the early stages of contagiousness [ ] . to date, despite some promising results associated with efficacy and safety of hcq in covid- , the evidence regarding its effect remains limited [ ] . infrequent and rare side effects include retinal toxicity, cardiac toxicity, qt interval prolongation and agranulocytosis have been reported in patients receiving hcq or cq. life threatening arrhythmias following use of cq and hcq appear to be rare but cardiac monitoring is necessary if the drug is being used more extensively [ , ] . hcq inhibits il- , tnf-α, il- β and nf-κb [ ] [ ] [ ] [ ] [ ] , and it has immunomodulatory and anti-inflammatory effects [ , , ] , which may be beneficial in patients with covid- who inflammatory response and storm cytokine production plays a major role in damaging the lung tissue [ ] . there are complex interactions between inflammation and thrombosis, that inflammation is causing a thrombotic tendency. the suspected contribution of thrombotic events to morbidity and mortality in covid- patients, it is recommended to use medicine with anti-inflammatory and antithrombotic properties for prevention or management of thrombosis in covid- [ ] . recently in the recovery trial found that use of dexamethasone was associated with mortality benefit in patients with severe form of covid- infection [ ] . nf-κb plays a central role in inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, and autoimmunity. nf-κb is activated through microbial products and also pro-inflammatory cytokines, as well as endogenous ligands that function as its trigger during tissue injury, the latter of which may promote inflammation in the absence of infection [ ] [ ] [ ] . in addition, viral infections cause nf-κb overexpression, which plays a crucial role in the production of pro-inflammatory cytokine storms and triggers various cellular responses including cell phagocytosis, dendritic cell maturation, chemotaxis and lipopolysaccharide-induced pulmonary inflammation [ , , ] . it seems that downregulation of nf-κb results in the attenuation of inflammatory cytokine signaling and this article is protected by copyright. all rights reserved may be a promising target for lung protection [ ] . fbx is in a class of medications called xanthine oxidase (ox) inhibitors leading to decrease in uric acid production. beside decreasing serum uric acid in gout by fbx, there are well documents demonstrating fbx suppresses pro-inflammatory cytokines such as il- β, il- mcp- and tnf-α as well as inhibits the oxidative stress and inflammatory responses through nf-κb pathway in animal models [ , , , ] . fbx is able to improve lung damage induced by toxic agents through down-regulation of oxidative stress pathway and suppression of inflammatory mediators [ , , , ] . to date, there is not any report associated to anti-viral activity of fbx. xanthine oxidase, which is responsible for the generation of oxygen free radicals, was elevated in serum and lung tissue of mice infected with influenza virus [ ] [ ] [ ] . due to the crucial roles of cytokines and pro-inflammatory mediators in severe acute respiratory syndrome induced by covid- , with respect to beneficial effects of fbx in blockading the activation of cytokines and nf-κb pathway, this medicine seems to be an effective drug for the prevention and treatment of lung inflammation in patients with covid- insignificant efficacy with hcq. however, further studies are needed to find the exact mechanism of fbx in treatment of covid- infection. the adverse effects associated with fbx therapy include nausea, diarrhea, arthralgia, headache, increased hepatic serum enzyme levels, rash and cardiovascular problems. these side effects were not observed in our study, which may be due to the short time consuming of fbx. it is also notable that we excluded patients with cardiovascular and chronic kidney diseases. the limitation of our study was the absence of placebo group. considering ethical issues, we designed this study without a placebo group due to the life-threatening nature of covid- infection. our trial suggests a beneficial effect of administration of fbx in patients with suspected mild-tomoderate covid- infection. the effects of fbx and hcq was not different in terms of need to hospitalization, improvement in clinical symptoms and ct findings pointing out the lung involvement. fbx may be considered in patients who are not a good candidate of hcq due to underlying cardiovascular diseases. this article is protected by copyright. all rights reserved this study was supported by a grant from mazandaran university of medical science, sari, iran (id# ). we thank all patients who participated in this trial and their families. we thank adel heidari for assistance in data gathering. we appreciated the health care staffs who lost their lives in the care of patients with covid- . the data that support the findings of this study are available on request from the corresponding author. the data are not publicly available due to privacy or ethical restrictions. there were not any significant differences between two groups in baseline demographic and clinical characteristics &: indicate the lung ct data on day compared to day ; p-value for between groups differences were not significant for any of variables both on day and day . clinical characteristics of hospitalized patients with evaluation and treatment coronavirus (covid- ) dysregulation of immune response in patients with covid- in wuhan, china accepted article this article is protected by copyright. all rights reserved coronavirus infections and immune responses the xanthine oxidase inhibitor febuxostat reduces tissue uric acid content and inhibits injury-induced inflammation in the liver and lung protective effects of febuxostat against paraquatinduced lung toxicity in rats: impact on rage/pi k/akt pathway and downstream inflammatory cascades febuxostat pretreatment attenuates myocardial ischemia/reperfusion injury via mitochondrial apoptosis febuxostat attenuates ulcerative colitis by the inhibition of nf-kappab, proinflammatory cytokines, and oxidative stress in mice effects of febuxostat on serum cytokines il- febuxostat modulates mapk/nf-kappabp /tnf-alpha signaling in cardiac ischemia-reperfusion injury protective effects of febuxostat against paraquatinduced lung toxicity in rats: impact on rage/pi k/akt pathway and downstream inflammatory cascades xanthine oxidase contributes to sustained airway epithelial oxidative stress after scald burn febuxostat protects rats against lipopolysaccharide-induced lung inflammation in a dose-dependent manner accepted article this article is protected by copyright. all rights reserved xor inhibition with febuxostat accelerates pulmonary endothelial barrier recovery and improves survival in lipopolysaccharide-induced murine sepsis hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial efficacy of hydroxychloroquine in patients with covid- : results of a randomized clinical trial characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention covid- in italy: momentous decisions and many uncertainties lymphopenia predicts disease severity of covid- : a descriptive and predictive study china medical treatment expert group for c. clinical characteristics of coronavirus disease in china breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies health commission of guangdong province for chloroquine in the treatment of novel coronavirus p chloroquine and hydroxychloroquine as available weapons to fight covid- clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in covid- patients with at least a six-day follow up: a pilot observational study should chloroquine and hydroxychloroquine be used to treat covid- ? a rapid review chloroquine and hydroxychloroquine in the era of sars -cov : caution on their cardiac toxicity off-label prescribing in the midst of a pandemic: the case of hydroxychloroquine increased myeloid dendritic cells and tnf-alpha expression predicts poor response to hydroxychloroquine in cutaneous lupus erythematosus hydroxychloroquine inhibits proinflammatory signalling pathways by targeting endosomal nadph oxidase hydroxychloroquine alleviates -fluorouracil-induced enteritis in mice and its mechanism accepted article this article is protected by copyright. all rights reserved hydroxychloroquine decreases th -related cytokines in systemic lupus erythematosus and rheumatoid arthritis patients cholesterolmodified hydroxychloroquine-loaded nanocarriers in bleomycin-induced pulmonary fibrosis the pharmacological mechanisms and therapeutic activities of hydroxychloroquine in rheumatic and related diseases mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology pharmacological agents targeting thromboinflammation in covid- : review and implications for future research recovery trial: the uk covid- study resetting expectations for clinical trials nf-kappab, inflammation, and metabolic disease the nuclear factor nf-kappab pathway in inflammation the role of nf-kb inhibitors in cell response to radiation accepted article this article is protected by copyright. all rights reserved barbaloin protects against lipopolysaccharide (lps)-induced acute lung injury by inhibiting the rosmediated pi k/akt/nf-kappab pathway osthole protects against acute lung injury by suppressing nf-kappab-dependent inflammation acute induction of inflammatory cytokine expression after γ-irradiation in the rat: effect of an nf-κb inhibitor use of xanthine oxidase inhibitor febuxostat inhibits renal interstitial inflammation and fibrosis in unilateral ureteral obstructive nephropathy febuxostat ameliorates doxorubicin-induced cardiotoxicity in rats the xanthine oxidase inhibitor febuxostat reduces tissue uric acid content and inhibits injury-induced inflammation in the liver and lung febuxostat protects rats against lipopolysaccharide-induced lung inflammation in a dose-dependent manner oxygen radicals in influenzainduced pathogenesis and treatment with pyran polymer-conjugated sod protective effect of n-acetylcysteine in a model of influenza infection in mice accepted article the authors declare no potential conflicts of interest with respect to authorship, and/or publication of this study. key: cord- -tsd npma authors: wilde, a. a. m.; offerhaus, j. a. title: the ‘president’s drug’ date: - - journal: neth heart j doi: . /s - - -x sha: doc_id: cord_uid: tsd npma nan the ongoing sars-cov- pandemic and the associated lung disease covid- have led to unprecedented apprehension worldwide. this has resulted in equally unprecedented preventive measures mandated by national governments as well as to an explosion of scientific activity in the search for, among many aspects of the disease, preventive therapy. central to this search for preventive therapy is the repurposing of chloroquine (cq) and hydroxychloroquine (hcq), a traditional antimalarial drug (cq) and a drug (hcq) also used in the treatment of systemic lupus erythematosus (sle) and rheumatoid arthritis (ra). these drugs have surfaced as a potential effective treatment option based on reasonable experimental data but poorly conducted (first) clinical trials. despite an unconfirmed efficacy and potential serious side-effects the presidents of france, brazil and the united states subsequently publicly promoted the use of hcq, resulting in an absolute run on hcq, a shortage of the drug for patients with traditional indications, and a lively debate in the respective countries [ , ] . cardiac side-effects of these drugs have been known for years but have been considered mild. one potentially lethal side-effect is qtc prolongation, caused by blocking of the human ether-a-go-go-related gene (herg) potassium channel, which results in a prolonged action potential duration [ ] . due to the potentially lethal nature of this side-effect, electrocardiogram (ecg) monitoring in patients treated with these drugs for malaria, sle or ra has been proposed in the past, but is currently not standard practice [ ] . actually, cq is estimated to be one of the drugs to which human beings are exposed most [ ] . yet, cq and hcq are listed on the website www. crediblemeds.org in the category 'known risk of causing torsades de pointes'. however, these cases are rare and mostly associated with an (intentional) overdose or in combination with other qtc-prolonging factors (e.g. other drugs, hypokalaemia) and, therefore, cq and hcq are generally considered safe with only relatively minor qtc prolongation [ , ] . yet, a recent survey of new user cohort studies ( - ) in almost a million hcq users revealed slight excess cardiovascular mortality most likely due to sudden death [ ] . although initial reports, on studies with serious methodological flaws and small sample sizes, seemed to show a beneficial effect on the disease course [ ] [ ] [ ] , evidence is accumulating that in hospitalised patients hcq is not effective in reducing mortality [ , ] or faster virus elimination [ ] . in fact, one retrospective study comprising patients found an increased risk of mortality for hcq, although this could be the result of baseline dissimilarities between the intervention and the control group [ ] . this negative effect does not necessarily mean that cq or hcq are useless in the setting of a sars-cov- infection. it is quite conceivable, based on their demonstrated in vitro efficacy, that treatment in earlier stages of infection might be beneficial. this reasoning underlies the many randomised trials that have been proposed and initiated, also focusing on prevention of infection (i.e. pre-exposure), mainly in healthcare workers, and post-exposure shortly after the onset of disease symptoms [ ] . in two studies in this issue of this journal, the effect of cq was studied in hospitalised patients with emphasis on the main side-effect of the drug, i.e. pro-the 'president's drug' longation of the qtc interval. outcome data were not included in these reports. in the study by van den broek et al., patients were treated with cq (loading dose mg, followed by × mg for days; % of patients in the intensive care unit), which resulted in a mean qtc prolongation of ms ( % confidence interval - ms) [ ] . in patients ( %) the qtc interval exceeded ms, which is generally regarded as the value at which to stop all qt-prolonging medication. interestingly, in addition to qtc prolongation, the authors also detected pr and qrs prolongation, a known effect of cq [ ] . this indicates a decrease in conduction, which is also potentially pro-arrhythmic by favouring re-entrant circuits. in the study by sinkeler et al. hospitalised patients were treated with cq (same dosage regimen) [ ] . after - h the qtc had increased by ms (± ms, mean ± sd); a second ecg in a subset of patients revealed a further increase to ± ms. in ± % of patients the qtc interval exceeded ms and/or the increase in qtc was more than ms. one patient developed a non-sustained ventricular tachycardia. although in both studies serum potassium levels were measured and the use of additional qtc-prolonging drugs was recorded, details are not given in relation to the qtc prolongation. also, in both studies the computer-measured ecg overestimated the qtc interval, so in patients where clinical decision-making depends on the qtc interval a manual measurement is mandatory. these results are in line with other published data summarised in a recent review [ ] . up to % of patients develop qtc prolongation into the range ≥ ms with cq-hcq monotherapy and slightly more when azithromycin is added [ ] . however, only rarely does a patient develop torsades de pointes; a nice example is described by szekely et al. [ ] . specific subgroups at high risk are recognised and should undergo extra-intense ecg monitoring [ ] . an important finding of both studies and other studies on this topic is that covid- patients are apparently much more sensitive to cq-or hcq-related qtc prolongation than patients with more conventional indications. this is potentially explained by a number of factors which may contribute to the qt prolongation. in hospitalised covid- patients there is an exaggerated immune response, resulting in high levels of cytokines, including interleukin , which has been shown to prolong repolarisation [ ] . furthermore, hypoxia may augment the late sodium inward current, and subsequently prolong the action potential duration [ ] . in addition, sick patients may use other qt-prolonging drugs, including the above-mentioned drug azithromycin, and may present with hypokalaemia. finally, genetic factors particularly present in black african individuals may predispose them to accumulation of all these effects [ ] . the hypothesis that qt-prolonging factors, as mentioned above, are imperative in covid- patients is underscored by the fact that the baseline mean qtc interval in covid- patients as presented in both dutch studies [ , ] is much longer (in the range - ms) than in studies with volunteers ( - ms) [ ] . how should we proceed? clearly it is a bad idea to provide every citizen with either cq or hcq before more information on their efficacy is known. instead, large-scale, randomised, double-blinded trials are the highest priority in order to prove the efficacy of either drug [ ] . these trials should focus on mildly affected patients early after disease onset in order to demonstrate whether these drugs can cure an early infection with sars-cov- . secondly, similar studies should be performed in individuals with a high level of exposure (e.g. healthcare workers) to demonstrate whether these drugs are effective in preventing an infection with sars-cov- . in both groups careful ecg monitoring is warranted to prevent excessive qtc prolongation and, with longer treatment in the preexposure group, conduction disturbances. for these prophylactic studies the same motto applies as for the aforementioned presidents: 'first, do no harm' [ ] . open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/by/ . /. finding effective treatments for covid- . scientific integrity and public confidence in a time of crisis inhibition of herg k+ currents by antimalarial drugs in stably transfected hek cells cardiac complications attributed to chloroquine and hydroxychloroquine: a systematic review of the literature randomized doseranging controlled trial of aq- , a candidate antimalarial, and chloroquine in healthy volunteers safety of hydroxychloroquine, alone and in combination with azithromycin, in light of rapid wide-spread use for covid- : a multinational, network cohort and self-controlled case series study chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label nonrandomized clinical trial efficacy of hydroxychloroquine in patients 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late na(+) current: proarrhythmic effects, roles in long qt syndromes, and pathological relationship to camkii and oxidative stress genetic susceptibility for covid- -associated sudden cardiac death in african americans covid- , clinical trials and qt-prolonging prophylactic therapy in healthy subjects: first, do no harm key: cord- -xc bxin authors: rolain, jean-marc; colson, philippe; raoult, didier title: recycling of chloroquine and its hydroxyl analogue to face bacterial, fungal and viral infections in the st century date: - - journal: int j antimicrob agents doi: . /j.ijantimicag. . . sha: doc_id: cord_uid: xc bxin chloroquine (cq) and its hydroxyl analogue hydroxychloroquine (hcq) are weak bases with a half-century long use as antimalarial agents. apart from this antimalarial activity, cq and hcq have gained interest in the field of other infectious diseases. one of the most interesting mechanisms of action is that cq leads to alkalinisation of acid vesicles that inhibit the growth of several intracellular bacteria and fungi. the proof of concept of this effect was first used to restore intracellular ph allowing antibiotic efficacy for coxiella burnetii, the agent of q fever, and doxycycline plus hcq is now the reference treatment for chronic q fever. there is also strong evidence of a similar effect in vitro against tropheryma whipplei, the agent of whipple's disease, and a clinical trial is in progress. other bacteria and fungi multiply in an acidic environment and encouraging in vitro data suggest that this concept may be generalised for all intracellular organisms that multiply in an acidic environment. for viruses, cq led to inhibition of uncoating and/or alteration of post-translational modifications of newly synthesised proteins, especially inhibition of glycosylation. these effects have been well described in vitro for many viruses, with human immunodeficiency virus (hiv) being the most studied. preliminary in vivo clinical trials suggest that cq alone or in combination with antiretroviral drugs might represent an interesting way to treat hiv infection. in conclusion, our review re-emphasises the paradigm that activities mediated by lysosomotropic agents may offer an interesting weapon to face present and future infectious diseases worldwide. cq is a -aminoquinoline known since , which emerged during the first part of the th century as an effective quinine substitute and the drug of choice against malaria [ ] . it proved to be among the most successful antimalarial drugs on a worldwide scale owing to its wide deployment coinciding with the geographical distribution of plasmodium and its high intrinsic antiparasitic efficacy and low toxicity. concomitant with a gradual decrease in its use for therapy and prophylaxis of plasmodium-induced disease worldwide, related to the emergence of cq-resistant parasites, cq and its hydroxyl analogue hcq have gained interest in the field of other infectious diseases [ ] . the mechanism of action of cq is multiple, differing according to the pathogen, and has not been well elucidated for all microorganisms. cq and hcq enter cells as non-protonated forms where they become protonated according to the henderson-hasselbach law, i.e. in a manner inversely proportional to the ph [ ] . they therefore concentrate within acidic organelles, including endosomes, lysosomes and golgi vesicles, in which they increase the ph [ ] . over the last decade, two main mechanisms of action of cq have been well described, i.e. alkalinisation of acid vesicles in cells infected by intracellular bacteria and fungi, and alteration of post-translational modifications of newly synthesised proteins in cells infected by viruses. the proof of concept of the use of cq as an anti-infectious agent, other than an antimalarial agent, has been fully demonstrated for the first time in vitro and in vivo with the model of chronic q fever. this concept was initially based on cellular biology findings, mainly by manipulation of the ph of acidic vacuoles where coxiella burnetii, the agent of q fever, live and multiply. demonstration of a negative effect on growth of c. burnetii by lysosomotropic agents [ ] was the first step, followed by demonstration that alkalinising c. burnetii-containing vacuoles could restore the intracellular activity of antibiotics ( fig. ) [ ] . this paradigm was later used to demonstrate the clinical efficacy of an association of doxycycline and hcq in the treatment of chronic q fever endocarditis and this regimen is now the reference treatment and is to date the only model of confirmed clinical efficacy ( fig. ) [ ] . there is strong evidence of such an effect in other intracellular bacteria, especially for tropheryma whipplei, the agent of whipple's disease, which multiply in phagosomes, since agents that increase the intravacuolar ph decrease bacterial viability [ ] . by analogy with c. burnetii, hcq restores the intracellular activity of doxycycline in vitro [ ] and a clinical trial using this regimen is under evaluation. several other bacteria and fungi live and multiply in acidic vacuoles and preliminary in vitro data are encouraging for the usefulness of cq in such infections. for viruses, cq led to an inhibition of low-ph-dependent entry steps or alteration of post-translational modifications of newly synthesised proteins, especially via inhibition of glycosylation. these effects have been well described in vitro for many viruses, with human immunodeficiency virus type- (hiv- ) being the most studied. moreover, preliminary in vivo clinical trials have suggested that cq alone or in combination with antiretroviral drugs might represent an interesting way to treat hiv infection. here we review available in vitro and in vivo data on the effects of cq/hcq on bacterial, fungal and viral infections, with the concept that manipulation of the intracellular ph in cells and modification of glycosylation of proteins by lysosomotropic agents instead of antimicrobial compounds is a powerful approach as new therapeutic strategies for the prevention and therapeutic management of several infectious diseases, including some of great public health concern worldwide. cq/hcq also have anti-inflammatory properties, however these will not be discussed in this review. the intracellular location of several bacteria and fungi has been known for decades as a critical point to explain failure of antibiotic treatment to eradicate these pathogens from host cells [ ] . intracellular pathogens evade the first-line antimicrobial defence, which includes attack by phagocytes (fig. ). after being internalised by the cell, usually there is formation of a phagosome that rapidly fuses with lysosomes. the bacteria are then killed by oxygen-dependent and oxygenindependent killing mechanisms, which leads to acidification of the phagolysosome (ph . ) and acidic activation of lysosomal enzymes. intracellular pathogens may evade this lysosomal pathway by several mechanisms: (i) lifestyle in lysosome free-cells such as erythrocytes (bartonella spp.); (ii) escape from the phagosome before fusion with lysosomes and multiplication in the cytosol (rickettsia and shigella) (fig. ) ; (iii) inhibition of phagolysosomal fusion and multiplication in the phagosome (chlamydia, ehrlichia, legionella, salmonella, yersinia, brucella, mycobacterium, francisella, histoplasma capsulatum and aspergillus fumigatus) (fig. ) ; and (iv) survival and multiplication in phagolysosomes (c. burnetii, t. whipplei, staphylococcus aureus, candida albicans and cryptococcus neoformans) (fig. ) [ , , ] . there are many arguments suggesting that the low ph environment within phagosomal compartments of the cell is critical for many intracellular pathogens to access cellular iron for growth [ , ] . cq treatment of different cells, including macrophages, could inhibit the growth of several of these intracellular bacteria by neutralising the phagolysosomal ph (table ; figs. and ). thus, two main mechanisms can explain intracellular bacterial inhibition by cq: ph-dependent iron deprivation [ ] ; and direct toxicity by increasing the phagolysosomal ph, which is harmful for the growth of several intracellular pathogens such as c. burnetii [ ] and t. whipplei [ ] . coxiella burnetii is the agent of q fever and is a strict intracellular bacterium that is able to survive in phagolysosomes where a low ph (ph . ) is necessary for its metabolism [ , , ] . q fever includes acute manifestations (mainly pneumonitis and hepatitis) and chronic forms (mainly endocarditis) [ ] . usually a regimen of doxycycline mg per day for weeks is recommended for patients with acute q fever. presently, this treatment of acute q fever is not sufficient to prevent the development of chronic q fever [ ] . thus, reliable antibiotic therapy for chronic infection is a more challenging problem since antibiotics are not bac-tericidal in vitro against c. burnetii [ ] . when the ph of c. burnetii-containing phagolysosomes was raised using basic lysosomotropic agents such as cq, methylamine and ammonium chloride, bacterial multiplication was inhibited, showing a direct negative effect on growth by lysosomotropic agents, including cq [ , ] . an original killing assay model developed by maurin et al. [ ] demonstrated that doxycycline, pefloxacin and rifampicin did not show any significant bactericidal activity. conversely, it was shown that in vitro intracellular antibiotic activity was restored and was correlated with modification of the ph by lysosomotropic agents (fig. ) . the lack of bactericidal activity was probably due to inactivation by the low ph of the phagolysosomes in which c. burnetii survives. addition of a lysosomotropic alkalinising agent, i.e. cq, to antibiotics improved the activities of doxycycline and pefloxacin, which then became bactericidal [ , ] . the model in which bacteria actively multiply only at acidic ph is a good paradigm to demonstrate that lack of bactericidal effect of antibiotics is due to intraphagolysosomal acidity. these in vitro findings have now been evaluated in many in vivo studies of the treatment of patients with chronic q fever endocarditis using a combination of doxycycline and hcq (at g/ml in serum) for - months. indeed, prior to this treatment, patients were treated with a long-term table bacteria, fungi and viruses inhibited by chloroquine and/or hydroxychloroquine (in vitro data) reference fungi reference virus reference coxiella burnetii [ , ] histoplasma capsulatum [ ] hiv [ , [ ] [ ] [ ] [ ] ] tropheryma whipplei [ , ] cryptococcus neoformans [ , ] sars-cov [ , ] legionella pneumophila [ ] paracoccidioides brasiliensis [ ] influenza viruses [ ] [ ] [ ] [ ] francisella tularensis [ ] penicillium marneffei [ , ] flavivirus, including yellow fever virus [ ] mycobacterium tuberculosis [ ] aspergillus fumigatus [ ] rubella virus [ tetracycline and quinolone regimen for at least years, with a high percentage of relapses [ ] . this regimen was compared with a combination of doxycycline and hydroxychloroquine sulphate in patients suffering from q fever endocarditis [ ] . of patients treated with a doxycycline and quinolone com- bination, died, relapsed ( were re-treated and switched to the new regimen), was still being treated and were considered cured using this regimen only. the mean duration of therapy for cure in this group was months (median months) [ ] . twenty-one patients received the doxycycline and hcq regimen: patient died of a surgical complication, were still being treated, were cured and was currently being evaluated. two patients treated for months but none of the patients treated for > months relapsed [ ] . the mean duration of treatment in this group was months (median months). this regimen allowed a reduction in the duration of therapy to months for many patients and also reduced the relapse rate to < % [ ] . this regimen is now the current therapy for the treatment of chronic q fever. cq used at therapeutic dosages may have some deleterious effects, including the risk of retinopathy, necessitating a regular ophthalmological examination [ ] . cq levels in serum should be monitored to ensure that they are maintained at ± . mg/l. similarly, an hiv-infected patient with q fever endocarditis was successfully treated with valvular replacement and a combination of doxycycline and hcq [ ] . this combined treatment is probably also indicated in cases of c. burnetii vascular graft infection, as reported recently [ ] . similarly, a retrospective study of patients diagnosed as having q fever during - evaluated the risk of developing endocarditis according to the regimen of antibiotics given to the patients [ ] . when these regimens were compared, ( %) of patients who did not receive treatment developed a chronic infection, of developed a chronic infection when receiving doxycycline alone for weeks to months, and none of the who received doxycycline and hcq for - months developed chronic infection. the regimen containing hcq was found to be significantly superior in preventing q fever endocarditis compared with doxycycline alone (p = . ) [ ] . no significant differences were found between treatment with doxycycline alone and no treatment [ ] . it is now established that development of q fever endocarditis may be prevented by searching for minor valvulopathies with echocardiography following diagnosis of acute q fever [ ] and by treatment with a combination of doxycycline and hcq for year [ ] . finally, four cases of q fever osteoarticular infection (two tenosynovitis and two spondylodiscitis complicated by paravertebral abscess) were eventually cured using the combination of doxycycline and hcq [ ] . whipple's disease was invariably fatal before the advent of antibiotics. however, current therapeutic recommendations are not based on therapeutic trials or adjusted according to the susceptibility of t. whipplei to various antimicrobial agents, since the bacteria was only isolated in [ ] . following isolation of the bacteria, it has been shown that vacuole acidification is critical to the survival of t. whipplei in phagosomes, since agents that increase the intravacuolar ph decrease bacterial viability ( fig. ) [ ] . by analogy with c. burnetii, we have demonstrated that doxycycline alone was not bactericidal against t. whipplei in an in vitro cell model and that alkalinisation with hcq may restore activity [ ] . a regimen based on this observation (doxycycline and hcq) has thus far been the only successful bactericidal regimen against t. whipplei in vitro. whether this regimen will work in a general clinical setting remains to be established, but it has been successful in four of our patients: two with classic whipple's disease and two with blood culture-negative endocarditis [ ] . staphylococcus aureus is a facultative intracellular bacterium that resides within phagolysosomes [ ] [ ] [ ] [ ] . the intracellular location of certain strains of s. aureus serves as a reservoir of bacteria that is thought to be important in therapy of recurrent infections in humans and in chronic staphylococcal mastitis in dairy cows [ ] . although aminoglycosides are bactericidal for extracellular staphylococci, they are ineffective in reducing the intracellular form of the microorganism [ ] . it was hypothesised that diminished susceptibility of intracellular s. aureus may be related to the acidic ph within phagolysosomes [ ] . it has been demonstrated that alkalinising s. aureus-containing vacuoles could restore the intracellular activity of aminoglycosides [ ] . furthermore, intracellular killing of s. aureus correlated well with increased lysosomal ph due to lysosomotropic alkalinising agents [ ] . recently, it has been demonstrated that cq and ammonium chloride significantly enhanced intracellular killing by levofloxacin [ ] . the bactericidal activity of levofloxacin was partially restored when the ph was neutralised from . to . [ ] . the bactericidal activity of moxifloxacin, abolished in the intracellular salt medium, was partially restored when the ph was raised from . to . [ ] . similarly, alkalinisation of phagolysosomes significantly enhanced intracellular killing by moxifloxacin [ ] . in a model of bovine mastitis due to s. aureus, it has been clearly demonstrated that low intraphagolysosomal ph affects the ability of an antibiotic to kill intracellular bacteria, since the activity of rifampicin was enhanced at ph . [ ] . similar reasoning probably explains why rifampicin, which both penetrates within eukaryotic cells [ ] and is more active at acidic ph [ ] , displays bactericidal activity against intracellular s. aureus [ ] . legionella pneumophila is a strict intracellular bacterium that multiplies in human mononuclear phagocytes and is responsible for legionnaire's disease [ ] . cellular iron metabolism is of critical importance to l. pneumophila since its multiplication is dependent upon the availability of intracellular iron. it has been demonstrated that cq and ammonium chloride inhibit the intracellular multiplication of l. pneumophila by limiting the availability of iron to the bacterium [ ] . thus, cq may interfere with intracellular iron metabolism by recycling iron from ferritin by blocking degradation of ferritin by acid proteases [ ] . francisella tularensis bv. tularensis and f. tularensis bv. palearctica are facultative intracellular bacteria responsible for tularaemia. it has been demonstrated that f. tularensis finds a successful niche for replication in an acidified vacuole where iron is concentrated [ ] . growth of f. tularensis in murine macrophages has been shown to be dramatically inhibited in vitro by cq in a dose-dependent manner [ ] . intracellular localisation in an acidic vesicle, which facilitates the availability of iron essential for francisella growth, is a survival tactic of this bacterium and iron depletion is one mechanism that macrophages use to inhibit its growth [ ] . cq has been reported to inhibit the intracellular multiplication of mycobacterium tuberculosis both in human monocyte-derived macrophages and mouse peritoneal macrophages [ ] . it seems possible that cq inhibits m. tuberculosis intracellular multiplication by raising intracellular ph and limiting the availability of iron to this bacterium, as it does for l. pneumophila. similarly, it has been demonstrated that addition of cq results in a significant reduction of the intracellular growth of mycobacterium avium in bone marrow-derived macrophages [ ] . in vitro activity of cq and/or hcq has been demonstrated for other bacteria, including salmonella enterica serovar typhi, escherichia coli, bacillus anthracis, bacillus subtilis, borrelia burgdorferi, brucella abortus and listeria monocytogenes (table ; fig. ). these bacteria may be good candidates for clinical use of cq/hcq. recent in vitro studies indicate that cq may also have interesting activity against fungal diseases ( fig. ; table ) , including mainly h. capsulatum [ ] and c. neoformans [ ] . intracellular h. capsulatum is adapted to survive within the mammalian phagolysosome and resides within a membrane-bound phagosome that does not fully acidify. histoplasma capsulatum is able to maintain a phagosomal ph of . [ ] by inhibition of phagolysosomal fusion [ ] and by expression of a unique endogenous h + -atpase that buffers the phagosomal ph [ ] . it has been demonstrated that cq induces an antihistoplasmal state in macrophages by restricting the ph-dependent release of iron within the phagolysosome [ ] . similarly, cq has been shown to kill c. neoformans, but by a mechanism independent of iron deprivation [ , ] . indeed, unlike h. capsulatum, c. neoformans is able to maintain a phagolysosome milieu at ca. ph . [ ] and addition of cq increases the phagolysosomal ph allowing inhibition of growth at alkaline ph [ ] . it has been reported that a. fumigatus has the ability to inhibit fusion of the phagosome with the lysosome and that cq may increase killing of this fungus in macrophages by a phdependent mechanism [ ] . cq has been shown to inhibit the intramacrophagic growth of penicillium marneffei [ ] , an opportunistic fungus that causes disseminated infection in acquired immune deficiency syndrome (aids) patients by increasing the intravacuolar ph and disrupting ph-dependent metabolic processes [ ] . the decrease in the intracellular iron concentration results in impaired functionality of several cellular enzymes with a subsequent deleterious effect on critical steps such as replication of cellular dna or gene expression [ ] . finally, the effect of cq on multiplication of paracoccidioides brasiliensis has been studied in human monocytes and in a murine paracoccidioidomycosis model [ ] . cq was demonstrated to be able to kill p. brasiliensis grown in human monocytes. the effect of cq was reversed by fenta, an iron compound that is soluble at neutral to alkaline ph, but not by holotransferrin, which releases iron only in an acidic environment. thus, cq inhibits p. brasiliensis survival in human monocytes by iron deprivation [ ] . (table ) . . mechanisms of antiviral activity (fig. ) the ph increase induced by cq/hcq within acidic organelles, including endosomes, lysosomes and golgi vesicles, is involved in its antiviral activity via two main mechanisms. first, these drugs might be responsible for inhibition of viruses requiring a ph-dependent step for entry into their host cell. indeed, many viruses have a low-ph-dependent conformational change that triggers fusion, penetration and/or uncoating, and for these viruses endocytosis is crucial due to acidification that occurs within the endosomal pathway [ ] . thus, in this mechanism the antiviral effect is dependent on the extent to which the virus uses endosomes for entry [ ] . for instance, shibata et al. [ ] found that cq might prevent the uncoating of influenza b virus by increasing the lysosomal ph above the critical value required for inducing fusion between the virus envelope and the lysosomal membrane. cq was also found to inhibit uncoating of the hepatitis a virus (hav) [ ] . second, cq/hcq might inhibit post-translational modifications of the virus envelope glycoproteins by proteases and glycosyltransferases within the trans-golgi network and endoplasmic vesicles. indeed, some of these enzymes require a low ph for their activity and cq/hcq might therefore lead to decreased viral infectivity through impaired envelope maturation. flaviviridae are examples of viruses for which cq could act as an antiviral by inhibiting their envelope maturation pathway through alteration of the proteolytic processing of the prm protein [ ] . also, a putative mechanism for anti-hiv- activity is through alteration of the glycosylation pattern and amino acid charge within several regions of the gp viral envelope protein [ , ] . for instance, a reduction in the number of potential n-linked glycosylation sites within the v region of gp , which might provide for altered immune escape and broadening of the antibody repertoire, has been observed [ ] . savarino et al. [ ] previously found that cq decreased the infectivity of newly produced hiv- as well as the ability of hiv- -infected cells to form syncitia, and this was associated with structural changes in gp . recent data further suggest that cq may be responsible for inhibition of the biosynthesis of sialic acid. indeed, it was recently observed that this drug inhibited cellular enzymes involved in sialic acid biosynthesis [ ] . this might represent a major antiviral mechanism, since sialic acids are a component of hiv- envelope glycoproteins. cq/hcq may also have indirect antiviral effects. indeed, cq was found to be effective in preventing the spread of severe acute respiratory syndrome (sars)-associated coronavirus (cov) in cell culture by interfering with terminal glycosylation of the cellular receptor, angiotensin-converting enzyme (ace ) [ ] ; and sialic acids, biosynthesis of which might be inhibited by cq/hcq, are component of receptors of sars-cov and orthomyxoviruses [ ] . in addition to these two main mechanisms, other possible mechanisms such as immunomodulatory and antiinflammatory properties have been suggested, however these will not be discussed in the present review. in vitro activity of hcq/cq has been reported for a wide range of viruses, most frequently in experiments aiming to study the cycle replication pathways using cq, especially the mechanism by which viruses penetrate host cells (table ; fig. ). inhibitory concentrations of cq fell within the . - mol/l range, depending on antiviral cq doses, the viruses that were targeted and the assays used for assessment of the antiviral effect. importantly, these concentrations are in the range that is clinically achieved in plasma during malaria therapy, varying from . mol/l to . mol/l [ ] . the most studied in vitro effect of cq/hcq has been against hiv. moreover, in vivo studies quasi-exclusively concerned hiv- . this is probably due to the high morbidity and mortality related to hiv- worldwide and the need for low-cost antiretroviral therapies in resource-poor countries. cq/hcq have largely been shown to inhibit hiv replication in vitro. importantly, this inhibition was observed in several cell line models, but also in lymphocytes and mono-cytes from peripheral blood [ ] . an anti-hiv effect has been demonstrated either in the presence of high concentrations of cq/hcq prior to infection of hiv- -permissive cells [ , , ] or during incubation of hiv-infected cells with cq concentrations similar to those found in peripheral blood from individuals chronically treated with cq [ ] . thus, these data suggest both a preventive and curative effect of this drug against hiv. the concentration inhibiting % of viral replication (ic ) ranged between mol/l and mol/l for various hiv strains and culture cells in the studies of savarino et al. [ ] . boelaert et al. [ ] described an additional inhibition of hiv- replication with hydroxyurea plus didanosine (ddi) or zidovudine, with a cq ic of . - . mol/l for the cell lines and . - . mol/l for the primary cells; no cq-induced toxicity or apoptosis was noted. interestingly, according to in vitro data, cq/hcq also appears to be active against hiv- , which mostly circulates in west africa, as well as against hiv- of different subtypes [ ] . this deserves attention since hiv- strains have established or suggested natural resistance to several antiretroviral drugs, such as non-nucleoside reverse transcriptase inhibitors and likely some protease inhibitors (pis) [ ] . anti-hiv- activity of cq/hcq has been observed in a few in vivo studies since [ ] [ ] [ ] [ ] [ ] . two small, phase ii, randomised, double-blind studies, including and patients with cd cell counts of - cells/mm , compared reduction of plasma hiv- rna levels in individuals treated with hcq versus either placebo or zidovudine [ , ] . in both trials, more than two-thirds of patients were antiretroviral-naive. in the first trial, weeks of treatment with mg hcq per day resulted in a significant mean . log reduction of hiv- load (p = . ), whereas no significant decrease was observed in the placebo arm ( patients in each arm) [ ] . concomitantly, the percentage of cd + lymphocytes remained stable in the hcq group, whereas it significantly decreased in the placebo arm (p = . ). in the second trial, and patients received hcq or zidovudine, respectively, for weeks [ ] . hiv- load was significantly reduced in both groups (by . log copies/ml and . log copies/ml, respectively) and, interestingly, of patients in the hcq group versus of patients in the zidovudine group showed an increase in hiv- rna levels. two other recent non-controlled studies have been reported. in singapore, patients with hiv- load < copies/ml and cd cell count > cells/mm received hcq ( mg), hydroxyurea and ddi twice daily for weeks, resulting in a . log decrease in plasma hiv- rna levels [ ] . hiv- rna levels were further reduced compared with baseline (mean decrease . log copies/ml) in all patients who completed a -week course of therapy, with drug resistance mutations detected in patients at this time point [ ] . in another study in india, patients with cd counts > /mm received lamivudine, hydroxyurea and cq ( mg) twice daily for months [ ] . hiv- load reduction was significant (− . log ), reaching undetectable levels in patients, and the median rise in cd count was cells/mm . altogether, these in vivo data in patients with non-severe immunosuppression (cd cell count > /mm ) at least suggest that hiv- resistance to cq/hcq alone or in combination with antiretroviral drugs might not develop easily [ ] . in contrast, addition of cq to a zidovudine and ddi regimen provided no significant improvement in viro-immunological parameters in hiv- -infected children in a recent study from thailand [ ] . it is interesting to note that a -fold accumulation of cq in colostrum cells of african mothers taking mg of cq per day has been observed. this suggests that this drug could be potentially active as an adjuvant to post-natal antiretroviral prophylaxis of mother-to-child transmission by decreasing hiv- load in milk in geographical areas where vertical transmission is of great concern [ , ]. in several settings, cq/hcq should be used in combination with other antiviral drugs, questioning whether associations may be additive or synergistic. the additive effect of cq and zidovudine has been shown, and might also exist in association with ddi or hydroxyurea [ ] . furthermore, the combined effect of cq and pis is synergistic in a dose-independent manner [ ] . savarino et al. showed that cq in combination with pis carries out a combined inhibitory effect on p-glycoprotein and multidrug resistance protein , which is involved in efflux of pis, a major class of antiretrovirals. interestingly, the synergism between cq and pis was associated with a decreased threshold of susceptibility to pis in resistant isolates [ ] . of note, cq is a major substrate of cytochrome cyp a [ ] , and nevirapine (a non-nucleoside hiv- reverse transcriptase inhibitor) and hiv pis are well recognised cyp a inducers and inhibitors, respectively [ ] . thus, these latter drugs might subsequently decrease or increase the levels/effects of cq. it has been shown that endosomal transport is needed for human coronavirus hcov- e and that cells treated with cq displayed reduced expression of viral antigens [ ] . more recently, cq was found to have strong antiviral effects on sars-cov infection in cell cultures when they were treated either before or after exposure to the virus (even - h following infection), suggesting both prophylactic and therapeutic effects [ , ] . in keyaerts et al.'s study [ ] , cq inhibited viral replication with a % effective concentration (ec ) of . mol/l. of note, the dose inducing % cytostatic activity was much higher ( . mol/l). potential mechanisms of action of cq against coronaviruses are through underglycosylation of ace , which has been identified as a functional cellular receptor of sars-cov spike protein [ , ] . alteration by cq of the sars-cov spike protein is controversial [ , ] . a ph-related reduction of the transduction of sars-cov pseudotype viruses has also been suggested [ , , ] . of note, biot et al. [ ] recently reported the design and synthesis of hydroxyferroquine derivatives with antimalarial, anti-hiv and anti-sars-cov activities. the activity of cq against orthomyxoviridae (influenza a and b viruses) has been described for several decades [ , , ] and in vitro assays on avian influenza virus strains are ongoing [ ] . shibata et al.'s [ ] results suggested that cq prevents the uncoating of influenza b virus. ooi et al. [ ] found that the ic values of cq against influenza a viruses h n and h n were . mol/l and . mol/l, respectively. in a recent study, blanchard et al. [ ] found that pretreating target cells with cq inhibited hepatitis c virus (hcv) clone jfh- propagation in cell culture, which suggests that hcv, like flaviviruses and pestiviruses, enters cells through clathrin-mediated endocytosis and fusion within an acidic endosomal compartment. cq was also found to be active against hepatitis b virus (hbv) and duck hbv [ , , ] , contrasting with another study in which infection of human hepatocyte cultures with hbv was found to be unaffected by cq [ ] . seven patients with histologically proven chronic active hepatitis b have been treated with - mg of cq for a median of months [ ] . in all patients, alanine aminotransferase (alt) returned to normal values and prothrombin time improved. interestingly, alt increased in three patients following inadvertent cq withdrawal and returned to prior levels on re-administration. in four patients, a repeat liver biopsy year later revealed inactive cirrhosis. recently, an enhancement by cq of human cd + t-cell cell response to hbv antigen has been observed [ ] . inhibition by cq of hepatitis a virus (hav) uncoating and replication has also been described [ , ] . a major advantage of cq/hcq is their limited and preventable toxicity. long experience of the use of these drugs in the treatment of malaria has already demonstrated the safety of short-term administration to humans. moreover, cq/hcq have been widely used for chronic administration in rheumatic diseases, chronic q fever and for antimalarial prophylaxis for up to several years with only a low incidence of adverse effects even during pregnancy [ , [ ] [ ] [ ] . the main adverse effect reported in long-term administration of these drugs was macular retinopathy due to the cumulative dose, which could be prevented with regular visual monitoring during the course of treatment [ , ] . in summary, cq/hcq have several advantages as antimicrobial agents, including multiple potential mechanisms and a broad spectrum of activity at clinically achievable plasma concentrations, together with well known and limited toxicity and low cost. two major concepts have emerged to explain the activity of cq/hcq, namely alkalinisation of phagolysosomes for intracellular bacteria and fungi, and inhibition of entry steps and protein glycosylation for viruses. the pioneer and the only model of an infectious disease that could be treated by manipulation of intracellular ph by a lysosomotropic agent was chronic q fever. this review re-emphasises that the c. burnetii paradigm and activities mediated by lysosomotropic agents could be generalised for other intracellular pathogens living in acidic vacuoles or that require a low ph for multiplication. this may offer an interesting weapon to face present and future infectious diseases worldwide. funding: none. competing interests: none declared. ethical approval: not required. chloroquine-resistant malaria anti-hiv effects of chloroquine: inhibition of viral particle glycosylation and synergism 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strategies in chronic hepatitis b virus infection: ii. inhibition of duck hepatitis b virus in vitro using conventional antiviral agents and supercoiled-dna active compounds hepatitis c virus entry depends on clathrin-mediated endocytosis early events in arenavirus replication are sensitive to lysosomotropic compounds mechanism of lymphocytic choriomeningitis virus entry into cells ammonium chloride and chloroquine inhibit rabies virus infection in neuroblastoma cells processing and presentation of cell-associated varicella-zoster virus antigens by human monocytes respiratory syncytial virus inhibits granulocyte apoptosis through a phosphatidylinositol -kinase and nf-b-dependent mechanism effects of lysosomotropic weak bases on infection of bhk- cells by sindbis virus mechanism of enhancement of the antiviral action of interferon against herpes simplex virus- by chloroquine inhibition of multiplication of herpes simplex virus type by ammonium chloride and chloroquine epstein-barr virus enters b cells and epithelial cells by different routes entry of poliovirus type and mouse elberfeld (me) virus into hep- cells: receptor-mediated endocytosis and endosomal or lysosomal uncoating chloroquine induces empty capsid formation during poliovirus eclipse mechanism of entry into the cytosol of poliovirus type : requirement for low ph endoproteolytic activation of newcastle disease virus fusion proteins requires an intracellular acidic environment mechanism of borna disease virus entry into cells inhibition of vesicular stomatitis virus infection by spike glycoprotein. evidence for an intracellular, g proteinrequiring step attenuation of recombinant vesicular stomatitis viruses encoding mutant glycoproteins demonstrate a critical role for maintaining a high ph threshold for membrane fusion in viral fitness inhibition of vesicular stomatitis virus glycoprotein expression by chloroquine studies on the mechanism of entry of vaccinia virus in animal cells inhibition of murine rna tumor virus replication and oncogenesis by chloroquine early steps in fmdv replication: further analysis on the effects of chloroquine weak bases affect late stages of mayaro virus replication cycle in vertebrate cells entry of feline calicivirus is dependent on clathrin-mediated endocytosis and acidification in endosomes effect of chloroquine on african swine fever virus infection t cell proliferative response to bovine leukaemia virus (blv): identification of t cell epitopes on the major core protein (p ) in blv-infected cattle with normal haematological values infectious entry pathway for canine parvovirus cytoplasmic trafficking of minute virus of mice: low-ph requirement, routing to late endosomes, and proteasome interaction ph dependence of the coxiella burnetii glutamate transport system coxiella burnetii: the 'query' fever bacterium. a model of immune subversion by a strictly intracellular microorganism risks factors and prevention of q fever endocarditis q fever endocarditis in hiv-infected patient coxiella burnetii vascular graft infection endocarditis after acute q fever in patients with previously undiagnosed valvulopathies from acute q fever to endocarditis: serological follow-up strategy q fever osteoarticular infection: four new cases and a review of the literature cultivation of the bacillus of whipple's disease whipple's disease the survival of staphylococci within human leukocytes intracellular survival of staphylococci the influence of extracellular and phagolysosomal ph changes on the bactericidal activity of bovine neutrophils against staphylococcus aureus modification of interactions between neutrophils and staphylococci by lysosomotropic week bases activity of antibiotics against staphylococcus aureus within polymorphonuclear neutrophils effect of low intraphagolysosomal ph on antimicrobial activity of antibiotics against ingested staphylococci phagolysosomal alkalinization and intracellular killing of staphylococcus aureus by amikacin factors compromising the activity of moxifloxacin against intracellular staphylococcus aureus evaluation of antibiotic effectiveness against staphylococcus aureus surviving within the bovine mammary gland macrophage intracellular distribution and activity of antibiotics killing of intraleukocytic staphylococcus aureus by rifampin: in vitro and in vivo studies chloroquine and the fungal phagosome determination of the ph of the cryptococcus neoformans vacuole histoplasma capsulatum modulates the acidification of phagolysosomes regulation of the macrophage vacuolar atpase and phagosome-lysosome fusion by histoplasma capsulatum cloning and sequence analysis of an h(+)-atpase-encoding gene from the human dimorphic pathogen histoplasma capsulatum cryptococcus neoformans resides in an acidic phagolysosome of human macrophages dissecting virus entry via endocytosis effect of chloroquine on reducing hiv- replication in vitro and the dc-sign mediated transfer of virus to cd + t-lymphocytes kinetic mechanism of quinone oxidoreductase and its inhibition by the antimalarial quinolines clinical pharmacokinetics and metabolism of chloroquine. focus on recent advancements the additive in vitro anti-hiv- effect of chloroquine, when combined with zidovudine and hydroxyurea susceptibility of hiv- , siv and shiv to various anti-hiv- compounds: implications for treatment and postexposure prophylaxis hydroxychloroquine treatment of patients with human immunodeficiency virus type comparison of hydroxychloroquine with zidovudine in asymptomatic patients infected with human immunodeficiency virus type hydroxychloroquine, hydroxycarbamide, and didanosine as economic treatment for hiv- hydroxyurea and didanosine as initial therapy for hiv-infected patients with low viral load: safety, efficacy and resistance profile after weeks low cost anti-retroviral options: chloroquine based arv regimen combined with hydroxyurea and lamivudine: a new economical triple therapy therapeutic potential of chloroquine added to zidovudine plus didanosine for hiv- infected children mother-tochild transmission of hiv- : timing and implications for prevention effects of chloroquine on viral infections: an old drug against today's diseases? drug interactions of hiv protease inhibitors in vitro metabolism of chloroquine: identification of cyp c , cyp a , and cyp d as the main isoforms catalyzing n-desethylchloroquine formation angiotensin-converting enzyme is a functional receptor for the sars coronavirus alteration of the ph dependence of coronavirus-induced cell fusion: effect of mutations in the spike glycoprotein characterization of severe acute respiratory syndromeassociated coronavirus (sars-cov) spike glycoprotein-mediated viral entry ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign design and synthesis of hydroxyferroquine derivatives with antimalarial and antiviral activities infectious cell entry mechanism of influenza virus inhibition of hepatitis b dna polymerase by intercalating agents ph-independent uptake of hepatitis b virus in primary human hepatocytes treatment of chronic active hepatitis b (cah b) with chloroquine: a preliminary report chloroquine enhances human cd + t cell responses against soluble antigens in vivo correlation between serum levels of doxycycline and serology evolution in patients treated for coxiella burnetii endocarditis correlation between ratio of serum doxycycline concentration to mic and rapid decline of antibody levels during treatment of q fever endocarditis ocular toxicity and antenatal exposure to chloroquine or hydroxychloroquine for rheumatic diseases ophthalmologic considerations and testing in patients receiving long-term antimalarial therapy key: cord- -ilu oskk authors: sattui, sebastian e.; liew, jean w.; graef, elizabeth r.; coler-reilly, ariella; berenbaum, francis; duarte-garcía, alí; harrison, carly; konig, maximilian f.; korsten, peter; putman, michael s.; robinson, philip c.; sirotich, emily; ugarte-gil, manuel f.; webb, kate; young, kristen j.; kim, alfred h.j.; sparks, jeffrey a. title: swinging the pendulum: lessons learned from public discourse concerning hydroxychloroquine and covid- date: - - journal: expert review of clinical immunology doi: . / x. . sha: doc_id: cord_uid: ilu oskk introduction: several months into the covid- pandemic, safe and effective treatments against this global health disaster have yet to be identified. clinical research trials around the world are underway testing a wide array of possible medications. in particular, the off-label use of hydroxychloroquine for covid- prophylaxis and treatment has created many unprecedented challenges for the scientific community and the public. areas covered: we critically assessed major events from february – may that contributed to widespread use of hydroxychloroquine for the treatment and prophylaxis of covid- . we aimed to explore how opinions toward hydroxychloroquine may shift from early enthusiasm (based on in vitro and preliminary clinical data) to the hope for a miracle cure (through communication and promotion of questionable results) and, finally, to a rise of skepticism as more in-depth analyses are emerging. expert opinion: mindful and rigorous acquisition of data, as well as its interpretation, are essential to an effective pandemic response. the rapid and premature promotion of results has had major implications for global crisis management, even creating distrust among the public. it is crucial for the medical and scientific community to incorporate the lessons learned from this situation. the coronavirus disease (covid- ) pandemic has disrupted all aspects of society including the economy, health care, and scientific research. in the midst of a global health disaster, there has been a collective race to find safe and effective treatments. the use of preprints as a medium of rapid scientific communication has surged in recent months in order to expedite the availability of study results, though not without peril. preprints have not been peer-reviewed at the time of online distribution, vary in quality, and some may never be published in a peer-reviewed scientific journal. the use of chloroquine (cq) and hydroxychloroquine (hcq) for covid- exemplifies the risks of both overinterpreting and amplifying preliminary data by those outside of the scientific community and was followed by swift corrective measures by researchers. this may represent the most rapid medical reversal in recent history, a full 'pendulum swing' from early enthusiasm to wide skepticism. in this report, we analyze the evolving waves of discourse regarding cq/hcq in relation to covid- , lessons learned, and implications for the future (figure ). the antiviral effect of cq on sars-cov- was published, showing biological plausibility [ ] . this data was announced by the state council of china on february regarding possible efficacy of cq in the treatment of covid- pneumonia [ ] . as reported by gao et al, this resulted in the development of multiple trials in cq [ ] . the authors also alluded to successful treatment of over patients using cq, however, no data was published at that time. similar in vitro efficacy against sars-cov- was demonstrated with hcq shortly thereafter [ ] . raoult and colleagues in france reported these findings as 'hot topics' in a showcase of their group's research interests [ ] . this was also promoted on social media by raoult in late february [ ] . by early march, interest in hcq abruptly transitioned from mechanistic plausibility that would support its study in a clinical trial setting to rapid off-label use in patients with covid- , primarily fueled by promotion on social media, lay press, and celebrity influence [ ] . on march , results of the first study from the ihu-méditerranée infection on the use of hcq and azithromycin (azm) were revealed on raoult's youtube channel (video no longer available). on march , president trump endorsed hcq for the treatment of covid- in a press conference and commented on the drug's presumed safety in this setting as 'if things don't go as planned, it's not going to kill anybody' [ ] . the study by raoult and colleagues was published as a preprint on march and received immediate and at times uncritical media attention, as well as criticism from the scientific medical community regarding the study design and outcomes [ , ] . following this, trump posted on twitter on march describing the combination of hcq and azm potentially being 'one of the biggest gamechangers in the history of medicine', also mentioning the federal drug administration (fda) efforts to approve combination and quoting data presented in the international journal of antimicrobial agents [ ] . concurrently, new hcq prescriptions for covid- surged for both therapeutic and prophylactic use [ ] . without prior stockpiling, drug shortages quickly ensued [ , ] . patients with rheumatic diseases appeared on news outlets and social media to shed light on the implications of hcq and cq drug shortages. in the same week, multiple state public health agencies and regulatory boards attempted to protect the hcq supply chain by restricting prescriptions, for both covid- and rheumatic diseases [ ] . multiple rheumatology organizations and governmental institutions also released guiding statements regarding scarce resource allocation during the pandemic [ ] [ ] [ ] [ ] . with the enthusiasm around hcq came reports of toxic ingestions, including at least one fatality, from chloroquine phosphate containing aquarium products as well as inappropriate prescriptions by healthcare providers possibly for hoarding purposes [ , ] . by late march, two new studies became publicly available: a second study from the group of ihu-méditerranée infection using hcq and azm in patients with mild covid- infection released on their webpage, and a preprint of the first randomized controlled trial of patients from wuhan reporting a difference in clinical time to recovery and radiologic findings with hcq treatment [ , ] . with the increasing interest and examples of the irrational use of hcq, physicians and rheumatologists raised concerns about the increasing use of hcq and the inevitable impact on patients who rely on this medication [ ] [ ] [ ] . however, the fda issued an emergency use authorization (eua) for the use of hcq and cq in hospitalized patients with covid- on march [ ] . on march , hcq was added to the fda drug shortage list with several companies reporting limited supplies [ ] . during a white house press conference on april , trump suggested that a study had shown lupus patients were not contracting covid- ; this hypothesis was unfortunately echoed by some scientists without supporting data [ , ] . these assertions that lupus patients were protected against covid- were swiftly countered with emerging data from the global rheumatology alliance and emerging data from italy [ ] [ ] [ ] . there were concerns that the use of hcq, particularly in combination with azm, might induce arrhythmias, as new onset cardiomyopathy had been reported with severe covid- . lane et al released a preprint in medrvix on april , in which they used claims data from multiple international sources to study hcq with or without azm versus active comparators [ ] . although their study did not find a difference in short-term outcomes with -day follow-up, they did detect a concerning safety signal: hcq combined with azm was associated with an increased risk of cardiovascular death, angina, and heart failure. further studies assessing hcq with or without azm on qt prolongation in covid- patients emerged [ ] . several observational studies were published on the potentially arrhythmogenic impact of hcq and azithromycin [ ] [ ] [ ] . on april , magagnoli et al. released a preprint of their study from the veterans health administration that showed an increased risk of death from any cause in those who had been treated with hcq for covid- versus those who did not [ ] . • the covid- pandemic has resulted in the rapid dissemination of research, both through scientific and non-scientific channels. • we highlight the narrative of antimalarial therapy for the treatment of covid- over the course of the pandemic from february through june . • researchers and clinicians should be mindful of the public's uptake of both pre-print and published data and provide meaningful interpretations. • premature use of unstudied therapies can delay clinical trial enrollment and potentially harm recipients. • well-designed rigorous clinical trials are possible and necessary during a public health emergency. although the observational design had limitations, it's release was followed by articles in the press highlighting concerns of harm with hcq use [ ] . additionally, silva-borba et al. published a study demonstrating that high dose cq was associated with a higher risk of mortality versus low dose [ ] . with mounting evidence, the national institute of health (nih) made an advisory on april , followed by an fda advisory on april [ , ] . by may, the pendulum's return swing gained force as multiple studies were published in high impact journals. on may and , two large observational studies of hospitalized covid- patients in new york found nonsignificant associations between the use of hcq and major outcomes such as intubation or death, or in-hospital mortality [ , ] . two studies were published on may : a negative open-label rct of hcq versus standard of care on the outcome of seroconversion at days, and an observational study of patients requiring supplemental oxygen for covid- that did not demonstrate a significant association between the use of hcq versus no hcq on the outcome of survival without icu transfer within days [ , ] . counter to this new evidence, on may trump announced at a press conference that he was personally taking hcq for the prevention of covid- . he cited anecdotes that frontline healthcare workers were doing the same [ ] . on may , an observational study using purported deidentified international registry data on nearly , patients with covid- found an increased risk of mortality and new arrhythmias for hcq or cq alone, and either antimalarial in combination with a macrolide, versus comparators, receiving neither an antimalarial nor a macrolide [ ] . these findings prompted near immediate safety reviews of multiple international trials involving hcq use. less than hours after publication, enrollment in the hcq arm of the solidarity trial was suspended pending an interim data safety monitoring board (dsmb) review [ ] . within days, france's public health agency recommended against hcq's use as treatment for covid- treatment and revoked authorization for its use in clinical trials [ , ] . similar measures were taken simultaneously by drug safety agencies in italy and belgium [ ] . however, the plausibility of surgisphere's data was soon questioned by the scientific community. for example, the study contained electronic data derived from the digital hospital records of almost a third of all covid- cases and % of covid- related deaths in africa at the time [ ] . clinicians and researchers working in africa found it implausible that these extensive digital platforms existed and that this proportion of covid- cases across the continent were admitted to hospitals with these digital facilities. multiple other questions were raised, including the lack of a data availability statement and the lack of ethical approval. after third party reviewers were not given access to verify the data, this study and a related nejm study by the same group were retracted [ , ] . in addition to friction from scientific dissent, results from multiple large rcts on hcq for prevention or treatment of covid- provided a restoring force to the pendulum's swing. on june , a double blind placebo controlled rct for hcq as post-exposure prophylaxis against covid- infection involving subjects showed no significant differences in subsequent infection rates between the treatment and control groups [ ] . most subjects were healthcare workers and infections were self-reported by participants as either pcr-confirmed or symptomatically compatible due to limited testing availability in the us. no arrhythmias or deaths were reported. the authors also included data on concurrent zinc use which has been a topic of considerable interest amongst the general public [ ] . on june , the lead investigators of the recovery trial announced the suspension of the hcq arm in a statement citing lack of efficacy [ ] . an interim dsmb review showed no significant difference comparing those treated with hcq alone to those who received supportive care regarding the primary endpoint of -day mortality or secondary outcomes such as length of hospitalization or mechanical ventilation. these data were also cited by the fda in a detailed memorandum which revoked the eua for cq/hcq on june [ ] . shortly thereafter, the who announced that the hcq arm of the solidarity trial, which included the french discovery trial data, had been stopped due to inefficacy on june [ , ] . following an interim dsmb review on june involving enrolled subjects, the nih suspended the orchid study due to lack of efficacy between the hcq arm and placebo in hospitalized patients [ ] . the discourse surrounding covid- in the us has forced patients to disseminate accurate information to the public and combat the politicization of the narrative. it has been extremely frustrating for patients to ensure that the general public, and especially patients with rheumatic diseases, have been receiving correct information. patients, especially those with chronic illness, tend to latch on to any hope they find. public support for the use of hcq as a cure for covid- has put patients on these medications in danger as a massive influx of people tried to access hcq who did not have prior prescriptions. patients already on hcq were facing shortages and some were questioning if they should alter their doses. some wanted to decrease the risks that they would contract covid- while others were seeking to lower their doses in the face of obstacles to receiving their prescriptions. these actions have important consequences, as this increased their risk of worsening disease activity. two things stand out to patients: ) the need to combat misinformation and ) coordination of physician-researchers, patient advocacy groups, and patient care partners. it is expected that some people will take the media, scientists, and politicians at their word with no further search for the truth. others will seek to read the published data, but when the data is uninterpretable due to study design or methodological issues, patients are left in a difficult position. most rheumatic disease patients go to advocates and patientoriented organizations as their source for information related to their health, sources that are outside of their care team. for these people, it is imperative that the groups have a wealth of accurate information. this is not only where precise lay summaries are helpful, but also references to the sources of this information. if nothing else, we are taught that going forward we need to have better systems in place and infrastructure to work together to disseminate factual and transparent scientific information to the public in a comprehensible way. this best helps us to avoid instances of people taking matters into their own hands. much of the early enthusiasm for hcq originated from preprints listed on medrxiv, a database launched in with the aims of enhancing scientific collaboration and increasing accessibility of scientific findings. however, unfettered access to preliminary reports has proven to be a double-edged sword with widespread dissemination via social media and the press serving as dangerous substitutes for peer review. communicating accurate and accessible scientific information, including the limitations of current understanding, is crucial in preserving the public's trust during the uncertainties inherent in public health emergencies. several authors have drawn comparisons between covid- and the - ebola virus disease outbreak in west africa where widespread investigational drug use outside of welldesigned clinical trials confounded the search for safe effective treatments [ ] . despite these reminders, close to clinical trials involving hcq for covid- had been registered on clinicaltrials.gov including which were actively enrolling participants as of late may. most of the trials are too small to detect a meaningful effect size despite the immeasurable resources required to develop and conduct these trials. the fda's eua, issued prior to the availability of rigorous clinical data demonstrating efficacy or safety, allowed widespread access to hcq/cq outside of clinical trials. consequently, enrollment in large rcts rapidly decreased, delaying the critical safety and efficacy data necessary to evaluate these treatments in a timely fashion. while the story of hcq for covid- is unique and has many unprecedented factors, it is also not the first time that humanity has pinned its hopes on this compound. discovered in the bark of the cinchona tree, high in the mountains of the andes in the s after the pope died of malaria, quinine and its derivatives have been an essential weapon in pandemics, wars, politics and empire building throughout history [ , ] . during the flu, it is reported that 'londoners refused to be fobbed off with advice to gargle with salt water, and besieged chemists and doctors' surgeries demanding quinine' [ , ] . although data sharing and dissemination of data are important and enabled now by current forms of communication, the role of peer review is more crucial now than ever. as much as social media plays a role in the dissemination of information and scientific findings, scientists need to adequately direct and discuss these non-scientists, especially in challenging times such as a pandemic. while research is at times difficult to translate to the public, we can improve communication by providing patient-centered summaries at the time of publication. in many ways, the swift uptake and downfall for the recommended use of antimalarials, particularly hcq, for covid- serves as a cautionary tale for the conduct of research during a pandemic. the timeline of events has been particularly illustrative of the various types of pitfalls of research and scientific communication. that these events have occurred in a compressed timespan of several months is even more remarkable. scientific rigor needs to be upheld even in critical situations, such as pandemics, where there is an urgent need for new and efficacious treatments [ ] . transparency and accessibility in the publication and dissemination of results must remain key parts of research. data availability, open peer review, and open access are emerging tools to improve scientific integrity. maintaining these principles of the scientific method is even more vital during critical times, since results and findings will rapidly influence decision making. as observed with the use of antimalarials, rapid clinical implementation was based on data with clear limitations, without an adequate understanding of either benefits or potential harms. furthermore, the impact of these decisions was not only restricted to its use in covid- but had repercussions on patients who depended on these medications for other data-driven indications. it also led to inadequate allocation of resources that could have been employed for other vital interventions or equipment. effective, timely, and open peer review is critical to the validity of the data and the interpretation of the results. the widespread use of pre-print publications has highlighted the importance of adequate peer review, but also highlighted the value of the 'community review' of pre-prints when formal peer review has failed. with the contracted process from data collection to publication, the continued promotion of publication ethics is important to protect scientific integrity and trust in the system. finally, it is important for clinicians and scientists to realize that their role as communicators is now more vital than ever. media, including social media, are powerful tools of dissemination. as experienced during this crisis, the handling of data dissemination is critical and can have an either positive or negative impact in the public's image of the scientific community. as the covid- pandemic persists in the coming months, the scientific community must reemerge as a reliable source for guidance in interpreting the evolving knowledge base. this should be accomplished in tandem with stakeholders, such as patient advocates and public health officials, to carefully and effectively communicate findings to a wide audience. drs. sattui, liew, and graef and contributed equally. drs. kim and sparks contributed equally. figure was created with biorender.com. this paper was not funded. research scholar fund as well as personal fees from bristol-myers squibb, gilead, inova, janssen, and optum outside of the submitted work. the authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. peer reviewers on this manuscript have no relevant financial or other relationships to disclose. francis berenbaum references papers of special note have been highlighted as either of interest (•) or of considerable interest (••) to readers middle east respiratory syndrome and severe acute respiratory syndrome: current therapeutic options 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?__blob= publicationfile&v= . statement: lupus foundation of america urges manufacturers of hydroxychloroquine and chloroquine to ensure supply to treat lupus. lupus foundation of america arizona man dies after taking a form of chloroquine used to treat aquariums [internet]: cnn; updated doctors are hoarding unproven coronavirus medicine by writing prescriptions for themselves and their families [internet]. propublica clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in covid- patients with at least a six-day follow up: a pilot observational study efficacy of hydroxychloroquine in patients with covid- : results of a randomized clinical trial use of hydroxychloroquine and chloroquine during the covid- pandemic: what every clinician should know patients given unproven drug in texas nursing home in 'disconcerting' move festina lente: hydroxychloroquine, covid- and the role of the rheumatologist request for emergency use authorization for use of chloroquine phosphate or hydroxychloroquine sulfate supplied from the strategic national stockpile for treatment of coronavirus disease food and drug administration. fda drug shortages remarks by president trump, vice president pence, and members of the coronavirus task force in press briefing sle patients with covid- : a comment rheumatic disease and covid- : initial data from the covid- global rheumatology alliance provider registries •• first report of the incidence of covid- infection in patients with rheumatic diseases from covid- global rheumatology alliance registry baseline use of hydroxychloroquine in systemic lupus erythematosus does not preclude sars-cov- infection and severe covid- analysis from the covid- global rheumatology alliance registry showing no difference in covid- outcomes in lupus patients with or without baseline hcq use clinical course of coronavirus disease (covid- ) in a series of patients with systemic lupus erythematosus under long-term treatment with hydroxychloroquine safety of hydroxychloroquine, alone and in combination with azithromycin, in light of rapid wide-spread use for covid- : a multinational, network cohort and self-controlled case series study risk of qt interval prolongation associated with use of hydroxychloroquine with or without concomitant azithromycin among hospitalized patients testing positive for coronavirus disease (covid- ) effect of chloroquine, hydroxychloroquine, and azithromycin on the corrected qt interval in patients with sars-cov- infection assessment of qt intervals in a case series of patients with coronavirus disease (covid- ) infection treated with hydroxychloroquine alone or in combination with azithromycin in an intensive care unit qt interval prolongation and torsade de pointes in patients with covid- treated with hydroxychloroquine/azithromycin outcomes of hydroxychloroquine usage in united states veterans hospitalized with covid- study finds no benefit, higher death rate in patients taking hydroxychloroquine for covid- effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus (sars-cov- ) infection: a randomized clinical trial covid- ) treatment guidelines [internet]. national institutes of health hydroxychloroquine or chloroquine for covid- : drug safety communication -fda cautions against use outside of the hospital setting or a clinical trial due to risk of heart rhythm problems observational study of hydroxychloroquine in hospitalized patients with covid- us-based observational study showing no decreased or increased risk of intubation or death with hcq in hospitalized covid- patients association of treatment with hydroxychloroquine or azithromycin with in-hospital mortality in patients with covid- in new york state based observational study showing no differences in inhospital mortality for patients treated with hcq, az or both hydroxychloroquine in patients with mainly mild to moderate coronavirus disease : open label, randomised controlled trial clinical efficacy of hydroxychloroquine in patients with covid- pneumonia who require oxygen: observational comparative study using routine care data • french multicenter observational study showing no differences in icu transfers, survival, incidence of ards in hospitalized covid- patients treated with hcq or standard of care trump says he is taking hydroxychloroquine to protect against coronavirus, dismissing safety concerns [internet]: the washington post retracted: hydroxychloroquine or chloroquine with or without a macrolide for treatment of covid- : a multinational registry analysis who director-general's opening remarks at the media briefing on covid- - covid- : l'ansm souhaite suspendre par précaution les essais cliniques évaluant l'hydroxychloroquine dans la prise en charge des patients -point d'information covid- : utilisation de l'hydroxychloroquine eu governments ban malaria drug for covid- , trial paused as safety fears grow [internet]. reuters retraction-hydroxychloroquine or chloroquine with or without a macrolide for treatment of covid- : a multinational registry analysis retraction: cardiovascular disease, drug therapy, and mortality in covid- a randomized trial of hydroxychloroquine as postexposure prophylaxis for covid- rct showing no benefit of hcq for post-exposure prophylaxis in prevention of illness or confirmed covid- infection after high-or moderate-risk exposure evidence yet to support its effects on coronavirus statement from the chief investigators of the randomised evaluation of covid- therapy (recovery) trial on hydroxychloroquine: university of oxford food and drug administration. coronavirus (covid- ) update: fda revokes emergency use authorization for chloroquine and hydroxychloroquine world health organization targeted update: safety and efficacy of hydroxychloroquine or chloroquine for treatment of covid- nih halts clinical trial of hydroxychloroquine treating covid- -off-label drug use, compassionate use, and randomized clinical trials during pandemics the miraculous fever tree: malaria and the quest for a cure that changed the world the chloroquine chronicles: a history of the drug that conquered the world what the flu pandemic can teach us about coronavirus drug trials living with enza: the forgotten story of britain and the great flu pandemic of . london against pandemic research exceptionalism key: cord- -sji u nu authors: cavalli, giulio; dagna, lorenzo title: large-scale use of hydroxychloroquine for covid- confirms safety, if not effectiveness date: - - journal: eur j intern med doi: . /j.ejim. . . sha: doc_id: cord_uid: sji u nu nan in this issue of the journal, di castelnuovo and colleagues report the findings of the observational multicentre italian corist study on the use of hydroxychloroquine (hcq) in hospitalised covid- patients [ ] . in this large, retrospective cohort of , covid- inpatients from clinical centers, the use of hcq was associated with a significant increase in survival. specifically, the authors reported an in-hospital death rate of . per , person-day for patients receiving hcq, and of . for patients not receiving hcq. using an inverse probability weighting approach for propensity matching, and adjusting for various possible confounders, the authors report a % reduction in the mortality risk in patients receiving hcq. while these findings may provide clinical evidence in support of the use of hcq therapy in patients with covid- , the study findings ought to be considered with caution, in light of several limitations, which are inherent to the retrospective, observational design of this study. these include bias by indication (why did some patients receive hcq in addition to standard management, whereas others did not?), a possible immortal time bias (patients who died before treatment administration tend to be included as controls in retrospective studies), and of the fact that residual confounders typically remain even after stringent propensity matching is applied (not all clinically relevant variables are included in or captured by covariate analyses). however, these inherent limitations should not discourage large, real-world observational studies, which are particularly informative on the safety, rather than the efficacy of medications. in these regards, the study by di castelnuovo and colleagues holds clear value. hydroxychloroquine (hcq) is broadly used for the treatment of autoimmune and rheumatologic conditions such as lupus and rheumatoid arthritis [ ] . it is orally administered, well tolerated, and safe, as there are no common adverse events typically limiting its use. a feared but utterly rare long-term side effect is retinal deposition leading to progressive visual impairment, whereas theoretical risks of arrhythmic disturbances are clinically negligible (i.e., no cardiac screening is required prior to initiation of hcq treatment). before use in autoimmune patients, hcq was effectively used as an antimalarial agent. of note, evidence accumulating since the late ′ s also indicates in vitro antiviral activity [ ] . specifically, hcq interferes with a key step of the infection lifecycle of different viruses, by inhibiting endosomal acidification and viral entry into the host cell. in vitro, this effect prevented infection of target cells by clinical isolates of sars-cov- [ ] . following these and other reports, empirical, large-scale use of hcq began with the wishful aim of preventing development of covid- or escalation to severe disease states. hcq was one of many agents used to treat rheumatologic conditions, which were repurposed for use in covid- patients. other notable examples include biologic cytokine inhibitors such as the interleukin il- blocker anakinra [ ] , the il- blockers tocilizumab and sarilumab [ , ] , and the gm-csf blocker mavrilimumab [ , , ] . in general, published studies as well as direct physician experience indicate that hcq is marginally or not effective for the treatment of covid- , regardless of the disease stage and the dose administered [ ] [ ] [ ] . however, a controversial, and later retracted publication raised concerns that hcq might be associated with an increase in the risk of death due to arrhythmia and cardiac events [ ] . this finding, which was in stark contrast with clinical experience with hcq in immune-rheumatologic conditions, was later debased by a retraction [ ] and refuted by subsequent, separate studies. in these regards, the study by di castelnuovo and colleagues is particularly valuable, as included patients had an acute, severe disease, were on concomitant therapies with other drugs, and had pre-existing comorbidities such as ischemic heart disease, cancer and severe chronic kidney disease. controlled, prospective investigations will determine the efficacy of hcq in covid- . properly conducted real-world, retrospective investigations can reassure physicians that this old-used medication is generally safe and well tolerated. of chloroquine and covid- therapeutic options for the novel coronavirus ( -ncov) interleukin- blockade with high-dose anakinra in patients with covid- , acute respiratory distress syndrome, and hyperinflammation: a retrospective cohort study interleukin- blockade with sarilumab in severe covid- pneumonia with systemic hyperinflammation: an open-label cohort study efficacy and safety of tocilizumab in severe covid- patients: a single-centre retrospective cohort study gm-csf blockade with mavrilimumab in severe covid- pneumonia and systemic hyperinflammation: a single-centre, prospective cohort study observational study of hydroxychloroquine in hospitalized patients with covid- association of treatment with hydroxychloroquine or azithromycin with in-hospital mortality in patients with covid- in new york state effect of hydroxychloroquine in hospitalized patients with covid- : preliminary results from a multi-centre, randomized hydroxychloroquine or chloroquine with or without a macrolide for treatment of covid- : a multinational registry analysis retraction-hydroxychloroquine or chloroquine with or without a macrolide for treatment of covid- : a multinational registry analysis [retraction of: lancet targeting gm-csf in covid- pneumonia: rationale and strategies targeting il- , il- or gm-csf in covid- key: cord- - kcturbn authors: fassihi, safa c.; nabar, neel r.; fassihi, reza title: novel approach for low‐dose pulmonary delivery of hydroxychloroquine in covid‐ date: - - journal: br j pharmacol doi: . /bph. sha: doc_id: cord_uid: kcturbn nan despite inconclusive evidence, chloroquine (cq) and hydroxychloroquine (hcq) are commonly used for the treatment of coronavirus disease in critically ill patients. the widespread use of hcq has had unintended consequences on the healthcare system by precipitating an increased need for cardiac monitoring (due to qtc prolongation) and straining the medication's existing supply chain [chorin, dai, et al. ( ) ; jakhar & kaur ( ) ]. in addition, numerous trials have demonstrated marked qtc prolongation (> ms) and trends towards higher lethality, especially when administered in larger doses ( mg/day hcq; - mg/day cq) [borba, val, et al. ( ) ; chorin, dai, et al. ( ) ; mahevas, tran, et al. ( ) ]. this is of particular concern in the critically ill covid- patient, who is likely elderly, has multiple medical comorbidities, may be taking other qtc-prolonging medications (i.e. azithromycin), and may have myocarditis secondary to viral infection [arentz, yim et al. ( ) ]. taken together, strategies that mitigate the risk of cq/hcq-related systemic adverse effects should be pursued in the critically ill covid- population. with respect to the treatment of covid- , cq/hcq are thought to block proteolytic processing and endosomal acidification, inhibit autophagosome-lysosome fusion, inactivate enzymes required for viral replication, inhibit formation of viral proteins, and block viral entry into host cells through impairment of terminal glycosylation of ace receptors [sanders, monogue et al. ( ) ]. furthermore, these drugs may decrease cytokine production, which may provide further theoretical benefits, as hyperinflammation is implicated in covid- 's pathogenesis. it remains unclear to what degree each of these mechanisms contributes to the potential clinical benefit of cq/hcq and whether the immunomodulatory effects need to be systemic or can be localized to the lungs. given that covid- replicates chiefly within the pulmonary system and induces significant morbidity through the pro-inflammatory cascade of adult respiratory distress syndrome, many have hypothesized that the anti-inflammatory effects of cq/hcq may be most beneficial when targeted within lung tissues [frie & gbinigie ( ) ]. as an alternative to high-dose ( - mg) oral therapy, we propose low-dose ( - mg) delivery of water-soluble hydroxychloroquine sulfate (hcqs) via controlled nebulization or inhalation using metering valves and commercially available metered-dose inhalers. as the ec of hcq against sars-cov- is . μm, physiologically-based pharmacokinetic modeling demonstrates that an effective oral hcq dose is mg twice daily for one day followed by a maintenance dose of mg twice daily for four days [yao, ye et al. ( ) ]. given that the recommended maintenance dose of hcq is . mg/kg/day and that the collective weight of the lungs is ~ kg, - mg/day of inhaled hcqs (equivalent to . - . mg/day free base) is a suitable alternative to the presently utilized orally-administered dosing regimens [mcchesney, banks et al. ( ) ; yao, ye et al. ( ) ]. this lower inhaled dose is possible due to the large volume of distribution ( , l) for orally administered hcq [browning ( ) ]. when given orally, only a small fraction of the drug is delivered to the lungs, while the remainder deposits in other tissues and may cause unintended adverse effects. drug administration via nebulization allows direct delivery to lung alveoli and pulmonary tissues to elicit local effects, necessitating far lower doses than those used orally. pharmacokinetically, hcq remains largely neutral at physiological ph and can freely diffuse into cells and lysosomes. upon encountering the acidic ph ( . - . ) of lysosomes, it is protonated into its ionized state, becoming membrane-impermeable and effectively "trapped" in the lysosome [browning ( ) ]. this phenomenon greatly limits systemic exposure from inhaled or nebulized hcq. the potential benefits of this delivery modality are numerous. cardiotoxicity and qtc prolongation from oral administration of hcqs may be circumvented with targeted pulmonary delivery, reducing the need for inpatient cardiac monitoring in an already overburdened healthcare system [chorin, dai et al. ( ) ]. higher lung-tissue concentrations of hcq may be achieved via this route, potentially increasing the therapeutic efficacy of the drug while minimizing systemic adverse effects, including potentially fatal cardiac arrhythmia in at-risk patients. furthermore, by using far lower doses of hcq via pulmonary delivery, concerns regarding drug shortages for patients previously prescribed hcq for rheumatic conditions may be alleviated [jakhar & kaur ( ) ]. targeted pulmonary delivery of hcq has been previously described for various conditions involving the upper respiratory system and bronchial tree. in animal models, aerosolized hcq was found to be effective in the management of reactive airway disease [ (charous, nemeth et al. ( ) ; barrett, rudolph et al. ( ) ]. given its anti-inflammatory properties, aerosolized hcq has also been investigated in human trials for the treatment of moderatepersistent asthma. following phase a clinical trials in asthmatic patients, nebulized hcq did not meet the pre-specified clinical efficacy endpoints but was found to be safe, with no serious adverse events reported [aradigm ]. this lack of clinical efficacy in treating asthma cannot be translated to covid- , which has a different etiology for pulmonary inflammation and is a disease process that stands to benefit from the anti-viral effects of hcq. after reviewing the prior literature, klimke et al. tested the safety and tolerability of inhaled hcq in two healthy human subjects by dissolving hcq in . % sodium chloride and administering it via commercial nebulizers. doses began at mg twice daily and increased in a stepwise fashion to mg/day over a period of one week. outside of a transient bitter taste following inhalation, nebulized hcq was well-tolerated without relevant adverse effects [klimke, hefner, et al. ( ) ]. although further evidence is needed to determine the efficacy of aerosolized hcq in the treatment of covid- , low-dose targeted pulmonary delivery represents a safe and potentially preferred delivery method, particularly given the purported mechanisms by which hcq acts against sars-cov- . hcqs inhalation solution can be routinely prepared in hospital pharmacies by combining sterile hcqs powder with either sterile water or . % sodium chloride ( . % weight/volume) as a vehicle under aseptic conditions. this concentration can be achieved due to the high water solubility of hcqs [pauli, joshi et al. ( ) ; "plaquenil" ( ) ]. benzalkonium chloride ( . % or . mg/ml), a component of several fda-approved products with clinically demonstrated safety at . %, can be added to the solution as a preservative [woolf & manzi ( ) ]. the resulting mixture can be used as stock solution from which doses may be administered at volumes deemed appropriate by treating clinicians. generally, - ml may be given by nebulization or inhalation in a closed environment, with a loading dose of twice daily inhalation on the first day of therapy. subsequently, once daily dosing of - ml should suffice, as hcqs has both a half-life and tissue residence time of at least days [browning ( ) ]. in light of the consequences seen with widespread use of high-dose, orally-administered hcq in the treatment of covid- , clinical testing of the pharmacological parameters of inhaled or nebulized hcq should be a high priority. it should be noted that the authors are not advocating for the use of hcq in treating covid- , as the existing clinical evidence is inconclusive and limited by study design. however, if hcq is to be administered in critically ill covid- patients, low-dose inhaled or nebulized therapy may confer the collective benefits of similar or greater drug concentrations in pulmonary tissues, less systemic adverse effects (including cardiotoxicity), decreased burden on the healthcare system, and diminished strain on the existing supply of hydroxychloroquine. aradigm announces the results of the aerx hcq asthma phase a study characteristics and outcomes of critically ill patients with covid- in washington state nasal hydroxychloroquine (hcq) decreases nasal congestion and inhibits mediator release after ragweed challenge in sensitized beagle dogs effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus infection: a randomized clinical trial pharmacology of chloroquine and hydroxychloroquine aerosolized hydroxychloroquine (ahcq) protects against antigen-induced early (ear) and late airway responses (lar) and airway hyperresponsiveness (ahr) in allergic sheep the qt interval in patients with covid- treated with hydroxychloroquine and azithromycin chloroquine and hydroxychloroquine: current evidence for their effectiveness in treating covid- potential of chloroquine and hydroxychloroquine to treat covid- causes fears of shortages among people with systemic lupus erythematosus hydroxychloroquine as an aerosol might markedly reduce and even prevent severe clinical symptoms after sars-cov- infection no evidence of clinical efficacy of hydroxychloroquine in patients hospitalized for covid- infection with oxygen requirement: results of a study using routinely collected data to emulate a target trial tissue distribution of chloroquine, hydroxychloroquine, and desethylchloroquine in the rat evaluation of an immediate-release formulation of hydroxychloroquine sulfate with an interwoven pediatric taste-masking system pharmacologic treatments for coronavirus disease (covid- ): a review benzalkonium chloride in albuterol solutions: time for a change? in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) key: cord- - b uk authors: ibáñez, sebastián; martínez, oriela; valenzuela, francisca; silva, francisco; valenzuela, omar title: hydroxychloroquine and chloroquine in covid- : should they be used as standard therapy? date: - - journal: clin rheumatol doi: . /s - - - sha: doc_id: cord_uid: b uk the pandemic of the new coronavirus, known as severe acute respiratory syndrome coronavirus (sars-cov- ), has urged the nations to an unprecedented world-wide reaction, including an accelerated exploration of therapeutic options. in the absence of a vaccine and specifically designed antivirals, the medical community has proposed the use of various previously available medications in order to reduce the number of patients requiring prolonged hospitalizations, oxygen therapy, and mechanical ventilation and to decrease mortality from coronavirus disease (covid- ). hydroxychloroquine and chloroquine are among the proposed drugs and are the most widely used so far, despite the lack of robust evidence on their usefulness. the objective of this article is to review and discuss the possible role of these drugs in the therapy of covid- . the severe acute respiratory syndrome coronavirus (sars-cov- ) pandemic has urged the nations to an unprecedented world-wide reaction, including drastic containment measures and the search of therapeutic options. the main focus has been to slow down the spread of this virus, but until now, this is an ongoing process. sars-cov- produces the coronavirus disease (covid- ) characterized by lung infection and many other possible manifestations, in humans [ ] , with potential high mortality. until a vaccine or specifically developed antiviral is available, the need to control the disease in those with a severe presentation, and to reduce mortality, has moved the medical community to evaluate empirically the use of previously available drugs. hydroxychloroquine (hcq) and chloroquine (cq) are among the drugs proposed. hcq and cq are weak bases that accumulate in acidic compartments, such as lysosomes and inflamed tissues, and have a large volume of distribution and long half-life, giving them a slow onset of action and effects that last after suspension. their mechanisms of action include the interference of lysosomal activity and autophagy, the alteration of membrane stability, and the disruption of signaling pathways and transcriptional activity. these actions mean that at the cellular level, these drugs can inhibit immune activation, by decreasing toll-like receptor (tlr) signaling and modulating other co-stimulatory molecules, and by reducing the production of cytokines [ ] . the objective of this article is to review and discuss the possible role of these drugs in the therapy of covid- . mechanisms of action with possible role in covid- therapy cq, which has been used to prevent and treat malaria and as an anti-inflammatory agent for the treatment of rheumatoid arthritis and lupus erythematosus, has shown a potential broad-spectrum antiviral activity [ ] . it can inhibit a pre-entry step of the viral cycle by interfering with the binding of viral particles to their receptors on the cell surface. cq inhibits quinone reductase [ ] , which participates in the biosynthesis of sialic acids that are critical components of ligand recognition. human coronavirus hcov-o and the orthomyxoviruses use sialic acids as receptors [ ] . if sars-cov- targets sialic acids, this could be affected by cq [ , ] . it can also be hypothesized that, in the presence of sars-cov- , cq could interfere with the glycosylation of the angiotensin-converting enzyme (ace ) receptor, preventing the virus from binding to its target cells. this hypothesis is based on in vitro evidence of reduced glycosylation of the sars-cov surface receptor, ace , on vero cells [ ] . for the sars-cov , dengue, and chikungunya viruses, a ph-dependent mechanism of entry into target cells that can be interfered by cq has also been reported [ ] [ ] [ ] [ ] [ ] . preliminary data indicates that cq may interfere with sars-cov- acidification of lysosomes and inhibit cathepsins, which require low ph for cleavage of sars-cov- spike protein [ ] , necessary for the formation of the autophagosome [ ] . another possible mechanism of action is the inhibition of phosphorylation (activation) of the p mitogen-activated protein kinase (mapk) in thp- cells by cq [ ] . this phosphorylation is required by various viruses to achieve their replication cycles [ ] . in the model of hcov- coronavirus, cq inhibition of the virus appears to occur by this mechanism [ ] . regarding sars-cov- , the inhibition of kinases such as mapk could also be a mechanism of action for cq. other proposed effect of cq is that it can increase the soluble viral antigens in the cytosol of dendritic cells and enhance a cytotoxic cd + t cell response against them [ ] . in the influenza virus model, cq improved the crosspresentation of non-replicating virus antigen by dendritic cells to cd + t-cells, eliciting a protective immune response [ ] . also, cq is capable of mediating an anti-inflammatory response [ ] . it inhibits interleukin- beta (il- β) mrna expression in thp- cells and reduces il- β release [ ] . cq-induced reduction of tumor necrosis factor-alpha (tnfα), il- , and il- cytokines has also been reported [ , [ ] [ ] [ ] [ ] [ ] . in the dengue virus model, cq was found to inhibit interferon-alpha (ifnα), ifnβ, ifnγ, tnfα, il- , and il- gene expression in u cells infected with dengue- virus [ ] . hcq, a less toxic aminoquinoline, has an n-hydroxyethyl side chain in place of the n-diethyl group that makes it more soluble than cq. as cq, hcq also increases ph and confers antiviral effects and has a modulatory effect on activated immune cells. the antimalarial activity seems equivalent to cq, but hcq is preferred because of its lower toxicity [ ] . hcq binds strongly to melanin and can deposit in melanincontaining tissues such as the skin and the eyes, which might explain the retinopathy risk. clinical observations suggest that hcq confers a lower risk of retinopathy than cq, and this could be explained by its lower volume of distribution and lower tissular accumulation [ ] . hcq was, in vitro, at least as effective as chloroquine in inhibiting sars-cov- infection, although it should be noted that studies on its mechanisms of action are not as extensive as with cq [ ] . clinical studies of cq and hcq in covid- the described preconceptions led quickly to studies in china. on february , , the chinese government recommended that antimalarials should be included in the guidelines for prevention, diagnosis, and treatment of covid- pneumonia, issued by the national health commission of the people's republic of china [ , ] , but it should be noted that all the positive reports available until march , were anecdotal reports and open-label studies without control groups. on that date, a report of a blinded, randomized, controlled trial of hcq from wuhan was published. it analyzed patients in the treatment group (hcq mg per day for days) and in the control group [ ]. the median age was . years, the male-female ratio was even, and all patients had pneumonia by computed tomography (ct) scan. both groups received a not well-defined standard-of-care (oxygen therapy, antiviral drugs, antibiotics and even immunoglobulin, with or without corticosteroids). at presentation, more patients in the treatment group had fever and cough as compared to the control group. the treatment group showed significant improvements in comparison to the control group in fever, in cough, and in pneumonia by ct scan. although this was the first controlled study to show any benefit from hcq, it should be noted that the original registered trial informed a design for control patients, patients to receive a low dose of hcq, and more to receive a higher dose. the trial design also mentions as endpoints results for viral rna, and for t cell recovery time [ ] . this was not shown in the final publication. a previous chinese controlled, pilot, study showed no benefit when treatment-naïve patients were randomized : to hcq mg per day for days or conventional treatment only [ ] . neither trial reported serious adverse events, but both excluded patients with cardiac arrhythmias, as high doses of hydroxychloroquine can induce qt interval prolongation. in a french clinical trial, patients that received mg of hcq per day were compared with a group of untreated patients from another center. viral load in nasopharyngeal swabs was tested daily. azithromycin was added in patients of the hcq group. results showed that the virus was not found after days in all the patients treated with hcq and azithromycin, in . % of the patients treated with hcq only, and in . % of the control group patients (p < . ). no side effects or clinical evolution of patients were described. the authors said that azithromycin was added by clinical decision to treat a possible bacterial infection, but they also mention in their discussion that azithromycin may have an antiviral effect based on in vitro studies [ ] . this study was questioned by a multinational team that reanalyzed its statistics performing a bayesian a/b test and reported that for the original data, there was a strong statistical evidence for the positive effect of hcq monotherapy on viral reduction, but that the level of evidence dropped to moderate when the deteriorated patients were included in the analysis, and to anecdotal evidence when the patients that were not tested on the day of the primary outcome (day ) were excluded [ ] . the same group recently reported the results of a cohort of patients that received hcq mg per day for days, and azithromycin mg the first day, and mg per day for the next days. only patients, according to their report, did not improve (an -year-old patient who died and a -yearold patient still in intensive care unit [icu] at the time of the report). in % of the patients, the virus was not found at the nasopharyngeal sample tested by pcr at day ( % at day ). virus cultures from patient respiratory samples were negative in . % at day . the mean length of hospital stay was days [ ] . these results support the original report in that between and days of treatment, few patients had detectable virus by nasopharyngeal swab. the lack of comparison (hydroxychloroquine monotherapy, or standard of care) is problematic. in contrast to these results, another french study evaluated consecutive patients treated with the same combination (hcq mg per day and azithromycin, mg the first day, and mg per day thereafter). the mean age was . years, and had significant comorbidities ( obese, with cancer, with hiv). in this group, the combination was ineffective as patient died, had to be admitted to the icu, and (of ) were still positive for the virus by nasal swab on day or after treatment. one patient had to discontinue therapy on day because of prolongation of the qt interval [ ] . also, a multicenter, open-label, randomized controlled trial from china analyzed patients receiving standard of care, and receiving mg of hcq per day for the first days and then mg per day for weeks or weeks (mild/moderate or severe disease, respectively). specimens from the upper or lower respiratory tract were analyzed for viral rna at screening, and then at days , , , , , and . the number of negative tests was similar between the two groups after days ( . % in the hcq group, . % in the standard of care group). post hoc analysis did not identify any subgroups that showed a difference in these results. the alleviation of symptoms was also similar, but the adverse events were more frequent in the hcq group ( % vs %), being diarrhea the most frequent [ ] . regarding the need to be admitted to icu, a retrospective study from france analyzed patients who were receiving oxygen therapy. eighty-four received hcq ( mg per day) and the rest did not receive hcq. the composite primary endpoint was transfer to an icu within days or death from any cause, and the secondary endpoint was the development of acute respiratory distress syndrome (ards). there were no statistical differences between the two groups. eight of the patients in the hcq group had electrocardiogram changes that required to stop the medication [ ] . in relation to the cardiovascular risk, it is worth mentioning a study from the usa where patients were treated with hcq plus azithromycin combination. a notable qt interval prolongation was found in % of the patients, and in %, the interval increased to > ms, with a high risk for arrhythmia. the mean age was , % of the patients were male, % had hypertension, and % were diabetic. the development of renal failure while on the drug combination was a strong predictor of qt interval prolongation [ ] . also, a multinational collaboration presented data from health care systems in germany, japan, netherlands, spain, uk, and the usa where the safety of hcq and azithromycin combination versus hcq and amoxicillin combination was compared. in users of the hcq and azithromycin combination, a % increased risk of angina/chest pain, % increased risk of heart failure, and times increased risk of cardiovascular mortality at days of treatment was found in , patients [ ] . finally, a study from brazil analyzed patients in two treatments arms, cq mg per day for days or low dose ( mg on the first day, mg for the next days). all patients also received azithromycin and ceftriaxone. the highdose arm showed more qt interval prolongation (> ms) and a trend toward higher mortality ( %) than the lower dose group. the overall mortality rate was . %, similar with their historical rate of patients not receiving cq. the authors had to stop recruiting patients for the high-dose arm due to the cardiovascular events. the authors mention that they did not use a placebo control group as the use of placebo in brazil in severe cases of covid- infections was not considered ethically acceptable by national regulatory health agencies [ ] . discovering therapeutic options is difficult, even more if most patients will recover with the current standard of care. it is important to observe the progression of the disease and standard outcomes, for example how many patients need mechanical ventilation, how many need supplemental oxygen and for how long, the length of stay in critical care units, and the length of hospital stay. these are patient-centered outcomes. substitute endpoints, as viral load, will not necessarily relate to patient-centered outcomes; it has to be proven and their usefulness should not be assumed in advance. the evidence for the use of hydroxychloroquine or chloroquine in covid- is not good so far, not only because of the negative results of most of the studies but also because of their design, when publishing results of a very low number of patients, when reporting favorable results but without having a control group that allows comparison, when choosing results for which it will be very difficult to find significant differences, such as mortality, or for which their clinical relevance is uncertain. some countries and healthcare centers have adopted the use of hydroxychloroquine as a norm in patients hospitalized for covid- , due to political and social pressure given the publicity it has received. this makes the study of its possible beneficial effects even more difficult, and it has increased the reporting of its adverse effects. although due to the proposed mechanism of action, it could be postulated that the use of these antimalarials should be in the early stages of the disease; there is no clinical evidence to support this, and it could lead to serious problems in the availability of these drugs for patients with diseases in which the usefulness of these antimalarials is confirmed, not to mention the cardiovascular risks to which we would expose patients by indicating high doses of antimalarials without adequate monitoring. it is hoped that the studies in progress can answer several of the questions that remain to be solved, such as what is the objective of treatment with antimalarials (decrease hospitalizations, decrease hospital stay, decrease the need for mechanical ventilation, etc.), what is the time suitable for its use, at what dose, for how long, what monitoring is necessary, and which patients are at the greatest risk of suffering adverse effects. until then, we believe that the use of hydroxychloroquine or chloroquine should be in the context of strict studies or records that allow the detection of possible benefits and adverse effects. disclosures none. new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid- ? mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies kinetic mechanism of quinone oxidoreductase and its inhibition by the antimalarial quinolines avian influenza and sialic acid receptors: more than meets the eye? structure of coronavirus hemagglutinin-esterase offers insight into corona and influenza virus evolution betacoronavirus adaptation to humans involved progressive loss of hemagglutinin-esterase lectin activity chloroquine is a potent inhibitor of sars coronavirus infection and spread ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign a randomized controlled trial of chloroquine for the treatment of dengue in vietnamese adults ph-dependent entry of chikungunya virus into aedes albopictus cells assessment of in vitro prophylactic and therapeutic efficacy of chloroquine against chikungunya virus in vero cells sars coronavirus entry into host cells through a novel clathrinand caveolae-independent endocytic pathway different host cell proteases activate the sars-coronavirus spikeprotein for cell-cell and virus-cell fusion remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro gold sodium thiomalate and chloroquine inhibit cytokine production in monocytic thp- cells through distinct transcriptional and posttranslational mechanisms the protein tyrosine kinase p lck is required for triggering nf-κb activation upon interaction of human immunodeficiency virus type envelope glycoprotein gp with cell surface cd inhibition of human coronavirus e infection in human epithelial lung cells (l ) by chloroquine: involvement of p mapk and erk chloroquine enhances human cd + t cell responses against soluble antigens in vivo enhancement of t cell-mediated immune responses to whole inactivated influenza virus by chloroquine treatment in vivo effects of chloroquine on viral infections: an old drug against today's diseases chloroquine inhibits production of tnf-α, il- β and il- from lipopolysaccharide-stimulated human monocytes/macrophages by different modes chloroquine-induced inhibition of the production of tnf, but not of il- , is affected by disruption of iron metabolism chloroquine inhibits processing of tumor necrosis factor in lipopolysaccharide-stimulated raw . macrophages chloroquine inhibits macrophage tumour necrosis factor-alpha mrna transcription chloroquine interferes with lipopolysaccharide-induced tnf-α gene expression by a nonlysosomotropic mechanism acidotropic amines inhibit proteolytic processing of flavivirus prm protein aminoquinolines against coronavirus disease (covid- ): chloroquine or hydroxychloroquine ophthalmologic safety profile of antimalarial drugs hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro province and health commission of guangdong province for chloroquine in the treatment of novel coronavirus pneumonia ( ) expert consensus on chloroquine phosphate for the treatment of novel coronavirus pneumonia efficacy of hydroxychloroquine in patients with covid- : results of a randomized clinical trial | medrxiv ) the world health organization international clinical trials registered organization registered platform a pilot study of hydroxychloroquine in treatment of patients with common coronavirus disease- hydroxychloroquine and azithromycin as a treatment of covid- : results of an openlabel non-randomized clinical trial reply to gautret et al. : a bayesian reanalysis of the effects of hydroxychloroquine and azithromycin on viral carriage in patients with clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in covid- patients with at least a six-day follow up: an observational study no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe covid- infection hydroxychloroquine in patients with covid- : an open-label, randomized no evidence of clinical efficacy of hydroxychloroquine in patients hospitalized for covid- infection with oxygen requirement: results of a study using routinely collected data to emulate a target trial the qt interval in patients with sars-cov- infection treated with hydroxychloroquine/ azithromycin safety of hydroxychloroquine, alone and in combination with azithromycin, in light of rapid wide-spread use for covid- : a multinational, network cohort and self-controlled case series study | medrxiv chloroquine diphosphate in two different dosages as adjunctive therapy of hospitalized patients with severe respiratory syndrome in the context of coronavirus (sars-cov- ) infection: preliminary safety results of a randomized, double-blinded, phase iib clinical trial (clorocovid- study) publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -vi fb t authors: gopinathannair, rakesh; merchant, faisal m.; lakkireddy, dhanunjaya r.; etheridge, susan p.; feigofsky, suzy; han, janet k.; kabra, rajesh; natale, andrea; poe, stacy; saha, sandeep a.; russo, andrea m. title: covid- and cardiac arrhythmias: a global perspective on arrhythmia characteristics and management strategies date: - - journal: j interv card electrophysiol doi: . /s - - - sha: doc_id: cord_uid: vi fb t background: cardiovascular and arrhythmic events have been reported in hospitalized covid- patients. however, arrhythmia manifestations and treatment strategies used in these patients have not been well-described. we sought to better understand the cardiac arrhythmic manifestations and treatment strategies in hospitalized covid- patients through a worldwide cross-sectional survey. methods: the heart rhythm society (hrs) sent an online survey (via surveymonkey) to electrophysiology (ep) professionals (physicians, scientists, and allied professionals) across the globe. the survey was active from march to april , . results: a total of respondents completed the survey with % of respondents from outside the usa, representing countries and continents. of respondents, ( %) reported having covid- -positive patients in their hospital. atrial fibrillation was the most commonly reported tachyarrhythmia whereas severe sinus bradycardia and complete heart block were the most common bradyarrhythmias. ventricular tachycardia/ventricular fibrillation arrest and pulseless electrical activity were reported by . % and . % of respondents, respectively. there were of ( . %) respondents who reported using anticoagulation therapy in all covid- -positive patients who did not otherwise have an indication. one hundred fifty-five of ( %) reported regular use of hydroxychloroquine/chloroquine (hcq) + azithromycin (azm); concomitant use of azm was more common in the usa. sixty of respondents ( . %) reported having to discontinue therapy with hcq + azm due to significant qtc prolongation and ( . %) reported cases of torsade de pointes in patients on hcq/chloroquine and azm. amiodarone was the most common antiarrhythmic drug used for ventricular arrhythmia management. conclusions: in this global survey of > ep professionals regarding hospitalized covid- patients, a variety of arrhythmic manifestations were observed, ranging from benign to potentially life-threatening. observed adverse events related to use of hcq + azm included prolonged qtc requiring drug discontinuation as well as torsade de pointes. large prospective studies to better define arrhythmic manifestations as well as the safety of treatment strategies in covid- patients are warranted. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. the novel coronavirus (sars-cov- ) and the resulting respiratory tract infection (coronavirus disease or covid- ) is a pandemic with over , , cases globally, resulting in , deaths at the time of this writing [ , ] . following initial reports in wuhan, china, viral progression culminated in over , cases in china during january/february [ , ] . the subsequent global spread has involved more than countries [ ] . the usa has reported > . million confirmed cases and over , deaths, the highest in the world [ ] . as this global pandemic continues to rage, cardiovascular, especially arrhythmic, manifestations associated with covid- have become evident [ ] [ ] [ ] . a recent report from wuhan, china, noted that . % of hospitalized and . % of icu patients with covid- had arrhythmias [ ] . although arrhythmias appear to be common in hospitalized covid- patients, arrhythmia mechanisms and characteristics as well as antiarrhythmic therapies and their outcomes have not been welldefined. to better understand the cardiac arrhythmic manifestations and treatment strategies employed in hospitalized covid- patients through a worldwide cross-sectional survey of arrhythmia professionals. the heart rhythm society (hrs) conducted a global survey that was developed by the hrs communications committee with input from the hrs covid- rapid response task force. the survey was active from march , , to april , . the audience for the survey was electrophysiology (ep) professionals (physicians, nurse practitioners, physician assistants, nurses, ep lab technicians, scientists, and other allied professionals) across the globe. the survey consisted of questions (supplemental appendix). demographic questions included primary occupation, practice setting, and practice location (state/province and country). the goal of the survey was to understand the cumulative experience as well as the variability in incidence and management strategies of arrhythmias associated with covid- . the survey was administered using surveymonkey (surveymonkey, palo alto, ca, usa), and the survey link was disseminated to the hrs membership through a dedicated email, keeping pace weekly email, and also through the covid- webpage on the hrs website and hrs member open forum. additionally, it was disseminated to other arrhythmia societies across the world through email to their leadership and to the larger arrhythmia community through social media (twitter, facebook, and linkedin). the first respondents were all hrs members who received the survey link through a dedicated email. the subsequent respondents represented a combination of hrs members as well as self-identified respondents who received the survey link either from their respective arrhythmia societies or through social media channels. continuous variables are reported as mean ± standard deviation or median (interquartile range). categorical variables are reported as frequency and percentages. student's t test, or mann-whitney u test, was used to compare continuous variables, and categorical variables were analyzed using χ tests. all tests are two-tailed and a p value < . indicates statistical significance. statistical analysis of the responses was performed using statistica . (tibco software, palo alto, ca). a total of respondents completed the survey. seventyfour percent of the respondents were physicians and % were allied professionals. twenty-six percent were in academic practice, whereas % and % were in private and hospital-based practices, respectively. fifty percent of respondents were from outside the usa and represented countries and six continents. practice locations of us respondents represented states. demographic characteristics are detailed in table . of the respondents, ( %) reported having covid- -positive patients in their hospital. for those who reported hospitalized covid- , the reported total number of hospitalized covid patients at the time of the survey was , , with a mean and median number of patients of . ± . and (interquartile range, - ), respectively. of the respondents who reported at least one covid- patient in their hospital, . % had < patients, % had - patients, . % had - patients, . % had - patients, and . % had ≥ patients. a variety of supraventricular and ventricular arrhythmias were reported in covid- patients. of the respondents, ( %) reported cases of atrial fibrillation, ( . %) reported atrial flutter, ( . %) reported sustained atrial tachycardia, and ( . %) reported paroxysmal supraventricular tachycardia. among ventricular arrhythmias, frequent monomorphic premature ventricular contractions were reported by ( . %) respondents, multimorphic premature ventricular contractions by ( . %), and non-sustained ventricular tachycardia (vt) by ( . %). sustained monomorphic vt was reported by ( . %), polymorphic vt/torsade de pointes by ( . %), vt/ventricular fibrillation (vf) arrest by ( . %), and pulseless electrical activity by ( . %) respondents respectively ( fig. and table ). of respondents, ( %) reported significant sinus bradycardia, ( %) reported complete heart block, ( . %) reported first-or second-degree av block, and ( . %) reported bundle branch block or intraventricular conduction delay in covid- patients ( fig. and table ). one hundred and forty of ( . %) respondents reported using empiric anticoagulation therapy in all covid- positive patients who did not have an indication otherwise. of those who used empiric anticoagulation, of ( %) reported using intravenous heparin or subcutaneous low molecular weight heparin, ( %) reported using novel oral anticoagulants, and ( %) reported using warfarin. reported use of empiric intravenous heparin or subcutaneous low molecular weight heparin was more common outside the usa ( . % vs . %, p = . ). of respondents, ( . %) reported acute pericarditis; small pericardial effusion was reported by ( . %), whereas moderate and large pericardial effusions were reported by ( . %) and ( . %) of respondents, respectively. of respondents, ( . %) reported having patients on hydroxychloroquine (hcq)/chloroquine. twenty-seven ( . %) reported using it only in - % of patients, whereas ( . %) reported using it in - % of covid- patients. of respondents, ( %) respondents reported using hcq/chloroquine in combination with azithromycin (azm). thirty-six ( . %) reported using the combination only in - % of patients, whereas ( . %) reported using it in - % of covid- patients. for hcq monotherapy, in the usa, % responded that they had not used hcq in any patients, % had used it in some, but not all, patients, and % reported using it in essentially all patients. outside the usa, % had not used it at all, % had used in some patients, and % reported using hcq in essentially all patients (p = . ). for hcq/azm combination therapy, in the usa, % reported not using at all, % in some, and % in essentially all patients. outside the usa, % of respondents had not used combination therapy at all, % had used in some patients, and only % responded using in essentially all patients (p = . ). based on these results, use of either hcq or hcq in combination with azm appears to be more common in the usa (fig. ) . of respondents, ( %) reported using a qtc monitoring protocol for patients on hcq/chloroquine, with no significant difference between those from the usa versus outside the usa ( % vs %, p = . ). among individuals using a qtc monitoring protocol in the usa, % were in academic practice whereas outside the usa, only % of those using a qtc monitoring protocol were in academic practice (p = . ). twenty percent of respondents reported using magnesium supplementation in all patients on hcq/chloroquine. qtc prolongation ≥ ms (≥ ms with qrs duration > ms) was reported by of respondents ( %) ( table ). sixty ( . %) of respondents reported having to discontinue combination therapy with hcq/chloroquine and azm due to significant qtc prolongation. twenty ( . %) respondents reported cases of torsade de pointes in patients on hcq/chloroquine and azm. prophylactic amiodarone use was rare (reported by only ( . %) of respondents). in covid- patients with ventricular arrhythmias, ( %) of the respondents reported not using any class i, ii, or iii antiarrhythmic agents whereas ( %) used amiodarone and ( . %) used lidocaine/mexiletine. sotalol and dofetilide use was infrequent, reported by ( %) and ( . %) of survey respondents, respectively. the major findings of this global survey include the following: (a) in hospitalized covid- patients, ep professionals across the globe reported a wide variety of arrhythmic manifestations, with several reporting potentially life-threatening ventricular arrhythmias (sustained monomorphic vt, polymorphic vt/torsade de pointes, vt/vf arrest) as well as fig. difference between us and non-us respondents regarding the percentage of hospitalized covid- patients being treated with hcq/chloroquine + azithromycin fig. characteristics of bradyarrhythmias observed in hospitalized covid- patients pulseless electrical activity. (b) atrial fibrillation was the most common cardiac arrhythmia noted in these patients. severe sinus bradycardia and complete heart block were the most common bradyarrhythmias. (c) twenty-two percent of respondents used therapeutic anticoagulation in covid- patients without established indications, with use of intravenous heparin/low molecular weight heparin more prevalent outside the usa. (d) there was wide variation in use of hcq/ chloroquine and azm, with concomitant use of azm more common in the usa. discontinuation of hcq/chloroquine + azm due to qtc prolongation and torsade de pointes was reported by . % and . % of respondents, respectively. (e) amiodarone was the most common antiarrhythmic drug used for managing ventricular arrhythmias. currently, limited information is available regarding arrhythmic manifestations associated with covid- . in one study of patients, . % reported palpitations at presentation [ ] . wang et al., in a single-center retrospective analysis of consecutive patients admitted with covid- in wuhan, china, reported arrhythmias in . % of hospitalized patients, with a much higher incidence ( . %) in those needing intensive care. however, no definition as to what constituted an arrhythmia was provided [ ] . guo et al., in another single-center retrospective study of patients from wuhan, china, evaluated the association of underlying cardiovascular disease and myocardial injury on fatal outcomes in patients with covid- . they noted that % of patients had myocardial injury as evidenced by elevated troponin t (tnt) levels. incidence of vt/vf was . % and increased to . % in patients with elevated tnt [ ] . the reported percentage of vt/vf in our survey is comparable with the data from guo et al. moreover, our survey also provides additional information on pulseless electrical activity (reported by . % respondents), underscoring the potential influence of covid- on life-threatening cardiac arrhythmias and likely pump failure. atrial fibrillation was the most common reported arrhythmia in covid- patients. although we do not have demographic details of patients and do not know how many had de novo versus pre-existing atrial fibrillation, this is not surprising as the majority of sicker covid- patients are older and have underlying comorbidities, predisposing to atrial fibrillation [ ] . multiple mechanisms could lead to the increased incidence of brady-and tachyarrhythmias associated with covd- infection. arrhythmic manifestations could be secondary to direct myocardial inflammation and injury. severe hypoxic lung disease from covid- can trigger atrial arrhythmias. viral infection and associated increased metabolic demand and cytokine activation can trigger atrial and ventricular arrhythmias in patients who develop acute myocarditis or inflammatory response and in those with underlying coronary or other structural heart diseases. our data show that % of respondents are using anticoagulation, either oral, subcutaneous, or intravenous, in patients who did not otherwise have an indication for anticoagulation. this highlights the concern regarding the prothrombotic potential of covid- . the relatively common presence of atrial fibrillation further raises the need to address anticoagulation. these aspects as well as duration of anticoagulation following recovery from covid- require further study. at the time of this writing, except for the emergency use authorization of remdesivir, no other fda-approved treatments are available for covid- . there has been great interest in hcq/chloroquine ± azm, for inpatient treatment of covid- ; however, available data have been conflicting and randomized studies are lacking [ ] [ ] [ ] . the combination, however, poses a significant risk of qtc prolongation and torsade de pointes [ ] . a randomized, double-blind, currently non-peer-reviewed study from brazil assigned patients to a low-and high-dose chloroquine regimen; all patients received ceftriaxone and azm. at -day follow-up, . % had a qtc > ms ( . % in low-dose and . % in high-dose arm (p = . )). two out of patients ( . %) had vt from qtc prolongation [ ] . in a retrospective study of patients given hcq + azm, % had a qtc > ms [ ] . information from this survey closely mirrors data from these recent studies [ , ] and shows that the risk for arrhythmic adverse events is not inconsequential, and suggests a cautious approach and close monitoring of qtc when using these, yet to be proven, therapies. this study has several limitations. the findings represent cross-sectional data from a survey completed by arrhythmia professionals. given that the survey was disseminated to the global arrhythmia community, it is difficult to assess a response rate. the opinions of the survey respondents may not fully represent the entire ep community and may not represent all practitioners who care for covid- patients. the survey findings are subject to recall bias as respondents may tend to remember the sicker patients. reports of arrhythmias are from ep professionals, likely representing selection bias for sicker covid- patients at a higher risk for developing arrhythmias. since the survey was disseminated to ep professionals and not to institutions, it is possible that respondents working in the same institution may be referencing the same patient or group of patients. although a thorough review of the data did not reveal any duplicate responses, the fact that the survey was also disseminated through social media, it is not possible for us to be absolutely sure that only one response was obtained per respondent. the information on various arrhythmias presented here may not represent the accurate incidence of arrhythmias in the covid- population, but simply serve to provide a broad overview of arrhythmic manifestations and therapeutic strategies employed by ep professionals across the globe. the way the survey questions were worded, it was not possible to determine whether a reported brady-or tachyarrhythmia was de novo or pre-existing. this survey did not assess for comorbid conditions or the level of medical care the covid- patients were receiving (regular ward vs intensive care, ventilator use); hence, no association could be made between underlying conditions or severity of illness and arrhythmic manifestations. with regard to empiric subcutaneous or intravenous anticoagulation, although the intent was to assess the use of therapeutic doses, the wording of the survey question may not distinguish between prophylactic and therapeutic use. these results should be confirmed by future prospective studies or registries. in this global survey of data from > ep professionals regarding hospitalized covid- patients, a variety of arrhythmic manifestations were observed, ranging from benign to life-threatening. observed adverse events related to use of hcq + azm included prolonged qtc requiring drug discontinuation as well as torsade de pointes. these findings underscore the need for large, prospective studies to better define arrhythmic manifestations as well as the safety and efficacy of treatment strategies in covid- patients. covid- interactive map a pneumonia outbreak associated with a new coronavirus of probable bat origin interim clinical guidance for management of patients with confirmed novel coronavirus ( -ncov) infection association of cardiac injury with mortality in hospitalized patients with covid- in wuhan, china clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china cardiac and arrhythmic complications in patients with covid- clinical characteristics of novel coronavirus cases in tertiary hospitals in hubei province cardiovascular implications of fatal outcomes of patients with coronavirus disease (covid- ) case-fatality rate and characteristics of patients dying in relation to covid- in italy breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe covid- infection chloroquine diphosphate in two different dosages as adjunctive therapy of hospitalized patients with severe respiratory syndrome in the context of coronavirus (sars-cov- ) infection: preliminary safety results of a randomized, double-blinded, phase iib clinical trial (clorocovid- study) the qt interval in patients with covid- treated with hydroxychloroquine and azithromycin publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments the authors would like to convey their sincere appreciation to all the arrhythmia professionals who shared their experience by completing this survey as well to the heart rhythm society for supporting this endeavor. key: cord- -h oyjz authors: scherf-clavel, oliver; kaczmarek, edith; kinzig, martina; friedl, bettina; feja, malte; höhl, rainer; nau, roland; holzgrabe, ulrike; gernert, manuela; richter, franziska; sörgel, fritz title: tissue level profiling of sars-cov- antivirals in mice to predict their effects: comparing remdesivir’s active metabolite gs- vs. the clinically failed hydroxychloroquine date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: h oyjz background and objectives remdesivir and hydroxychloroquine are or were among the most promising therapeutic options to tackle the current sars-cov- pandemic. besides the use of the prodrug remdesivir itself, the direct administration of gs- , the resulting main metabolite of remdesivir, could be advantageous and even more effective. all substances were not originally developed for the treatment of covid- and especially for gs- little is known about its pharmacokinetic and physical-chemical properties. to justify the application of new or repurposed drugs in humans, pre-clinical in vivo animal models are mandatory to investigate relevant pk and pd properties and their relationship to each other. in this study, an adapted mouse model was chosen to demonstrate its suitability to provide sufficient information on the model substances gs- and hcq regarding plasma concentration and distribution into relevant tissues a prerequisite for treatment effectiveness. methods gs- and hcq were administered intravenously as a single injection to male mice. blood and organ samples were taken at several time points and drug concentrations were quantified in plasma and tissue homogenates by two liquid chromatography/tandem mass spectrometry methods. in vitro experiments were conducted to investigate the degradation of remdesivir in human plasma and blood. all pharmacokinetic analyses were performed with r studio using non-compartmental analysis. results high tissue to plasma ratios for gs- and hcq were found, indicating a significant distribution into the examined tissue, except for the central nervous system and fat. for gs- , measured tissue concentrations exceeded the reported in vitro ec values by more than -fold and in consideration of its high efficacy against feline infectious peritonitis, gs- could indeed be effective against sars-cov- in vivo. for hcq, relatively high in vitro ec values are reported, which were not reached in all tissues. facing its slow tissue distribution, hcq might not lead to sufficient tissue saturation for a reliable antiviral effect. conclusion the mouse model was able to characterise the pk and tissue distribution of both model substances and is a suitable tool to investigate early drug candidates against sars-cov- . furthermore, we could demonstrate a high tissue distribution of gs- even if not administered as the prodrug remdesivir. due to threat of the current sars-cov- pandemic, scientists around the world work vigorously on the development of therapeutic options, be it new chemical entities, antibodies, or repurposed known drug molecules. in order to advance quickly from pre-clinical to clinical studies, successful in vivo experiments in animal models are mandatory, a fact recently acknowledged by dinnon and colleagues [ ] . an adapted mouse model could be a suitable proof-of-concept for new compounds as demonstrated for remdesivir (rem) [ ] . however, to step up to humans, the relationship between pharmacodynamics (pd) and pharmacokinetics (pk) needs to be assessed in such a model to calculate an appropriate dose. two prominent examples of drug candidates against covid- are rem and hydroxychloroquine (hcq). rem represents a prodrug developed to quickly convert to the corresponding nucleoside (gs- ) intracellularly or even in plasma ( figure ). however, using gs- would be a more direct treatment strategy which does not rely on enzymatic conversion, and thus be more suitable to demonstrate the applicability of a model. gs- is the major circulating plasma metabolite of rem but only very limited data on the pk of this compound are available. furthermore, the efficacy of rem for covid- should be reevaluated in comparison to its main metabolite (gs- ), which was highly effective against feline coronavirus (fcov) causing feline infectious peritonitis (fip) [ ] [ ] [ ] . in fact, theoretical considerations come to the conclusion, that the prodrug rem might not be the most appropriate nucleoside strategy for the treatment of covid- . after all, it was specifically designed as prodrug to target the ebola virus [ ] [ ] [ ] , while the broad and extensive multi-organ pathology of covid- could actually benefit from direct application of gs- . the aim of this study was to demonstrate the applicability of mice to assess in vivo pharmacokinetics of two pharmacologically extremely different compounds with regards to their pharmacokinetic and physical-chemical properties. moreover, not only plasma concentrations, but also the amount and levels in relevant tissue should be assessable to obtain information on the distribution, an important aspect regarding the question whether or not the foci of infection could even be reached, in vivo. in the present work, we chose a mouse model using gs- and hcq as model compounds. we further investigated the degradation of remdesivir in human plasma and blood in vitro in order to add data on the aspect of its short plasma half-life and origin of gs- after administration as the phosphoramidate prodrug. animal care was provided in accordance with the guidelines of the eu directive / /eu and the german animal welfare agency. all experiments were approved by an ethics committee and the governmental agency (lower saxony state office for consumer protection and food safety; laves; protocol number: az a ). all efforts were made to minimize both the suffering and the number of animals. a total of naive male mice (n= for hcq, n= for gs- ) at - weeks of age and . - . g body weight on a bdf (c bl /dba hybrid) background bred and housed in the institute's facility were used. animals were group housed ( - animals per cage) on standard bedding (shredded wood) and maintained on a reversed -h light/ -h dark cycle (lights off at a.m.). room temperature in the mouse holding room was °c ± °c and relative humidity was about %; values were recorded during the daily animal check. food (altromin standard diet) and water were available ad libitum and material for nest-building (paper rolls) was provided in addition to red plastic houses as enrichment. all drugs or vehicle were administered intravenously as a single injection at a volume of ml/kg via the tail vein. for pharmacokinetic studies in mice, gs- was dissolved in sterile aqua ad injectabilia with . % ethanol, . % propylene glycol, and . % peg . the ph of the drug solution and the vehicle was adjusted to . with sodium hydroxide (naoh) and hydrochloric acid (hcl), respectively. mice were randomly assigned to gs- at mg/kg (n= ) or the respective vehicle (n= ). hcq was dissolved in sterile aqua ad injectabilia. isotonic saline solution served as vehicle control. solutions were prepared just prior to injection, and the final concentrations of compounds were verified from aliquots using lc-ms/ms. a cohort of mice was randomly assigned to hcq at mg/kg (free base, n= ) or the respective vehicle (n= ). the target and actual time of blood sampling were recorded relative to the time of injection. blood samples were collected into microvette cb potassium-edta tubes, briefly mixed by slow inversion and placed on ice immediately. within hour after collection, plasma was separated by centrifugation at , g for minutes at °c. blood samples were collected from all mice immediately prior to injection of drugs or vehicle (baseline control) from the tail vein ( - µl) , and immediately after injection from the facial, sub-mandibular, vein ( - µl) . the plasma or whole blood was protected from light and stored at - °c until analyzed by lc-ms/ms for quantification. the animal group for testing gs- was split in half to allow further repeated blood sampling at , , , and min as follows: n= (and n= vehicle) were bled at and min post injection from the facial vein, and at min after sacrifice by decapitation from the vena cava; the other n= (and n= vehicle) were bled at and min post injection from the facial vein, and at min after sacrifice from the vena cava. from gs- or respective vehicle injected mice, the following organs were taken at sacrifice min post injection: cerebrum, cerebellum, lung, liver, kidneys (partly separated in cortex and medulla), spleen, heart, muscle (quadriceps). for animals sacrificed min post injection, additional samples were taken as follows: stomach, intestine (separated in small intestine and colon), nasal mucosa, and a sample of the cortex separated from cerebrum. the animal group for testing hcq was also split in half to allow further blood sampling. in addition to the immediate sample, blood sampling was repeated at and min from the facial vein, and at min after sacrifice from the vena cava in one group (n= plus n= vehicle) and at , min and hrs from the facial vein, and at hrs after sacrifice from the vena cava in the other group (n= plus n= vehicle). an additional µl of whole blood was taken at sacrifice from the vena cava and transferred into plastic tubes without additives. from hcq and respective vehicle injected mice, all the above mentioned organ samples (gs- ) were taken, with the addition of pancreas. surgical equipment was carefully cleaned with ddh o and dried between samples. in both cases, organs were quickly dried with moistened gauze to remove excess water, blood or content in case of intestines, placed in cryotubes and snap frozen in liquid nitrogen and kept at - °c until analyzed by lc-ms/ms for quantification. gs- and hcq concentrations were quantified in mouse plasma and several mouse tissue homogenates by two liquid chromatography/tandem mass spectrometry (lc-ms/ms) methods using an sciex api tm triple quadrupole (gs- ) and an api triple quadrupole (hcq) mass spectrometer (sciex, concord, ontario, canada). both instruments were equipped with turbo ion spray interface (sciex, concord, ontario, canada). details about the methods including quality data are described in two separate publications currently in preparation. data acquisition and processing of raw data was performed using analyst software version austria, ) using the packages 'ggplot ', 'noncompart', and 'tidyverse' [ ] [ ] [ ] [ ] . auc was calculated using the linear up, logarithmic down method. the terminal slope ( z ) was calculated by log-linear regression of the last three data points. published pk-data was imported using the webplotdigitizer version . [ ] . commercially available edta-plasma was spiked with remdesivir at a concentration of μg/ml. a sciex x qtof mass spectrometer (sciex, darmstadt, germany) was coupled to an agilent infinity uhplc (agilent, waldbronn, germany) and operated in esi negative mode. chromatographic separation was achieved on a hypercarb µm x . mm column (thermofischer scientific, germany) as stationary phase using mm ammonium acetate at ph . (nh oh %) and acetonitrile containing . % (v/v) nh oh % as mobile phase a and b, respectively. the gradient was programmed as follows: - . min: % a, . - min:  % a, - . min:  % a, . - . min:  %a, . - min: % a. the flow rate was set to µl/min. a sciex x qtof mass spectrometer was coupled to an agilent infinity uhplc and operated in esi positive mode. chromatographic separation was achieved on a kinetex f . µm x . mm column (thermofischer, germany) as stationary phase using water containing . % (v/v) formic acid and acetonitrile containing . % (v/v) formic acid as mobile phase a and b, respectively. the gradient was programmed as follows: - . min: % a, . - min:  % a, - . min:  % a, . - . min:  %a, . - min: % a. the flow rate was set to µl/min. a sciex triple quadrupole mass spectrometer was coupled to an agilent hplc and no obvious adverse effects were observed after injection of gs- and hcq, respectively. the pooled data of mice was used to estimate the pk properties of gs- in wildtype mice (see figure a). based on the estimated  z and auc -∞ , the volume of distribution (v z ) was estimated at . l/kg, whereas clearance was estimated at . l/h/kg. the pooled data of mice was used to estimate pk parameters of hcq in wildtype mice. v z and clearance were estimated at . l/kg and . l/h/kg, respectively. the mean blood-toplasma ratio (b/p) and hours after infusion of mg/kg hcq was . ± . (n = ) and . ± . (n = ), respectively. the obtained plasma concentration-time profile is presented in figure b. one hour after administration of mg/kg, mean tissue-to-plasma ratio (t/p) ranged from . (brain) to . (adrenal cortex). four hours after i.v. administration, mean t/p was as high as . and . in brain and adrenal cortex, respectively (see figure a). highest mean t/p were observed for liver ( . ), kidneys ( . - . ) and intestines (> . ). obtained absolute tissue concentrations are presented in table . the full summary is presented in table s . six hours after administration of mg/kg, mean t/p ranged from . (cerebellum) to . (lungs). with exception of tissue found in the cns, fat, muscle, colon and stomach wall, mean t/p was at least for all tissues (see also table . the full summary is presented in table s . tof-ms and tof-ms/ms experiments revealed a rapid decrease of rem at elevated temperature in plasma in vitro. at the same time, the peak due to the alanine metabolite appeared and increased accordingly. gs- was not detected in those experiments ( fig. s -s ). in the low resolution lc-ms/ms experiments, gs- and the alanine metabolite were present at about the same level, expressed as peak area ratio (analyte/internal standard) prior to incubation. after incubation at °c the peak ratios increased by a factor of . and . for the alanine metabolite and gs- respectively. more than % of rem were degraded within hours at °c. gs- is the major circulating plasma metabolite of rem but only very limited data is available on the pk of this compound. most on the information obtainable from published sources refers to the pk after administration as the prodrug rem (gs- ) either in animal models [ , ] or healthy human subjects [ ] . there is also a case report of two covid- patients receiving rem [ ] . further hints on the pk of gs- can be found in the assessment report for gilead sciences` veklury (rem) [ ] . to our knowledge, the only other publications investigating the pk after administration in the form of the nucleoside are investigations by murphy and colleagues in cats [ ] . in comparison to the estimated pharmacokinetics in cats, our results are in the same order of magnitude however suggesting increased clearance and reduced volume of distribution (see table ). these discrepancies could be due to interspecies differences, further highlighting the importance of thorough pharmacokinetic studies in species used to model covid- . it was suggested by yan and muller, that rem in vivo is predominantly hydrolysed in plasma to yield gs- rather than being activated intracellularly [ ] . however, this theory seems to contradict the increasing, dose dependant half-life of gs- after administration as rem (see table ). the dose dependency furthermore suggests that in this case half-life is not defined by the elimination rate constant. instead, the liberation of gs- from tissue/organs saturated with the prodrug is suggested to be the rate limiting process at higher doses. this is underlined by the fact that renal clearance increases with increasing half-life ( table ). the mass-balance study for rem (gs-us- - ) disclosed that renal clearance is in addition to hepatic extraction (via alanine metabolite and the monophosphate) the most relevant elimination pathway and gs- is the predominant metabolite detected in urine ( %), followed by rem ( %). it is therefore not surprising that in one human patient with renal impairment receiving rem for the treatment of covid- , all gs- plasma concentrations were significantly elevated compared to another critically ill patient without renal impairment [ ] . gs- exposure in an patient suffering from end stage renal disease receiving rem was dramatically increased compared to healthy volunteers or non-impaired patients [ ] . being a comparatively old drug, the pk of hcq has been studied extensively. however, there is still limited information on its pharmacokinetics especially with regard to distribution. reports considering the terminal half-life in plasma or blood are inconclusive and range from approx. to days [ , ] . these values refer to an elimination half-life calculated after discontinuation from steady state. due to the high degree of tissue distribution these long halflives include redistribution from tissue and thus are considerably longer compared to a halflife calculated from a single dose or within steady state. the plasma half-life calculated from our data is comparable to the half-lives obtained in mice and humans (table ) . hcq exhibits complex pharmacokinetics with extremely high volume of distribution ranging from approx. to l for a kg adult. the value obtained from our experiments falls in that range and is comparable to the v z obtained in mice from blood concentrations and to plasma v z measured in humans (see table ). the substance is subject to renal and hepatic elimination. three major metabolites are known: bis-desethyl chloroquine, desethyl chloroquine, and monodesethyl hydroxychloroquine. the latter two are considered active metabolites [ ] . hcq is predominantly metabolized via cyp c , cyp d , and cyp a [ ] and approximately % of hcq are eliminated renally as parent drug by filtration and most likely also tubular secretion [ , ] . due to the structural similarity to chloroquine, which is a mate- substrate, secretion via this transporter seems probable [ ] . tett et al. were the first to report bioavailability and terminal half-life of hcq. they described the pharmacokinetics of a single i.v. and oral dose using a four-and tri-exponential equation, respectively [ ] . the estimated oral systemically available fraction ranged from . to . . later investigations by carmichael and colleagues confirmed this finding with their estimate of . [ ] . in plasma, the compound is bound to serum albumin and  -acid glycoprotein (aag), which is not unusual for alkaloids [ ] . the plasma protein binding ranged from . to . %, was dependant on the concentration of (aag), and was higher (approx. . -fold) for the senantiomer [ ] . thrombocytes and especially leukocytes have been identified as deep compartment for hcq leading to a blood-to-plasma ratio (b/p) ranging from . to [ , ] . due to the acidic environment and organelles in those cells, hcq is trapped in the protonated form within those kinds of cells, but not in erythrocytes. to our knowledge, b/p of hcq has not been studied in mice before and seems to be lower according to our results. this might be explained by the fact that the murine cellular immune system differs from the humane. whereas human blood is particularly rich in neutrophils ( - % of leukocytes) this cell type plays a minor role in the murine blood ( - % of leukocytes) which is dominated by lymphocytes [ ] . pharmacokinetics, including the metabolites of hcq, in mice [ ] . however, their method was not validated for mouse plasma and therefore not suitable to determine t/p or b/p of hcq. it is noteworthy, that the blood concentrations in the terminal phase obtained by chhonker et al. furthermore, it could be agreed, that for the purpose of tdm, whole blood concentrations might be more suitable compared to plasma concentrations, due to the lower variability in whole blood. on the other hand, the determination of pharmacokinetic parameters on the basis of whole blood concentrations might not be the best choice, since distribution and clearance processes usually only affect (unbound) drug molecules in the plasma and redistribution from blood cells might be slow. in cynomolgus monkeys rem ([ c]gs- , mg/kg), showed a tissue-to-plasma ratio (t/p) for testes and epididymis of about to . after h. a considerably lower amount of radioactivity was recovered from eyes and brain (t/p: ~ . and ~ . , respectively) indicating towards a low permeability for the blood-brain barrier for rem or any of the downstream metabolites [ ] . furthermore, t/p < or > indicate involvement of influx and efflux transporters which can play an important role regarding rate and extent of tissue distribution [ ] . t/p < is due to efflux processes as it is mediated especially in the brain via p-gp. as rem was found to be a substrate for p-gp, low t/p values found in the brain of cynomolgus monkeys are plausible. to our knowledge, gs- itself was not investigated as a transporter substrate but according to our data, tissue-to-plasma ratio in the brain is far below . a hypothetical affinity of gs- to p-gp, besides its low membrane permeability, would therefore also be conceivable. in covid- patients receiving rem, a csf to plasma ratio of . % was reported by tempestilli et al. [ ] . furthermore, they found gs- concentrations in bronchoalveolar aspirate of . and . ng/ml on day and . and . ng/ml on day , respectively ( patients). our data revealed a significant distribution of gs- into numerous organs, such as liver and kidney with high k t/p values both after one and after four hours. hence, the phosphoramidate prodrug structure in the form of rem might not be necessary to achieve sufficiently high tissue and even intracellular concentrations. the high efficiency of gs- in treating fip corroborates this finding [ ] . given the multi-organ pathology observed in covid- , successful treatment strategies require broad distribution. lack of penetration into brain tissue after could explain limited efficacy of most therapeutics against neurological symptoms which are frequent and debilitating in severe covid- patients [ ] . as gs- represents the nonphosphorylated nucleoside form of rem, an active uptake process into the cell via equilibrative (ent) or concentrative (cnt) transporters is plausible [ ] . yan and muller also brought up the idea that, besides slow passive diffusion, gs- crosses membranes using nucleoside transporters [ ] . a variety of nucleoside based antiviral drugs (e.g. ribavirin) and their corresponding transporter proteins is presented by pastor-anglada et al. [ ] . due to the adenosine structure in gs- , purine-preferring cnt is one of the most probable nucleoside transporter (k m value of µm for adenosine) to mediate intracellular uptake [ ] . in comparison, k m values for adenosine uptake by ent (k m µm) and ent (k m µm) are much higher [ ] . nevertheless, they should not be overlooked as several nucleoside transporters can be expressed by a single cell. in mcchesney described the distribution of chloroquine and hcq in albino rats in a qualitative and quantitative way. they found organ concentrations in the following order: bone, fat, and brain < muscle < eye < heart < kidney< liver < lung < spleen < adrenal tissue [ ] . it was also demonstrated, that tissue accumulation did not reach steady state before seven months into treating albino rats with mg/kg hcq per day orally (stomach tube). after weeks of treating albino rats with . mg/kg per day orally (stomach tube), t/p ranged from (muscle) to (spleen) with the values for heart, kidney, liver, and lung in between ( , , , ) [ ] . we could confirm the extensive tissue distribution of hcq, explaining the frequently reported high v z . in general, a high t/p would be desirable for an antiviral drug, since penetration into tissue is a necessary step for the intracellular uptake. accumulation in internal organs like liver, kidney, lungs, and spleen was also observed in our experiments. the disparity between our t/p and the reported values in albino rats could be mostly contributed to the fact, that our experiment was based on a single dose and steady state pharmacokinetics have not been attained. parts of the central nervous system did not show accumulation of hcq, which is in accordance with earlier investigations [ ] and may limit its use for the neurological manifestations potentially caused by sars-cov- penetration into the brain [ ] . reported in vitro ec values for hcq against vero e infected sars-cov- cells range from . to . - . µm [ , ] . with respect to the pronounced t/p, the comparatively high ec values (~ µm) could be reached in our mouse model in some tissues (e.g. lungs and adrenal cortex). however, a multiple of the ec value might be necessary for a reliable antiviral effect. due to the long terminal half-life and potentially slow tissue distribution (time to reach steady state in tissue!) sufficiently high concentrations in tissue might not be achieved in time. this could be one reason for the failure of the -day hcq therapies tested [ ] . a quicker tissue saturation is not feasible due to the comparatively high toxicity of hcq. a prophylactic effect of hcq could be studied infecting the current mouse model after long term ( - weeks) treatment with hcq ( mg/kg per day) in order to reach high hcq tissue concentration at the time of infection. it could be demonstrated, that rem quickly degrades to two major products in plasma in vitro. one of those degradation products is gs- , the other is the alanine metabolite as an intermediate. gs- could only be found in the more sensitive lc-ms/ms approach. due to the lack of external standards for the alanine metabolite, we cannot make a statement regarding the sensitivity in lc-ms/ms (the transition could not be optimized) and therefore not give a measured concentration. however, using a mass balance approach assuming that no other degradation products were formed, only a minor percentage (approx. . %) of remdesivir was converted to gs- in plasma. if this finding translates to in vivo, the major part of plasma gs- originates from redistribution after cellular uptake of remdesivir and intracellular metabolism rather than hydrolysis in plasma as suggested by yan and muller (vide supra). we could demonstrate that the mouse model platform is suitable to characterise the pk and tissue distribution of two extremely different compounds. we could demonstrate that gs- is distributed into tissue even if not administered as the prodrug rem. thus, we believe that the mouse model and general procedure presented here is a useful tool in the early investigation of drug candidates targeted against sars-cov- . this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. none of the authors of this work has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. a mouse-adapted sars-cov- model for the evaluation of covid- medical countermeasures. biorxiv remdesivir inhibits sars-cov- in human lung cells and chimeric sars-cov expressing the sars-cov- rna polymerase in mice oral mutian(r)x stopped faecal feline coronavirus shedding by naturally infected cats efficacy and safety of the nucleoside analog gs- for treatment of cats with naturally occurring feline infectious peritonitis the nucleoside analog gs- strongly inhibits feline infectious peritonitis (fip) virus in tissue culture and experimental cat infection studies 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simulated assessment of pharmacokinetically guided dosing for investigational treatments of pediatric patients with coronavirus disease a dose-ranging study of the pharmacokinetics of hydroxy-chloroquine following intravenous administration to healthy volunteers molecular mechanism of renal tubular secretion of the antimalarial drug chloroquine population pharmacokinetics of hydroxychloroquine in patients with rheumatoid arthritis hematologic disposition of hydroxychloroquine enantiomers of mice and not men: differences between mouse and human immunology simultaneous quantitation of hydroxychloroquine and its metabolites in mouse blood and tissues using lc-esi-ms/ms: an application for pharmacokinetic studies physiologically-based pharmacokinetic (pbpk) modeling and simulations neurological associations of covid- . the lancet neurology metabolic efficacy of phosphate prodrugs and the remdesivir paradigm cell entry and export of nucleoside analogues kinetic and pharmacological properties of cloned human equlibrative nucleoside transporters, ent and ent , stably expressed in nucleoside transporter-deficient pk cells coronavirus susceptibility to the antiviral remdesivir (gs- ) is mediated by the viral polymerase and the proofreading exoribonuclease. mbio pharmacokinetics and tissue distribution of remdesivir and its metabolites nucleotide monophosphate, nucleotide triphosphate, and nucleoside in mice review of the basic and clinical pharmacology of sulfobutylether-beta-cyclodextrin (sbecd) animal toxicity and pharmacokinetics of hydroxychloroquine sulfate tissue distribution of chloroquine, hydroxychloroquine, and desethylchloroquine in the rat hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) pharmacokinetics of hydroxychloroquine and its clinical implications in chemoprophylaxis against malaria caused by plasmodium vivax population pharmacokinetics of hydroxychloroquine in japanese patients with cutaneous or systemic lupus erythematosus pharmacokinetics of hydroxychloroquine in pregnancies with rheumatic diseases we are grateful to martina gramer and larsen kirchhoff for technical assistance. key: cord- -bccrvapy authors: szente fonseca, silvia nunes; queiroz de sousa, anastasio; wolkoff, alexandre giandoni; moreira, marcelo sampaio; pinto, bruno castro; valente takeda, christianne fernandes; rebouças, eduardo; vasconcellos abdon, ana paula; nascimento, anderson l.a.; risch, harvey a. title: risk of hospitalization for covid- outpatients treated with various drug regimens in brazil: comparative analysis date: - - journal: travel med infect dis doi: . /j.tmaid. . sha: doc_id: cord_uid: bccrvapy background: for the past few months, hmos have faced crowded emergency rooms and insufficient hospital and intensive-care-unit beds, all from the worst pandemic of this century, covid- . methods: in a large hmo in brazil, our approach was to allow treating physicians to prescribe antiviral medications immediately at presentation, and prednisone starting on day- of symptoms to treat pulmonary inflammation. we implemented this covid- protocol for outpatients and studied consecutive sars-cov- -positive patients age years or older presenting at our emergency rooms. results: use of hydroxychloroquine (hcq), prednisone or both significantly reduced hospitalization risk by - %. ivermectin, azithromycin and oseltamivir did not substantially reduce risk further. hospitalization risk was doubled for people with type- diabetes or obesity, increased by two-thirds for people with heart disease, and by % for each decade of age over age . similar magnitudes of reduced risk with hcq and prednisone use were seen for mortality risk, though were not significant because of only deaths among the patients. no cardiac arrhythmias requiring medication termination were observed for any of the medications. conclusions: this work adds to the growing literature of studies that have found substantial benefit for use of hcq combined with other agents in the early outpatient treatment of covid- , and adds the possibility of steroid use to enhance treatment efficacy. for the past few months, hmos have faced crowded emergency rooms and insufficient hospital and intensive-care-unit beds, all from the worst pandemic of this century, covid- . in a large hmo in brazil, our approach was to allow treating physicians to prescribe antiviral medications immediately at presentation, and prednisone starting on day- of symptoms to treat pulmonary inflammation. we implemented this covid- protocol for outpatients and studied consecutive sars-cov- -positive patients age years or older presenting at our emergency rooms. use of hydroxychloroquine (hcq), prednisone or both significantly reduced hospitalization risk by - %. ivermectin, azithromycin and oseltamivir did not substantially reduce risk further. hospitalization risk was doubled for people with type- diabetes or obesity, increased by two-thirds for people with heart disease, and by % for each decade of age over age . similar magnitudes of reduced risk with hcq and prednisone use were seen for mortality risk, though were not significant because of only deaths among the patients. no cardiac arrhythmias requiring medication termination were observed for any of the medications. this work adds to the growing literature of studies that have found substantial benefit for use of hcq combined with other agents in the early outpatient treatment of covid- , and adds the possibility of steroid use to enhance treatment efficacy. mankind has been facing one of the greatest challenges of the xxi century: a pandemic ( ) caused by a new virus, sars-cov- , thought to be transmitted by airborne particles and droplets and contact with contaminated surfaces or objects ( ) . clinical manifestations of coronavirus disease (covid- ) patients range from asymptomatic to mild nonspecific signs and symptoms to severe pneumonia with organ function damage and eventual mortality ( , ) . there is a clear need to try to stop disease progression as early in the disease process as possible. infected patients with comorbidities such as heart failure, type- diabetes, asthma or chronic obstructive pulmonary disease and obesity, and patients over sixty years of age are at substantially higher risk to develop severe disease and tend to have higher risks of death ( - ). many drugs have been tried in hospitalized patients, with largely discordant results ( ) ( ) ( ) ( ) . randomized double-blind controlled trials demonstrating benefit or lack of benefit of drugs in high-risk outpatients will not be available any time soon, as many clinical sites are still recruiting patients ( ) . early outpatient illness is very different than hospitalized severe disease and treatment therefore will differ between these two distinct groups. relatively little is established about utility of medications in early outpatient treatment. currently ( , ) it is understood that covid- is at least a four-phase illness: phase is viral replication, followed by pulmonary inflammation in phase , "cytokine storm" and acute respiratory distress in phase , and disseminated multi-organ involvement in phase . for treatment at the beginning of the illness, there are indications that chloroquine and especially hydroxychloroquine (hcq) may be beneficial ( ) ( ) ( ) ( ) , but no specific antiviral medications have demonstrated proven efficacy as yet ( , ) . recently, the brazil federal committee for medicine has approved the prescription of chloroquine and hcq for clinically suspected covid- patients at the physician's discretion with informed consent ( ) and the health ministry has also endorsed the use of these medications ( ) . brazil has the highest rate in south america in the ranking of covid- deaths, with more than . million people infected in the country ( ) in circumstances of a large population still to be affected and with economic difficulties resulting in inadequate social distancing. data over march-may from the federal health ministry ( ) show that more than % of hospitalized patients j o u r n a l p r e -p r o o f with severe respiratory distress who were tested were positive for sars-cov- , with less than % detected with influenza. therefore, we assumed in clinical practice that most patients coming to the emergency room with influenza-like symptoms would have covid- . with all that, we developed a protocol for early recognition and treatment of high-risk patients (in our population, age greater than years because of generally poorer health standards, or with comorbidities) who would come to our outpatient network of emergency rooms with influenza-like symptoms: fever, cough, myalgia and headache, among others, and receive early treatment, provided to patients at the first doctor visit, using physician discretion from among hcq, azithromycin, ivermectin, oseltamivir, zinc sulfate, nitazoxanide and prednisone (the last starting on day- of symptoms). we evaluate here risks of subsequent hospitalization based upon outpatient use of these various medications. methods: patient data were analyzed from electronic charts of health maintenance organization (hmo) hapvida saúde, the largest brazilian hmo with million members spread over five regions of the country. data were collected after informed consent and institutional ethics committee ( . . cep-university fortaleza unifor) approval for this study. to-date, during the pandemic, more than , monthly emergency room (er) consults have occurred. patients were all seen at the ers of the widespread country hospital network and admitted if indicated. at the beginning of the pandemic in brazil, late march-april , the north and northeast cities were more affected, with a great number of er consults and hospital and intensive-care-unit admissions. a protocol for early treatment of covid- was developed by a team of senior hmo medical staff and started in early may; it included clinical recognition of the commonly described main covid- signs and symptoms, and protocol criteria assessment for hospital admission vs outpatient care. patients coming with influenza-like symptoms such as fever, sore throat, myalgia, arthralgia or coryza would enter the covid- protocol. patients presenting with hypoxia, defined as the need of oxygen to maintain an oxygen saturation greater than %, respiratory rate of or greater than respirations/minute, hypotension defined as systolic pressure less than j o u r n a l p r e -p r o o f mm hg or diastolic pressure less than mm hg, or with confusion or extreme lethargy were immediately admitted to the hospital. the remaining patients over age or with comorbidities were defined as high-risk and treated as outpatients. the protocol specifics were chosen by the attending physician, and all of its steps were monitored for quality assurance. the protocol was largely automated through on-screen suggestions and physician choice boxes leading to successive screens, medication prescription choices, etc. after discharge from the er, patients received paper charts instructing them on isolation, symptoms to expect and medications to use, and qr codes for telemedicine, chat or phone consults. patients were instructed to return if symptoms of dyspnea, confusion or lethargy occurred. telemedicine was also always available to hmo patients on the hmo website. for discharged patients, the covid- protocol included (all as oral medications), as chosen by doctors and patients: hcq as first-line treatment, if used ( mg bid day , mg qd days - ), prednisone ( mg/kg qd x days, maximum mg/day, no taper), azithromycin ( mg qd x days), ivermectin ( mg qd x days), plus symptom relievers. zinc sulfate, oseltamivir and nitazoxanide were also available to be prescribed but were used infrequently. as doctors quickly found that most of the prescribed hcq was not available at common drugstores, if prescribed it was decided to offer the drug free of charge to all patients who only had to sign informed consent to receive it. data were collected from the hmo database for consecutive patients registered from may th to june rd , . we selected all patients years and older who tested positive for sars-cov- using a realtime reverse-transcriptase-polymerase-chain-reaction (rt-pcr) assay of nasal and pharyngeal swab specimens ( ) . to be clear, while all relevant patients with clinically likely covid- were offered treatment by the hmo, for the present report, we analyzed all those patients whose infections were subsequently confirmed by laboratory assay. the collected data included patient characteristics and comorbidities, age, gender, history of type- diabetes, hypertension, cardiac illness, pulmonary disease, other conditions, and facts of hospital admission and death. collected data were analyzed with multivariate unconditional logistic regression models to determine associations with medication use as well as other j o u r n a l p r e -p r o o f risk factors for hospital admission and death. age (in decades) and presentation delay (days) were treated as continuous covariates whereas all other variables were dichotomous. in addition to the medications, all of the presentation characteristics and comorbidities in table were examined for statistical significance and for confounding adjustment. death outcomes were those considered to be due to complications associated with covid- . a two-sided p-value less than . was considered statistically significant. results: from may to june, , patients were included in the covid- protocol, % from the northeast brazil states of ceará, bahia and pernambuco. seven hundred seventytwo patients ( . %) were admitted to the hospital and died ( . % of those hospitalized, . % of the whole cohort). within the cohort of , patients, because of scarcity of the tests and without selection by disease severity, , had testing for sars-cov- performed; , were age years or over and ( %) of these patients had positive rt-pcr assays for sars-cov- . we also included patients who had positive sars-cov- serology (table ) . three hundred seventy-two patients were female ( %); the mean age was . years (range - years). the average delay from the start of symptoms to er visit was . days. common presenting symptoms included shortness of breath ( , %), cough ( , %), fever ( , %), myalgia ( , %) and sore throat ( , %); ( %) patients had histories of cardiovascular disease, ( %) had diabetes type , ( %) were obese and ( . %) had chronic pulmonary disease. there were hospital admissions ( %) and of these, ( %) patients required mechanical ventilation and ( . %) patients died. the median time between start of symptoms and hospital admission was eight days; between hospital admission and death was seven days. one hundred twenty-two of the patients received none of the medications, and ( %) of them required hospitalization. associations with fact of eventual hospitalization are given in table . the multivariate logistic regression model presented in the table shows that age, obesity (bmi > ) and dyspnea were very substantial risk factors for hospital admission. each additional decade of j o u r n a l p r e -p r o o f age over age multiplied the risk of admission by a factor of . . use of prednisone and use of hcq were both associated with significantly reduced risk, and both drugs used together seemed to perform slightly better than either one alone. when the analysis was restricted to exclude patients hospitalized within five days, thus not eligible to receive prednisone, the results were essentially unchanged. history of pulmonary disease, presentation delay, or presentations with cough, myalgias, sore throat, headache or diarrhea were not associated with risk of hospitalization. presentation with fever, however, had or= . ( %ci . - . ), p=. , but did not change the associations seen in table , and with consideration for multiple comparisons of the various patient characteristics, may not be statistically significant. based on the model of table , we also examined use of azithromycin, or= . ( %ci . - . ) and use of ivermectin, or= . ( %ci . - . ). zinc prescription was not given on its own and where prescribed was highly correlated with other medication use and had little independent information for estimation of its own association in the adjusted model. when the model of table was performed including only individuals who had a history of at least one condition of obesity, diabetes or heart disease ( hospitalized patients and not hospitalized), the associations with the medications largely remained: for both hcq+prednisone, or= . ( %ci . - . ), p=. ; for hcq alone, or= . ( %ci . - . ), p=. ; and for prednisone alone, or= . ( %ci . - . ), p=. . we also examined the model of table discussion sars-cov- will cause greater mortality than any recent contemporary pandemic; only when the pandemic ends it will be possible to assess the full health, social and economic impact of this global disaster ( ) ( ) ( ) . preliminary data show that in developed countries, the impact will be huge. but in developing countries, where public health systems already face great challenges to provide basic health care to all in need, the impact will be several times greater ( ) ( ) ( ) . these problems will not be solved anytime soon. in the midst of the sars-cov- pandemic, a feasible approach, with inexpensive drugs, relying on syndromic signs and symptoms rather than scarce laboratory tests may help many patients and will be even more important in developing countries. around the world there are already over million confirmed covid- cases ( ) . brazil has the third-largest number, with . million cases and , deaths as of september th ( ) . if this trend continues, in about six months, brazil will have the worldwide largest number of deaths of any country. in march , the world health organization recommended the use of medications oseltamivir and antibiotics ( ). on march , , the fda issued an emergency use authorization for remdesivir and hcq for patients in both clinical trials and with severe hospitalized disease ( ) . since then, pharmacological treatments have been controversial. on june the fda retracted its earlier authorization and on july posted warnings about its use, leaving hcq outpatient use not supported ( ) . countries such as china and india have issued guidelines supporting the use of chloroquine or hcq in covid- ( , ) . evidence of the real-world unimportance of arrhythmia and other cardiovascular adverseevent endpoints of hcq and hcq+az use is given in the large oxford-based record-linkage study ( ) and in a study of % of the english population ( ) . understanding the pathophysiology of covid- in the different clinical stages of the disease is important, as treatments will change according to progression of the disease ( ) . our study showed that hcq alone, prednisone alone, and hcq plus prednisone did better than standard treatment for early stage covid- . it may be that the corticosteroid benefit involves low levels of type j o u r n a l p r e -p r o o f i and iii interferons juxtaposed to elevated chemokines and high expressions of il- . reduced initial innate antiviral defenses allow the virus to multiply, followed after a few days by relatively excess inflammatory cytokine production, allowing for steroids to reduce the latter in the early features of covid- , before appreciable pneumonia has occurred ( ) . hydroxychloroquine has a number of suggested beneficial actions for early covid- , not least of which is its non-immunosuppressive immunomodulatory activity ( ) . because all treatments have costs and benefits, treating all high-risk patients early would take a major effort from brazil's universal public system (sus) and its private hmos, but would be much less expensive than hospital-based inpatient treatment, which would probably be impossible on the scale needed. our study showed that about % of high-risk outpatients over age treated with prednisone still required hospitalization, which is substantially better than the % among untreated patients, thus even this treatment plan could create a large hospital-bed demand. however, we found that even in hospital, these treated patients do better and their mortality is much lower. in an ideal world, large randomized double-blinded controlled clinical trials establish evidence, but take time to complete and many are not large enough for the randomization to be sufficiently effective in reducing biases. to-date, treatment protocols have proposed drugs with antiviral activity, and with anti-inflammatory responses, such as therapeutic regimens of ifn-α+lopinavir/ritonavir and ifn-α+lopinavir/ritonavir+ribavirin, among others. while cost-effectiveness of these regimens have been challenged, hcq is generic and has been prescribed for malaria for decades, as it has antiviral and anti-inflammatory properties. on march th , the brazilian federal health authority issued a note saying that it would treat severely ill patients in the public system with hcq ( ). on may th, the same authority issued another note that hcq would be available for physicians to prescribe for outpatients and mild cases, according to symptoms and severity ( ) . prednisone is also generic and inexpensive and has been used for many decades and does not interact adversely with hcq. our results demonstrate a positive benefit of hcq and prednisone in decreasing hospital admissions in a high-risk population over years of age with rt-pcr-positive sars-cov- infection when started at first doctor visit. a high-risk outpatient benefit of hcq use has been summarized elsewhere ( ) but to our knowledge this is the first time that efficacy of outpatient prednisone use has been reported. use of these medications also showed some evidence of reduced mortality in the study group, and larger studies of mortality will be needed to validate this finding. we observed that outpatient hospitalizations of the larger group of suspected covid- er patients, from the same hmo database before vs after the protocol started, march-april vs may, decreased significantly, % vs %, and mortality declined from . % to . %. for may, our hmo data also show that the mortality was less than covid- mortality for brazil as a whole. our study has several limitations. this is a retrospective, chart-based study, and even though our initial sample of patients was large, with almost , patients, few of these patients were tested due to the scarcity of rt-pcr tests. then, we chose to study only tested-positive sars-cov- patients to make sure we were dealing with confirmed cases of covid- . limiting analyses to patients greater than years of age further reduced our sample size. nevertheless, our experience of approaching and treating patients with influenza-like symptoms in this era of pandemic sars-cov- is useful and more generally applicable. in one state hospital network of the cohort this spring, more than % of patients admitted to the hospital with appreciable respiratory distress had positive rt-pcr for sars-cov- ( ), so it seems reasonable to infer that it would be similar for patients with influenza-like illness presenting at the emergency room. also, our study involved a range of treatment medications assigned by hmo physicians using their clinical judgements, rather than mandated by study design. clinical treatment decisions allow for the possibility that sicker patients get more or more aggressive treatments, creating the potential of confounding by indication. the comorbidity distributions of the various treatments as shown in table suggest that except for shortness of breath, patients not treated with hcq or j o u r n a l p r e -p r o o f prednisone may have been slightly less symptomatic than treated patients. however, this would if anything have tended to reduce the magnitude of risk lowering that we found for these medications toward the null. a pattern of chronic comorbidity differences is not apparent in the table; nevertheless, our results were adjusted for those comorbidities where associations with risk of hospitalization were observed ( table ). in spite of the aforementioned, our study was large enough to have observed statistically significant results and was based on actual clinical conditions and data recorded in active clinical charts, to enable reasonable inference about lack of reporting biases in the analyzed data. our analyses thus show that it is possible to give hcq with companion medications in an early stage protocol that proves to be safe, and warnings about cardiac arrhythmia adverse events are unnecessary unless significant contraindications are known. treatment-failure mortality, while small, is still the major concern of patient management. our new protocol is continuing in clinical practice in our hmo, and we hope for it to be more generally applied across the rest of brazil as quickly as possible. we found early outpatient use of hcq and prednisone, both as individual prescriptions and used together, to lower the risk of hospitalization in symptomatic high-risk covid- patients presenting for primary care at the emergency rooms of our large hmo in brazil. other than the small numbers of treatment failure, no potentially life-threatening adverse events were recorded with medication treatment. these medications were found to be safe and beneficial for early high-risk outpatient treatment of covid- . j o u r n a l p r e -p r o o f more than manufacturers of the various medications analyzed herein. this past work was not related to any of these medications and was completed more than two years ago. he has no ongoing, planned or projected relationships with any of these companies, nor any other potential conflicts-of-interest to disclose. none of the other authors have any potential conflicts of interest to disclose. funding: none. j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f covid- ) outbreak -who announces covid- outbreak a pandemic interim infection prevention and control recommendations for patients with suspected or confirmed coronavirus disease (covid- ) in healthcare settings downloadedseptember the epidemiology and clinical information about covid- clinical characteristics of coronavirus disease in china severe outcomes among patients with coronavirus disease (covid- ) -united states characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease 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effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) conselho federal de medicina. processo-consulta cfm n o / -parecer cfm n o / . tratamento de pacientes portadores de covid- com cloroquina e hidroxicloroquina orientações do ministério da saúde para manuseio medicamentoso precoce de pacientes com diagnóstico da covid- infogripe-monitoramento de casos reportados de síndrome respiratória aguda grave (srag) hospitalizados variation in false negative rate of rt-pcr based sars-cov- tests by time since exposure the benefits and costs of social distancing in rich and poor countries united nations development programme. socio-economic impact of covid- the socioeconomic implications of the coronavirus pandemic (covid- ): a review johns hopkins coronavirus resource center. covid- map -johns hopkins coronavirus resource center. johns hopkins coronavirus resource world health organization. clinical management of severe acute respiratory infection j o u r n a l p r e -p r o o f ( sari) when covid- disease is suspected: interim guidance based on fda's continued review of the scientific evidence available for hydroxychloroquine sulfate (hcq) and chloroquine phosphate (cq) to treat covid- , fda has determined that the statutory criteria for eua as outlined in section (c)( ) of the food, drug, and cosmetic act are no longer met fda news release. coronavirus (covid- ) update: daily roundup national china health office medical letter ( ) . notice on issuing the new coronavirus pneumonia diagnosis and treatment plan (trial version ) government of india, ministry of health and family welfare, directorate general of health services (emr division) early outpatient treatment of symptomatic, high-risk covid- patients that should be ramped-up immediately as key to the pandemic crisis hydroxychloroquine for prevention of covid- mortality: a population-based cohort study can steroids reverse the severe covid- induced 'cytokine storm immunomodulators in sle: clinical evidence and immunologic actions perfil epidemiologico dos pacientes hospitalizados por sindrome respiratória aguda grave (srag) no estado do ceará. de maio de /pagina / acknowledgements: dr. risch acknowledges past advisory consulting work with two of the declarations travel medicine and infectious disease requires that all authors sign a declaration of conflicting interests. if you have nothing to declare in any of these categories then this should be stated. a conflicting interest exists when professional judgement concerning a primary interest (such as patient's welfare or the validity of research) may be influenced by a secondary interest (such as financial gain or personal rivalry). it may arise for the authors when they have financial interest that may influence their interpretation of their results or those of others. examples of potential conflicts of interest include employment, consultancies, stock ownership, honoraria, paid expert testimony, patent applications/registrations, and grants or other funding. all sources of funding should also be acknowledged and you should declare any involvement of study sponsors in the study design; collection, analysis and interpretation of data; the writing of the manuscript; the decision to submit the manuscript for publication. if the study sponsors had no such involvement, this should be stated. signature (a scanned signature is acceptable, dr. risch acknowledges past advisory consulting work with two of the more than manufacturers of the various medications analyzed herein. this past work was not related to any of these medications and was completed more than two years ago. he has no ongoing, planned or projected relationships with any of these companies, nor any other potential conflicts-of-interest to disclose. none of the other authors have any potential conflicts of interest to disclose.no funding involved. j o u r n a l p r e -p r o o f key: cord- -u lx h authors: biguetti, claudia; marrelli, mauro toledo; brotto, marco title: primum non nocere – are chloroquine and hydroxychloroquine safe prophylactic/treatment options for sars-cov- (covid- )? date: - - journal: revista de saude publica doi: . /s - . sha: doc_id: cord_uid: u lx h chloroquine (cq) and its analog hydroxychloroquine (hcq) were recently included in several clinical trials as potential prophylactic and therapeutic options for sars-cov- infection/covid- . however, drug effectiveness in preventing, treating, or slowing the progression of the disease is still unknown. despite some initial promising in vitro results, rigorous pre-clinical animal studies and randomized clinical trials have not been performed yet. on the other hand, while the potential effectiveness of cq/hcq is, at best, hypothetical, their side effects are factual and most worrisome, particularly when considering vulnerable groups of patients being treated with these drugs. in this comment, we briefly explain the possible mechanisms of action of cq/hcq for treating other diseases, possible actions against covid- , and their potent side effects, in order to reinforce the necessity of evaluating the benefit-risk balance when widely prescribing these drugs for sars-cov- infection/covid- . we conclude by strongly recommending against their indiscriminate use. brought the world to an unimaginable halt. nevertheless, there is a strong need for up-to-date information about the evolution of the pandemic and the disease, the viability and testing of new antiviral therapies, and any drugs in consideration for prophylaxis and treatment of sars-cov- /covid- . among several alternatives, chloroquine (cq) and its analog hydroxychloroquine (hcq) gained special attention from governments after the publication of in vitro studies followed by limited clinical results , with vast news dissemination, particularly in the us and brazil. cq and hcq are antimalarial agents and have been prescribed for treatment of autoimmune diseases (e.g. rheumatoid arthritis and lupus) for almost years . the prescription of cq/hcq in malaria is for prophylaxis and/or for when drugs accumulate in malaria-infected erythrocytes and interfere with the toxic heme formation during the parasite growth. in rheumatic diseases, cq/hcq might exert multiple anti-inflammatory effects, which are associated with the drug affinity to the autophagosomes and lysosomes of leukocytes . cq/hcq could affect the autoantigen presentation of leukocytes along the lysosomal pathway in autoimmune diseases, by disrupting the mhcii pathway . in addition, cq and hcq alter the production of potent pro-inflammatory cytokines, such as tnfα and il , by interfering with the endosomal ph during the activation of toll-like receptors , . in the context of the new sars-cov- infection and covid- pathogenesis, figure shows possible mechanisms of action based on sars (severe acute respiratory syndrome) . for sars-cov- , both cq and hcq perform antiviral activity in entry and post-entry stages of the -ncov in kidney epithelial cells (vero e cell, atcc- ) , . sars-cov- uses the ace (angiotensin-converting enzyme- ) receptor for cell entry by receptor-mediated endocytosis, similarly to sars-cov. the entry of coronavirus into cell cytoplasm and figure. possible mechanisms of action for cq/hcq on sars-cov- /covid- infection. a) in vitro antiviral activity of cq and hcq have been shown for sars-cov and sars-cov- , viruses. both drugs can inhibit viral entry and viral genome delivery to the cell cytoplasm, by interfering with the ph of endocytic pathway along with the formation of early endosomes and its maturation into a lysosome, respectively. b) proposed anti-inflammatory role of cq and hcq against covid- inflammatory pathogenesis relies on the ability of the drug to inhibit the production of potent pro-inflammatory cytokines, such as tnfα and il , by interfering with the endosomal ph in leukocytes anti-inflammatory effects the release of virus content depend on the endocytic machinery, which cq/hcq could disrupt due to its ability to neutralize the ph in these compartments ( figure a) . the immunomodulatory activity of cq/hcq might interfere with the strong inflammatory response triggered by sars-cov- during development of the infection, but these ideas are hypothetical and not confirmed in pre-clinical or clinical studies. in fact, a recent gold standard, randomized, double-blind, placebo-controlled trial across the united states and parts of canada tested hydroxychloroquine as post exposure prophylaxis in subjects. this trial reported that hydroxychloroquine did not prevent illness compatible with covid- or confirmed infection when used as post exposure prophylaxis within days after exposure . it has been demonstrated that the rapid virus infection and replication leads to a dysregulated immune response that progress to a cytokine storm response in severe covid- , followed by the development of ards, septic shock, and eventual multiple organ failure . since cq/hcq might act through the inhibition of cell signaling mechanisms resulting in blunting of pro-inflammatory cytokines (figure b) , it might be tempting to propose that both drugs would reduce the immunopathological tissue damage caused by viral infection . the major problem is that no rigorous pre-clinical cell-based, animal, nor randomized clinical studies were conducted to investigate these potential new mechanisms for antimalarial drugs or their clinical effectiveness to treat covid- . however, there is bounteous evidence of their very detrimental side effects, and non-appropriated prescription can cause acute poisoning and even death. this is especially important when, in face of the recent spread of news for supposed benefits of cq and hcq against sars-cov- /covid- , individuals can self-medicate to prevent sars-cov- infection with off-label medicines, or obtain prescriptions from unethical practitioners. on march , , the world health organization published specific guidance about the use of "off-label" drugs for covid- in clinical trials, especially concerning the use of cq and hcq without proper medical prescription . on april , , the national institutes of health published a strong rebuttal against their use for the treatment of covid- . on may , , the world health organization temporarily suspended cq and hcq use in the solidarity trial . due to their lysosomal affinity, cq and hcq accumulate in cells from several tissues with consequent tissue injury in the liver , retina , skeletal , and cardiac muscle cells . as announced by the fda on april , , side effects of hcq include irreversible cardiac effects (including cardiomyopathy and qt prolongation), proximal myopathy and neuropathy . importantly, cq and hcq have long half-lives after oral administration ( - days) . the resolution of cardiac and skeletal muscle symptoms is frequently slow after hcq or cq discontinuation . patients with cq-or hcq-induced myopathy frequently present progressive proximal muscle weakness, dyspnea, absent leg reflexes, and ventilator failure in severe cases , , . specifically for hcq-induced cardiotoxicity, higher risk groups are older adults, cardiovascular patients, and patients with renal insufficiency . moreover, reported side effects of cq/hcq on skeletal and cardiac muscles are abundant for patients suffering from rheumatic diseases receiving these medications . the us center for diseases control and prevention (cdc) recently updated the nine key symptoms for sars-cov- /covid- , and among these, three are related to the musculoskeletal system. therefore, to prescribe prophylactic drugs that can trigger these problems in populations such older adults or obese/ diabetic patients that have sub-optimal musculoskeletal health defies scientific reasoning. the concept of using cq/hcq as prophylactic or therapeutic alternatives for sars-cov- infection is, at best, hypothetical, but their side effects are factual. in fact, cq/hcq could contribute to the exacerbation of musculoskeletal diseases in older adults at risk for developing severe covid- . also, some of the characteristics of rheumatic patients at risk for developing cq-and hcq-induced myopathies are old age and other remarkable underlying http://doi.org/ . /s - . medical conditions , , which are also found at the higher-risk patients for developing severe covid- . thus, we end this comment by invoking the foundation of medical ethical treatment for over , years: primum non nocere-first do no harm! remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial hydroxychloroquine -how much is too much? chloroquine interference with hemoglobin endocytic trafficking suppresses adaptive heme and iron homeostasis in macrophages: the paradox of an antimalarial agent effects of chloroquine on viral infections: an old drug against today's diseases? hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro a randomized trial of hydroxychloroquine as postexposure prophylaxis for covid- epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study the use of anti-inflammatory drugs in the treatment of people with severe coronavirus disease (covid- ): the perspectives of clinical immunologists from china coronavirus disease (covid- ): current status and future perspectives solidarity clinical trial for covid- treatments. geneva: who us national institutes of health. covid- treatment guidelines. overview and spectrum of covid- : summary recommendations. bethesda, md: nih world health organization. q&a: hydroxychloroquine and covid- . geneva: who hydroxychloroquine-induced toxic hepatitis in a patient with systemic lupus erythematosus: a case report hydroxychloroquine retinopathy. eye (lond) antimalarial myopathy: an underdiagnosed complication? prospective longitudinal study of patients hydroxychloroquine-induced cardiomyopathy: case report, pathophysiology, diagnosis, and treatment silver spring, md: fda; hydroxychloroquine neuromyotoxicity fatal antimalarial-induced cardiomyopathy: report of cases the effect of malaria and anti-malarial drugs on skeletal and cardiac muscles cardiovascular implications of fatal outcomes of patients with coronavirus disease (covid- ) hydroxychloroquine cardiotoxicity presenting as a rapidly evolving biventricular cardiomyopathy: key diagnostic features and literature review funding: cb, mm, mb are supported by nih grants: national institutes of aging (nia) -po ag nia-r ag ; national institutes of neurological disorders and stroke (ninds) -r ns ; national institutes of diabetes, digestive, and kidney diseases kidney and niddk-r dk jay and evanston research endowments (mb), and the uta college of nursing & health innovation bone-muscle research center the authors declare no conflict of interest. key: cord- -b yqek authors: sridhar, arun r.; chatterjee, neal a.; saour, basil; nguyen, dan; starnes, elizabeth; johnston, christine; green, margaret l.; roth, gregory a.; poole, jeanne e. title: qt interval and arrhythmic safety of hydroxychloroquine monotherapy in coronavirus disease date: - - journal: heart rhythm o doi: . /j.hroo. . . sha: doc_id: cord_uid: b yqek background: observational studies have suggested increased arrhythmic and cardiovascular risk with the combination use of hydroxychloroquine (hcq) and azithromycin in patients with coronavirus disease (covid- ). objective: the arrhythmic safety profile of hcq monotherapy, which remains under investigation as a therapeutic and prophylactic agent in covid- , is less established and we sought to evaluate this. methods: in consecutive patients with covid- admitted to the university of washington hospital system between march and may , , we identified treated with hcq monotherapy. patients treated with hcq underwent a systematic arrhythmia and qt interval surveillance protocol including serial electrocardiograms (ecg) (baseline, following second hcq dose). the primary endpoint was in-hospital sustained ventricular arrhythmia or arrhythmic cardiac arrest. secondary endpoints included clinically significant qtc prolongation. results: a total of patients with covid- underwent treatment with hcq monotherapy (mean age ± years, women [ %], serum creatinine . [interquartile range . ] mg/dl). there were no instances of sustained ventricular arrythmia or arrhythmic cardiac arrest. in patients with serial ecgs, clinically significant qtc prolongation was observed in a minority (n= [ %]). in patients with serial ecgs, there was no significant change in the qtc interval in pre-specified subgroups of interest, including those with prevalent cardiovascular disease or baseline use of renin-angiotensin-aldosterone axis inhibitors. conclusion: in the context of a systematic monitoring protocol, hcq monotherapy in hospitalized covid- patients was not associated with malignant ventricular arrhythmia. a minority of patients demonstrated clinically significant qtc prolongation during hcq therapy. severe acute respiratory syndrome coronavirus (sars-cov- ) is the pathogenic cause of coronavirus disease (covid- ) which is an ongoing global pandemic. given its immunomodulatory and antiviral effects against sars-cov- in-vitro, hydroxychloroquine (hcq) is being evaluated in multiple randomized clinical trials as a therapeutic and prophylactic strategy for covid- . recent observational cohort data have suggested a neutral effect of hcq on overall in-hospital mortality for patients with covid- , although there remain concerns regarding its cardiovascular risk profile. hcq can block the kcnh -encoded herg/kv . potassium channel and thereby carries a risk of drug-induced qt prolongation and cardiac arrest. , recent single-center studies evaluating the electrical effects of hcq in combination with azithromycin (azm) have identified clinically significant qt prolongation in up to % of patients , and combination therapy was associated with an increased risk of inhospital cardiac arrest in one observational study. the majority of these data, however, have focused on combination hcq/azm therapy, and thus may not generalize to hcq monotherapy which remains a point of active investigation in several ongoing clinical trials. therefore, in this study, we sought to evaluate the arrhythmic safety profile of hcq monotherapy in patients with covid- . we identified patients admitted to the university of washington medical system with sars-cov- infection on the basis of quantitative reverse transcriptase-polymerase chain reaction (qrt-pcr) testing between march , and may , . after excluding patients who were not treated with hcq, the final study cohort included patients. the study was approved by the institutional review board (irb) of the university of washington. informed consent was not obtained from the patients in accordance with uw irb, as this was a retrospective chart review study. hcq was administered in accordance with a pre-specified protocol developed by the university of washington covid- task force ( mg twice a day for one day followed by mg twice a day for four days). all qt prolonging medications were discontinued prior to initiation of hcq therapy. qtc monitoring was pre-specified ( figure ) with a baseline (pre-hcq) -lead electrocardiogram (ecg) and follow-up on-treatment qt assessment after the nd dose as captured by -lead ecg or telemetry. ontreatment ecg timing was selected based on physiologically-based pharmacokinetic (pbpk) modeling of the dosing regimen which identified steady-state concentrations of hcq following the initial loading dose (i.e. after the nd dose of mg). for patients with clinically significant prolongation of the qtc (> msec or absolute increase > msec), caution was advised regarding continued hcq therapy. detailed retrospective chart review of patient demographics, baseline cardiovascular and pulmonary comorbidities, and ecg parameters was performed. qtc interval was manually adjudicated and defined using the bazett correction (qtc = qt / √ rr) employing the longest measured qt (lead ii, v , or v ). the primary outcome was the incidence of sustained ventricular arrhythmia and/or arrhythmic cardiac arrest (i.e. ventricular tachycardia and/or fibrillation). the primary outcome was evaluated from the date of admission to the first occurrence of death, in-hospital cardiac arrest, hospital discharge or may , . secondary outcomes included clinically significant qtc prolongation (absolute on-treatment qtc > msec or increase in qtc > msec following hcq therapy). clinical outcomes were adjudicated until may , . change in electrical parameters were compared using a paired t-test. qtc change was evaluated in pre-specified subgroups of interest including patients with baseline cardiovascular disease, elevated serum creatinine on admission, baseline use of renin angiotensin aldosterone system (raas) blockade pharmacotherapy, and initial admission to an intensive care unit. as bazett correction may overestimate qt intervals at faster heart rates, a sensitivity analysis was performed using the hodges correction [qtch = qt + . (hr- ) = qt + *( /rr- )] which is the least sensitive to heart rate amongst qt correction methods. all probability values were two sided, and a p-value cutoff of ≤ . was used to determine statistical significance. all statistical analysis was performed using sas version . (sas institute inc, cary, nc). the study cohort was comprised of patients with covid- treated with hcq ( over days of person-time follow-up in patients treated with hcq, there were deaths ( %) in which none were attributable to ventricular arrhythmia. incident arrhythmia was observed in patients ( %) including premature ventricular ectopy (n= [ %]) and atrial tachycardia or atrial fibrillation (n= [ %]). there was one instance of sinus bradycardia with an accelerated idioventricular rhythm in a critically-ill patient. there were no instances of high-grade atrio-ventricular block (mobitz ii, complete heart block) or sustained ventricular tachyarrhythmia (either monomorphic or polymorphic). of patients with covid- treated with hcq, patients had serial ecgs including baseline and follow-up after the second dose of hcq (figure ) . in this subset, the mean change in qtc was - ± msec (range - to + msec; p, for ∆ qtc = . ). the mean relative change in qtc was ± %. in subgroup analysis, there was no significant change in qtc in patients with prevalent cardiovascular disease (n= ; ∆qtc = - ± msec; p= . ), baseline raas inhibitor use (n= ; ∆ qtc - ± msec; p= . ), admission serum creatinine ≥ . mg/dl (n= ; ∆ qtc - ± msec; p= . ), or intensive care unit location on admission (n= ; ∆ qtc - ± ; p= . ). a minority of patients demonstrated clinically significant lengthening of the qtc (n= [ %]) reflected by either on-treatment qtc ≥ msec (n= ) or increment in absolute qtc of greater than msec (n= ). the three patients with on-treament qtc of ≥ msec ( , , msec) had baseline qtc prolongation (qtc , , msec respectively) whereas the two patients with an increment in qtc > msec had on-treatment qtc intervals that were within a normal range ( and msec). there were no instances of discontinuation of hcq on the basis of qt or arrhythmic monitoring. given the risk of qt interval overestimation with the use of bazett's correction, and the observed decline in heart rate from baseline to follow-up ecg in our study (- ± beats per minute), we performed a sensitivity analysis employing the hodges correction for qt interval (qtch) which is the least sensitive to heart rate compared to other qt correction methods. similar to the findings using the bazett correction, the mean change in qtch in patients with paired ecgs was not significant ( ± msec, range - to + msec; p, for ∆ qtc = . ). subgroup analysis similarly demonstrated nonsignificant changes in qtch in patients with prevalent cardiovascular disease (∆ qtch = - ± msec; p= . ), baseline raas inhibitor use (∆ qtch = - ± msec; p= . ), admission serum creatinine ≥ . mg/dl (∆qtch = . ± msec; p= . ), or intensive care unit admission (∆qtch = ± msec; p= . ). the incidence of clinically significant qtch lengthening was similarly rare (n= [ %]) reflecting ontreatment qtch ≥ msec (n= ) or increment of absolute qtch of greater than msec (n= ). in this multi-hospital retrospective cohort study of covid- patients, hcq monotherapy was not associated with malignant ventricular arrhythmias or arrhythmic cardiac arrest. in patients with paired ecg evaluation, there was no significant increment in the qt interval in the majority of patients. these findings extended to patients with elevated admission serum creatinine, baseline raas inhibitor use, prevalent cardiovascular disease, and initial admission to an intensive care unit. importantly, nearly onethird of patients were treated with a concomitant qt prolonging pharmacotherapy prior to initiation of hcq. taken together, in patients with covid- treated with hcq monotherapy, implementation of a systematic qt monitoring protocol demonstrated a low rate of clinically actionable qt prolongation and no instances of malignant arrhythmias or arrhythmic death. in the ever-changing landscape of covid- pharmacotherapy, there are several agents under active investigation which have the potential for qt prolongation including hydroxychloroquine, chloroquine, azithromycin, lopinavir-ritonavir and fingolimod. recent cohort studies have demonstrated variable impact of hcq with or without azm on qt prolongation and electrical risk in patients with covid- . rosenberg and colleagues identified an increased risk of in-hospital cardiac arrest in patients treated with hcq/azm, but not hcq monotherapy, when compared to those treated without either. in patients treated with hcq alone, they observed a % incidence of qt prolongation, although ecg assessment was not protocolized or adjudicated, hcq dosing was heterogeneous, detailed information regarding quantitative qt measures were lacking, candidate ecgs were considered at any point during hospitalization, and qt prolongation was defined broadly. mercuro and colleagues evaluate patients treated with hcq (maintenance dose mg daily), in whom were additionally treated with azm, and noted that % of patients treated with hcq monotherapy and % of those treated with hcq and azm demonstrated a qtc interval longer than msec. of note, the majority of these patients were taking two or more qtc prolonging medications. in a smaller study of patients with covid- admitted to the intensive care unit, bessiere and colleagues observed a prolonged qtc (> msec or increase > msec) in % of patients treated with hcq monotherapy ( patients) or hcq + azm ( patients). finally, saleh and colleagues evaluated patients with covid- treated with hcq or chloroquine monotherapy and patients treated with hcq + azm. in patients treated with hcq or chloroquine monotherapy, on-treatment qtc > msec was observed in % although given the potential differential effects of chloroquine versus hcq on qt prolongation, the specific risk of hcq monotherapy was less certain. our study adds to this evidence base in several meaningful ways. first, these data represent the largest series to date of adjudicated arrhythmic and electrical safety of hcq monotherapy in covid- . in keeping with previously published work, , , our data highlights the low rate of malignant ventricular arrhythmias -specifically torsades de pointes -in covid- patients treated with a delimited course of hcq. our finding of clinically actionable qtc prolongation in % of our cohort is closely aligned with the findings of saleh and colleagues whose study employed a similar hcq dosing protocol, but substantially lower than other series with higher absolute daily drug dosage of hcq, higher rates of concomitant qt prolonging medication use, and higher rates of intensive care unit status. , in contrast to discrete qtc safety thresholds (i.e. absolute increment > msec, on-treatment ∆ > - msec), previous data evaluating the presence of any qt prolongation associated with hcq monotherapy are likely to overestimate arrhythmic risk. larger cohorts will be necessary to better delineate the continuous relationship between qt interval and malignant arrhythmia. second, nearly one-third of patients in our cohort were treated with a qt prolonging medication prior to initiation of hcq. given the overlap in clincal presentation of covid- and community acquired pneumonia, the most common qt prolonging drug therapy was either fluoroquinolone or azithromycin. the discontinuation of concomitant qt prolonging drug therapy and correction of underlying electrolyte abnormalities is a critical feature of the systematic protocol employed in this study and important context for the identified electrical and arrhythmic safety profile identified here. we would hypothesize that at least some of the decrease in qt interval observed in some patients in this study was related to discontinuation of qt prolonging drugs and correction of electrolyte abnormalities. third, we did not identify any specific subgroups at increased risk of malignant arrhythmia or excessive qt prolongation. while a previous study suggested that baseline use of loop diuretics and baseline qtc > msec may be risk factors for subsequent electrical risk with hcq, those findings were exploratory given the limited sample size within which they were evaluated. of note, other groups -similar to our study -have found no specific risk factors for arrhythmic risk following hcq monotherapy in covid- . there are, nonetheless, specific subgroups including those with genetic long qt syndrome, in whom the risk-benefit of hcq use should be carefully considered. there are presently more than clinical trials evaluating the safety and efficacy of hcq as either treatment or prophylaxis for patients with covid- . our study, in concert with previously published cohort data regarding the arrhythmic safety profile of hcq monotherapy, , , provides meaningful information for patients and clinical investigators. we would highlight that these data are specific to the dosing regimen employed in this study and may not generalize to higher-dose protocols. these safety data are also of particular relevance to patients and healthcare providers practicing in resource-limited healthcare settings, where availability of serial ecgs may be limited. important ancillary features of the systematic protocol employed here include systematic discontinuation of concomitant qtc prolonging medications and correction of electrolyte abnormalities. while our study was not specifically designed to evaluate the timing of hcq initiation following discontinuation of qtc prolonging medication, this decision would likely be guided by the half-life of the qtc prolonging medication in question and the clinical implications of delaying hcq therapy. practically, acquisition of a lead ecg or use of routine cardiac telemetry to evaluate the qt interval in covid- patients may be challenging given broader attempts to minimize caregiver exposure and maximize personal protective equipment supplies. use of continuous telemetry in patients treated with hcq may be reasonable during the initial loading dose of the protocol, during which time qt monitoring accrues. while our study cannot specifically guide the duration of continuous telemetry monitoring in hcq-treated covid- patients, our data would suggest that the risk of de novo arrhythmias during hcq therapy is low. future work identifying other risk factors for in-hospital arrhythmias is warranted to guide the indication for both in-hospital and post-hospital arrhythmia surveillance in covid- . looking ahead, novel technology -including the use of ambulatory event monitors (mobile continuous telemetry) or smart-phone based applications -will be important to integrate into workflows for arrhythmic safety monitoring in these patients. as pharmacokinetic simulation data indicate that hcq drug concentrations may exceed the % effective concentration (ec ) for several days following completion of a delimited hcq dosing protocol (e.g. hcq concentrations > ec on day following a day treatment course), there may be rationale to continue to avoid qt prolonging medications during this period, though further study is warranted. to the extent that hcq concentrations are anticipated to reach steady state following the loading dose used in this study ( mg bid x day), the residual arrhythmic risk attributable to hcq following the dosing protocol would be anticipated to be captured by ontreatment qt monitoring. strengths of our study include the implementation of a systematic protocol including serial ecg assessment, adjudication of electrical parameters including qt interval, and evaluation of arrhythmic risk within pre-specified subgroups at increased potential risk. there are, however, limitations. first, not all patients receiving hcq underwent baseline ecg. we do not anticipate that these patients would have had systematically different baseline or follow-up electrical responses to hcq, though we cannot rule this out. second, ascertainment of in-hospital arrhythmia was contingent on clinical documentation and availability of surveillance (ecg, telemetry). while the overall incidence of arrhythmia may be underestimated, we believe our findings related to clinically-actionable and malignant arrhythmias (cardiac arrest, torsades de pointes) are less likely to be impacted by this limitation. second, given the low incidence of qt prolongation and incident arrhythmia, our study was not able to specifically comment on the relationship between qt prolongation and incident arrhythmia. third, in order to maximize the generalizeability and applicability of our study to real-world applications of qt monitoring, we employed the bazett correction of the qt interval for baseline and follow-up ecg. as the bazett correction may overestimate the qt interval at faster heart rates, given the overall decline in heart rate from baseline to follow-up ecg in our study, the magnitude of qtc prolongation may have been biased towards the null. importantly, we demonstrate that the findings of the study were robust in a sensitivity analysis using a qt correction method (hodges) that is the least sensitive to heart rate when compared to other methods (bazett, friderica, framingham). finally, given the overall low incidence of qt prolongation and malignant arrhythmia, our study may have been underpowered for these endpoints within pre-specific subgroups. in this retrospective, cohort study of covid- patients treated with hcq monotherapy, there were no instances of malignant ventricular arrhythmias or arrhythmic cardiac arrest. a minority of patients ( %) demonstrated clinically significant on-treatment qtc prolongation (> msec increment) or long qtc (> msec). future studies should focus on identification of those at highest arrhythmic risk with hcq therapy to more optimally guide arrhythmia surveillance and prevention in patients with covid- . hashem am, alghamdi bs, algaissi aa, alshehri fs, bukhari a, alfaleh ma, memish za. therapeutic use of chloroquine and hydroxychloroquine in covid- and other viral infections: a narrative review. travel medicine and infectious disease may . • in patients hospitalized with coronavirus disease (covid- ), a day treatment regimen of hydroxychloroquine (hcq) monotherapy was not associated with in-hospital malignant ventricular arrhythmia over days of person-time follow-up. • hcq use was not associated with clinically significant qtc prolongation in the majority of patients ( %). this finding was robust when using different qt correction methods (bazett, hodges) and consistent across several subgroups of interest (prevalent cardiovascular disease, baseline renin angiotensin aldosterone system inhibitor use, and elevated admission serum creatinine). • using a standardized arrhythmic and qt surveillance protocol which includes discontinuation of qt prolonging medications and correction of electrolyte abnormalities, hcq monotherapy demonstrated a reasonable arrhythmic safety profile with a low incidence of clinically significant qt prolongation. a novel coronavirus emerging in china -key questions for impact assessment. the new england journal of medicine in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) observational study of hydroxychloroquine in hospitalized patients with covid- urgent guidance for navigating and circumventing the qtc prolonging and torsadogenic potential of possible pharmacotherapies for covid- chronic hydroxychloroquine use associated with qt prolongation and refractory ventricular arrhythmia assessment of qt intervals in a case series of patients with coronavirus disease (covid- ) infection treated with hydroxychloroquine alone or in combination with azithromycin in an intensive care unit risk of qt interval prolongation associated with use of hydroxychloroquine with or without concomitant azithromycin among hospitalized patients testing positive for coronavirus disease (covid- ) association of treatment with hydroxychloroquine or azithromycin with in-hospital mortality in patients with covid- in chloroquine and hydroxychloroquine for the prevention or treatment of novel coronavirus disease (covid- ) in africa: caution for inappropriate off-label use in healthcare settings. the american journal of tropical medicine and hygiene optimal qt interval correction formula in sinus tachycardia for identifying cardiovascular and mortality risk: findings from the penn atrial fibrillation free study a comparison of commonly used qt correction formulae: the effect of heart rate on the qtc of normal ecgs the effect of chloroquine, hydroxychloroquine and azithromycin on the corrected qt interval in patients with sars-cov- infection. circulation arrhythmia and electrophysiology prevention of torsade de pointes in hospital settings: a scientific statement from the american heart association and the american college of cardiology foundation continuous variables are expressed as mean±standard deviation or median renin angiotensin aldosterone system. *anti-psychotic or anti-depressants discontinued included mirtazapine, olanzapine, sertraline, venlafaxine, and aripiprazole. †tacrolimus there was no external funding for this research. none to declare key: cord- -kakxmc authors: achutha, a. s.; pushpa, v. l.; suchitra, surendran title: theoretical insights into the anti-sars-cov- activity of chloroquine and its analogs and in silico screening of main protease inhibitors date: - - journal: j proteome res doi: . /acs.jproteome. c sha: doc_id: cord_uid: kakxmc [image: see text] corona virus disease (covid- ) is a dangerous disease rapidly spreading all over the world today. currently there are no treatment options for it. drug repurposing studies explored the potency of antimalarial drugs, chloroquine and hydroxychloroquine, against sars-cov- virus. these drugs can inhibit the viral protease, called chymotrypsin-like cysteine protease, also known as main protease ( cl(pro)); hence, we studied the binding efficiencies of -aminoquinoline and -aminoquinoline analogs of chloroquine. six compounds furnished better binding energies than chloroquine and hydroxychloroquine. the interactions with the active site residues especially with cys and his , which are involved in catalytic diad for proteolysis, make these compounds potent main protease inhibitors. a regression model correlating binding energy and the molecular descriptors for chloroquine analogs was generated with r( ) = . and q( ) = . . this model was used to screen new analogs of primaquine and molecules from the asinex compound library. the docking and regression analysis showed these analogs to be more potent inhibitors of cl(pro) than hydroxychloroquine and primaquine. the molecular dynamic simulations of the hits were carried out to determine the binding stabilities. finally, we propose four compounds that show drug likeness toward sars-cov- that can be further validated through in vitro and in vivo studies. in december , patients were admitted in hospitals in wuhan, china due to pneumonia with fever and other symptoms. , in january , it was discovered that a novel coronavirus, which is similar to sars-cov and mers-cov genomes, is responsible for this disease. this virus is named as severe acute respiratory syndrome corona virus- (sars-cov- ) and the disease is called corona virus disease- . , it was declared as pandemic on march by the world health organization (who). as of september , , there are , , confirmed cases with , deaths worldwide according to who reports. coronavirus is a single-stranded positive sense rna with a membrane envelope composed of four types of viruses: α, β, γ, and δ coronaviruses. sars-cov- belongs to β-coronavirus family. , its genome encodes for structural proteins mainly spike glycoprotein (s), envelope glycoprotein (e), membrane glycoprotein (m), nucleocapsid protein (n), and nonstructural proteins consisting of chymotrypsin-like cysteine protease also known as main protease ( cl pro ) and papain-like protease (pl pro ). , , the viral rna is released to the host cell, which is then translated to polyproteins inside the cell. these viral polyproteins are cleaved to functional proteins by proteolysis, which is catalyzed by cl pro and pl pro , , . since the main protease or cl pro cleaves at sites of the polyprotein to produce smaller proteins for viral replication and because of its nonsimilarity with human proteins, cl pro is a potential target in anti-covid- drug design. , the main protease of sars-cov- has a cys-his catalytic diad involving cys and his and has four conserved binding subsites s , s , s , and s ′. main chains of cys , phe , and leu and side chains of asn , glu , his , and his residues are present in the s subsite. the side chains of his , met , tyr , and met , along with the alkyl side chain of asp forms the s subsite. the s subsite consists of met , leu , phe , and gln side chain residues and the main chain of gln . s ′ includes thr and thr . therefore, a molecule to show cl pro inhibitory property should interact with one or more residues among these amino acids. also, the inhibitory action can be assumed to enhance if they interact with cys or his , which is involved in the catalytic diad of main protease ( figure ). cysteine acts as a nucleophile and histidine as an acid/base catalyst in this catalytic reaction. , currently there are no specific approved treatment options for covid- . researches are in search of vaccine development, druggable small molecule, monoclonal antibodies and cell-based therapies. several drug repurposing studies have been conducted so far, and many of them gave positive results. the -aminoquinolines, chloroquine (cq), and its hydroxyl analog hydroxychloroquine (hcq) have been found to disrupt the viral replication and infectivity. their broad spectrum of activity as antibacterial, antiviral, and antifungal infections has also been reported. , hcq has been found out to be less toxic than cq, and it is currently being used to treat covid- patients. the present study deals with theoretical perspectives on the anti-sars-cov- activity of cq and hcq analogs by observing their main protease inhibitory property. molecular docking studies with the cl pro protein were performed to analyze the drug likeness as well as to correlate the binding energy of the docked complex with various physicochemical properties of the active molecules, which will aid in the design of new anti-covid- medicatives. analogs of cq and hcq as well as primaquine derivatives were obtained through literature search, from the database of small molecules pubchem as well as created manually. − all the molecules were drawn in marvin sketch and minimized their energy using avogadro software. , the structure of cl pro protein having pdb id lu was retrieved from rcsb protein data bank. , characterization studies of the protein were done by using expasy-protparam, an online server, which provides all the details regarding the protein. the energy minimization of the retrieved protein was done by employing swiss pbd viewer. , molecular docking was done using autodock . . . both protein and ligands were prepared in pdbqt format. polar hydrogens and gasteiger charges were added to the receptor. grid was prepared with × × Å with spacing . Å and centered at − . , . , and . Å along x, y, and z axes, respectively. grids were prepared for every atom present in the ligand data set. the genetic algorithm was employed as search parameter with runs, and , population size and number of generations, respectively. a multiple linear regression (mlr) model was generated to find the correlation with binding energy obtained from docking and the physicochemical properties using qsarins. , the molecular descriptors for the ligands were calculated utilizing padel-descriptor. to build the model, % of the data set were randomly divided as training set and test set in : ratio. thirty-five compounds ( training and test) including cq and hcq were employed for modeling purpose (supplementary tables s and s ). the models were generated using training set with mlr analysis of − variables. the generated models were examined by internal validation and external validation. internal validation was done by cross-validated leave-one-out (loo) method, which involves iteratively leaving one compound from the training set and generating regression model with the remaining molecules and predicting the value of response for the excluded one. for a good model, the regression coefficient (r ) as well as the cross validated r , that is, q has to be close and > . . , the model was validated using test set, which was not included in model generation. , the best model was used to predict the binding energy of novel set of compounds. all the md simulations were done using the namd package (version . ) developed by the theoretical and computational biophysics group in the beckman institute for advanced science and technology at the university of illinois at urbana−champaign with the charmm force field. , the docked structures were used as the initial coordinates for md simulations. tip p water box was used to solvate the complex with Å padding. the solvated system was minimized for ps with a time step of fs. the simulations were done with particle mesh ewald electrostatics and periodic boundary conditions. atom-based cutoff of Å was applied for nonbonding interactions. the temperature and pressure were kept constant at k and atm, respectively, using langevin dynamics. the system was equilibrated for ps followed by a production simulation for ns using the npt ensemble. all the preparation and analysis steps were done using the vmd package . . (available at http://www. ks.uiuc.edu/research/vmd/) (see figure ). being an inevitable enzyme for replication and transcription of the virus, the -chymotrypsin-like-cysteine protease ( cl pro ) having pdb id lu was retrieved from rcsb pdb and characterized by primary and secondary structure analysis as shown in table . the pi value (isoelectric point) of . shows the slightly acidic nature of the protein. the aliphatic index (ai) is a measure of the relative volume occupied by the side chains ala, leu, ile, and val, which are the aliphatic residues in a protein. high ai ranging from . to . implies high thermal stability and hydrophobicity, which help it for membrane penetration in biological system. , the value for grand average of hydropathy (gravy) indicates the hydrophilicity of the protein. the gravy value of − . , which is close to zero, shows that the protein is hydrophobic. instability index < shows that in vivo half-life is greater than h, which indicates the stability of the protein. , all the parameters show that the lu is a stable, hydrophobic protein. the lu protein consists of three domains in which the catalytically active site is present between domain i and ii. figure s (supporting information) shows the domains and active site of lu protein bound with its cocrystallized inhibitor n . , the antimalarial drugs chloroquine (cq) and its hydroxy analog hydroxychloroquine (hcq) are currently used as drugs for the treatment of covid- . , to compare its efficiency as an anti-covid- drug to that of an antimalarial drug, docking studies were carried out for both targets, cl pro and pfdhfr-ts. plasmodium falciparum dihydrofolate reductase thymidylate synthase (pfdhfr-ts) is one of the important targets for antimalarial drugs , (supplementary table s ). it was identified that for both the targets hcq has lower binding energy owing to its higher binding affinity to the protein. both the drugs are more active on pfdhfr-ts than cl pro . the d interaction diagrams and docked poses of cq and hcq with the target cl pro are given in figures s and s , respectively. hcq fits more perfectly to the active site of the target than cq, and hence, hcq has high negative binding energy, which imparts more stability to the docked complex (supplementary figure s ) . cq forms three hydrogen bonds with gly , cys , and his ; van der waals interaction with asn and gln ; πalkyl interaction with his ; and two π-donor hydrogen bond interactions with the −sh group of cys at distances . Å, . Å, . Å, . Å, . Å, . Å, . Å, and . Å, respectively (supplementary figure s a , table ). the cys -his diad is blocked by these interactions. the interaction of alkyl group of the cq with the π-cloud of imidazole ring of histidine may disturb the delocalization of π electrons. the basicity of the imidazole ring is due to the resonance stabilization of the positive imidazolium ion ( figure ). the alkyl−π interaction of the alkyl donor and aromatic acceptor increases the stability of the docked complex as well as reduces the ability of the imidazole ring to accept h + from the cys residue, which is the initial step in the catalytic cycle in proteolysis, thereby reducing the activity. , hcq forms two hydrogen bonds with phe , and one each with asn , ser , and glu at distances . Å, . Å, . Å, . Å, and . Å, respectively. it forms two van der waals interactions with phe , and one each with glu and his at distances . Å, . Å, . Å, and . Å, respectively. it also forms a π−σ interaction with asn and two π−alkyl interactions with cys (supplementary figure s b , table ). the π−alkyl interaction with cys may disturb the conformation required for the interaction with polypeptide in the catalytic cycle. there have been reports about the entry of sars coronavirus as acidic ph dependent in which the activation occurs by the fusion of the membranes of virus and cellular endosomes and viral genome enters the cytoplasm. , , the presence of amino side chain in cq and hcq makes them basic and increases the ph of cell organelles like endosomes and lysosomes, which interferes with the replication and inhibits the viral activation. , to find more potent inhibitors of cl pro , analogs of chloroquine were subjected to molecular docking, which were obtained from literature, pubchem database, by replacement with isosteres and functional group variations − of chloroquine (supplementary tables s and s ). the binding energy thus obtained from docking with lu protein is given in table . all the chloroquine analogs show some of the key interactions with the active site residues. the negative value shows a release of energy while forming a protein−ligand docked complex, which imparts stability. the more negative the binding energy, the higher will be the stability and binding affinity. the compounds , , , , , and have more negative binding energy than cq and hcq. their d ligand interaction diagrams are shown in supplementary figure s . from the structures of these six compounds, we can infer that if the side chain has an extra −nh group, it can introduce additional hydrogen bonds, which results in a decrease in binding energy. in compound , the tail of side chain has become a part of a ring, which can interact with met residue via π−alkyl interaction. because of the presence of an additional −nh group, the basicity of these compounds is higher than cq and hcq, and thus, they can act as better inhibitors against sars-cov- . compounds , , , , and form π-donor hydrogen bonds with cys , which will make the −sh hydrogen less available for catalytic cycle. compounds , , , , and have a π−alkyl interaction with cys . overall, these interactions may affect the catalysis of proteolysis reaction journal of proteome research pubs.acs.org/jpr article and hence block viral replication. compound is primaquine (an -aminoquinoline), which is an antimalarial drug. , the effect of the side chain in the chloroquinoline scaffold was studied by changing the chain length and methyl substitution of the side chain. compounds and are obtained by the removal of the branching methyl group from cq and hcq structures, which results in an increase in the binding energy to − . and − . kcal/mol, respectively. therefore, it is understood that the branching next to the −nh− group in -aminopentyl side chain is important. isosteric substitution of −ch group in compounds , , and with −oh, −nh , and −cf results in binding energies − . , − . , and − . kcal/mol, respectively. substitution with nh resulted in a decrease in binding energy compared to hcq by a factor of − . kcal/mol, which means that an extra . kcal/mol of energy is released while forming a docked complex, which is more stable than that of hcq. an increase in binding energy due to the variation of side chain length in cq and hcq structures of compounds , , , , , , , , and makes them less stable complexes with the protein than with cq and hcq. therefore, the chain length of -aminopentyl group is optimal for showing biological activity. when an additional −oh group is added to the ethyl chain of hcq resulting in -[( aminopentyl)( -hydroxyethyl)amino]ethan- -ol side chain as in compound , as a result, the binding energy is decreased to − . kcal/mol. therefore, the −oh group at one terminal ethyl chain only is required for their inhibitory activity. rearrangement of the amino side chain from the fourth position to fifth position of quinoline ring in hcq (molecule ) increased the binding energy by . kcal/mol. replacement of quinoline scaffold by isoquinoline scaffold also increased the binding energy by an amount of . kcal/ mol. the cq, hcq, and its analogs taken for this study were subjected to mlr analysis. regression models were generated with binding energy as the response and molecular descriptors as the variables. many models were generated by varying − variables at a time from a set of descriptors. the fitness criteria of a model include r ≥ . , r adj ≥ . , q loo ≥ . , high value for fisher ratio (f), low regression standard deviation (s), lower correlation between descriptors (k xx ), positive value for δ k , which is the difference in correlation between descriptors and the response (y) value and correlation among descriptors, and smaller root−mean−squared error for training calculation (rmse tr ). , the parameters of the best five models calculated are given in table . considering all the models generated, model has the highest fitness score. however, upon inspecting its external validation parameters rmse ext = . and r ext = . , the values did not meet the cutoff and hence show poor predictability. for a good prediction, rmse ext should not vary much from rmse tr (difference should be less) and r ext should be greater than . . model was selected for further studies since it was the best fit model with next highest r and q loo , which represents the fitness, robustness of the model, and the proximity of actual and predicted binding energy values. the model was built by incorporating the compounds , , , , , , and in the test set and all others in the training set. thus, the binding energy, the response value (y), can be expressed in terms of the descriptors considered for the generation of model in the form of a linear relationship y = Σm i x i + c where m i is the coefficient obtained for each descriptor in mlr model and x i is the value for the selected descriptor. the internal and external validation parameters determine the feasibility and predictivity of the model , (table s ). the linear equation correlating binding energy and descriptors generated by model can be written as shown in eq : the root−mean−square error for cross-validation rmse cv = . is greater than rmse tr = . , making the model stable and predictive. the criteria for y scrambling r y scr < . , q y scr < . , and r y scr > q y scr implies that there is no correlation between descriptors, and hence, the model is not simply correlated by chance. model was assessed with external validation parameters r ext > . and small difference between rmse ext and rmse tr and was hence acceptable due to its high predictivity. the predicted values of binding energy for both training and test set of molecules are given in table s . the plot of observed versus predicted binding energy of training and test set molecules is given in figure . substitution of −cl with −nh in quinoline scaffold in compound led to inaccurate prediction and hence was detected as an outlier. model shows that the binding energy is a linear combination of the descriptors sch- , nhsnh , minhbint , fp , and krfp . sch- is simple seventh order chi chain that is a topological descriptor based on interatomic distances calculated by the bonds between them representing molecular connectivity as a chemical graph like a bond-line formula of chemical structures. it considers the specificity of the structures at a fragment level rather than the whole molecule. the order represents the number of edges in the graph, which indicates the branching. , the negative value of its coefficient indicates its negative impact on biological activity. thus, a low value for sch- is required for a compound to show low binding energy, which makes a restriction on branching. nhsnh represents the number of nh groups and amine hydrogens. the positive coefficient of . indicates that as the number of nh groups increases, the binding energy decreases, which is in perfect agreement it is an atom-type electro-topological state descriptor in which both electronic and topological functions are considered. there is a positive dependence of minhbint with the binding energy because as the chances of h-bond increases, the binding energy will be more negative. fp and krfpc are fingerprint descriptors, which account for structure, specific properties, connectivity, or pharmacophores. krfpc accounts for the count of substructures also. both of these fingerprints are inversely related to binding energy. designing molecules in such a way that having high values for nhsnh and minhbint and low values for sch- , fp , and krfpc will help to reduce the binding energy considerably. compound , primaquine, shows the least binding energy among the cq analogs. it is the only antimalarial drug that can interfere with a majority of the life cycle stages of the malarial protozoan species. , the combined usage of chloroquine and primaquine may mask chloroquine resistance. however, its toxic effect is mainly due to the hemolytic lesions caused by methemoglobin production. , it is reported that introduction of a tert-butyl group at c position (r in scaffold given in table ) will increase the efficiency and is completely drained of from methemoglobin production as in compound pq . , by using the formulated regression model , we predicted the binding energy of some primaquine analogs obtained from the literature and pubchem database and then carried out their molecular docking studies on cl pro target to check the inhibitory potency of the ligands, given in table . also screened, , asinex compounds having antiviral properties using the regression model . it was observed that the predicted binding energy of some compounds obtained from journal of proteome research pubs.acs.org/jpr article the asinex database differed largely from its actual value, which was obtained from docking analysis. this may be due to their difference in scaffold from that of the compounds used to build the regression model. even though the predicted binding energy was less accurate, they have furnished better binding energies on docking with the target protein implying the reliability of the model. thirty molecules that showed lower binding energies were subjected to molecular docking analysis to identify the potent cl pro inhibitors (supplementary figure s ) . binding energies obtained through both the methods and their interactions with the receptor protein are given in table . all the primaquine analogs showed lower binding energies than cq and hcq. all the compounds except pq furnished binding energies lower than that of primaquine. the binding energies calculated using the regression model are in good agreement with the value obtained from docking analysis. all the compounds exhibited interactions with some of the active site residues including the catalytic diad residues either cys or his or both, which make them potent cl pro inhibitors. the compounds pq , pq , and pq exhibited lower binding energies of − . , − . , and − . kcal/mol. the d interaction diagrams of these ligands with the protein are given in figure . in accordance with the findings based on docking studies, the regression analysis of cq analogs reveals the presence of one extra −nh bond in their structure. inspecting the d interaction diagrams of compounds a , a , a , a , a , a , a , a it can be seen that there are unfavorable donor−donor or acceptor-acceptor interactions present between the compounds and target protein which may negatively affect the stability of the docked complexes (supplementary figure s ). hence these can be considered as poor inhibitors of cl pro protein even though they have binding energies less than hcq. from these results it is clear that binding energy as well as interactions play decisive roles in determining the druggability of compounds. asinex database compounds a , a , a , a , a , a , a , a , a , and a obtained lower binding energies among all ( figure and figure s ). these compounds form interactions with cys , his , or both along with other active site residues, which are responsible for their inhibitory property. the compounds a , a , a , a , a , and a form hydrogen bonding interactions with cys , which will obstruct the initial deprotonation step in the catalytic cycle of cl pro as shown in figure . the π−π interaction of his with π systems of the compounds a and a will affect the proton accepting ability of his . the π−sulfur interactions are present in aromatic rings with −sh group of the compounds a , a , a , a , a , and a with cys residue. it involves the interaction with the aromatic π orbitals and σ* acceptor orbital of sh bond of cys . s−h/π interactions are the important contributors to binding energy. the thiol group of cys is engaged in s−h/π comparing the structures of the hit compounds pq , pq , pq , and a with the rest, it was clear that the high flexibility of the structures owing to more single bonds and less compact rings makes them able to undergo conformational changes at the active site of the target leading to more interactions with cys and his along with other active site residues. hence, these four compounds were selected for further analysis. even though docking studies give the best binding poses, they do not account for the conformational changes taking place upon ligand binding as the docking analysis preferably treats protein as rigid. the conformational changes affecting the binding affinity could be analyzed using molecular dynamic simulations. four top ranked compounds via docking (pq , pq , pq , and a ) were selected for ns molecular dynamics simulation studies to understand the conformational changes in the protein on binding of the ligand. rmsd, rmsf, and radius of gyration plots were used to evaluate the stability of the complexes. the binding conformations of the compounds at the last frame of simulation are given in figure . the root−mean−square deviation (rmsd) plot of apoprotein cl pro , pq , pq , pq , and a with average rmsds . , . , . , . , and . Å, respectively, is given in figure a . it can be inferred that for the apo structure as well as the docked complexes except pq , rmsd values rose to . Å and then formed a plateau after ns of simulation, which indicates that the system has reached stable equilibrium state and has been confined in the active site. the rmsd of pq requires an extension of stabilization time to reach the root−mean−square fluctuation (rmsf) plot gives the fluctuations of individual residues in the protein backbone. higher rmsf denotes higher flexibility and vice versa. the rmsf plot (figure b ) showed greater residue flexibility for pq and pq compared to the unbound form. pq and a have comparable rmsf values with that of the apo form. the cys and his residues involved in the catalytic cycle have fluctuations with greater than . Å in apoprotein. in pq and pq , it fluctuates more than Å, while in pq and a , there is fluctuation less than . Å. in all the systems, the active site residues from to have the highest fluctuations. the structural flexibility or compactness of the protein molecule is analyzed by the radius of gyration. the protein is supposed to maintain a relatively steady value for r g . from inspection of figure c , it can be seen that r g has almost no significant variance with the average r g values of Å. the ligand bound form has lower r g values compared to apoprotein, which indicates that the bound structures are more compact. molecular docking analysis of chloroquine and hydroxychloroquine on main protease ( cl pro) protein has shown that hcq is more efficient than cq. more potent inhibitors of cl pro have been identified through docking studies. the interactions with the active site residues, especially with cys and his , which are the key residues involved in catalytic diad for proteolysis in the enzyme, are responsible for the activity of these compounds. a regression model with r = . and q = . was developed correlating binding energy with the molecular descriptors. this model was used to predict the binding energies of novel molecules. the primaquine derivatives and antiviral compounds obtained from asinex compound library were screened using the regression model, and the hit compounds thus obtained were further docked with cl pro protein. they showed less binding energies and proper interactions with the target, main protease ( cl pro ). molecular dynamics studies on these top four compounds pq , pq , pq , and a revealed their binding stability and conformational changes associated with the protein−ligand complexes. hence, these four compounds are proposed for validation and further studies as more efficient medication for covid- . the supporting information is available free of charge at https://pubs.acs.org/doi/ . /acs.jproteome. c . binding energy obtained for docking cq and hcq with cl pro and pfdhfr-ts; structure of cq analogs with -aminoquinoline scaffold; structure of chloroquine analogs with quinoline scaffold, values for internal and external validation criteria, binding energy values of training and test molecules predicted with model ; domains and active site of lu bound with inhibitor n ; d interaction diagrams of docked complexes of cq, hcq with cl pro ; docked poses of cq and hcq in active site of lu ; d interaction diagrams of , , , , , , and with lu ; structures of screened compounds from asinex compound library; d interaction diagram of compounds having unfavorable interaction with target; d interaction diagrams of asinex compounds with lu protein (pdf) research and development on therapeutic agents and vaccines for covid- and related human coronavirus diseases diagnosing covid- : the disease and tools for detection a review of the novel coronavirus (covid- ) based on current evidence coronavirus disease : coronaviruses and blood safety severe acute respiratory syndrome coronavirus (sars-cov- ): an overview of viral structure and host 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physicochemical characterization and functional analysis of some snake venom toxin proteins and related non-toxin proteins of other chordates abstract: background: bioinformation genetic involvement of interleukin for asthma and identification of potential phytochemical scaffold through molecular docking studies understanding the relationship between the primary structure of proteins and its propensity to be soluble on overexpression in escherichia coli structure of mpro from covid- virus and discovery of its inhibitors structure -activity relationship and comparative docking studies for cycloguanil analogs as pfdhfr-ts inhibitors chalcone analogue as potent anti-malarial compounds against plasmodium falciparum: synthesis, biological evaluation, and docking simulation study. asian pac pi-interactions in proteins ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign primaquine or other -aminoquinolines for reducing plasmodium falciparum transmission primaquine therapy for malaria analysis for antioxidant activity of dipicolinic acid derivatives qsar study of anti-human african trypanosomiasis activity for -phenylimidazopyridines derivatives using dft and lipinski's descriptors. heliyon qsar with electro topological state atom index: antialirenergic activi ty of n, n-dimethyl- -bromo- -phenylethylamines the molecular connectivity chi indexes and kappa shape indexes in structure-property modeling. reviews in computational chemistry ) yap, c. w. padel-descriptor: an open source software to calculate molecular descriptors and fingerprints investigation of , -dihydro- -pyrones derivatives as potent anti-hiv agents inhibitors quantitative regression models for the prediction of chemical properties by an efficient workflow synthesis and blood-schizontocidal antimalarial activities of -substituted/ , -disubstituted- -quinolinamines and some of their amino acid conjugates discovery of a bulky -tert -butyl group containing primaquine analogue that exhibits potent blood-schizontocidal antimalarial activities and complete elimination of methemoglobin toxicity blood stage antimalarial efficacy of primaquine in plasmodium vivax malaria insights into thiol-aromatic interactions: a stereoelectronic basis for s -h/π interactions docking studies and molecular dynamics simulations of the binding characteristics of waldiomycin and its methyl ester analog to staphylococcus aureus histidine kinase understanding the structural features of jak inhibitors: a combined d-qsar, dft and molecular dynamics study molecular docking and molecular dynamics to identify a novel human immunodeficiency virus inhibitor from alkaloids of toddalia asiatica molecular docking and molecular dynamics simulation studies to identify potent aurka inhibitors: assessing the performance of density functional theory, mm-gbsa and mass action kinetics calculations the authors declare no competing financial interest. the authors gratefully acknowledge generous funding from kerala state council for science, technology and environment (kscste) for the junior research fellowship (no. / /kscste). key: cord- - hj hzt authors: yang, jianling; wu, meng; liu, xu; liu, qi; guo, zhengyang; yao, xueting; liu, yang; cui, cheng; li, haiyan; song, chunli; liu, dongyang; xue, lixiang title: cytotoxicity evaluation of chloroquine and hydroxychloroquine in multiple cell lines and tissues by dynamic imaging system and pbpk model date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: hj hzt chloroquine (cq) and hydroxychloroquine (hcq) have been used in treating covid- patients recently. however, both drugs have some contradictions and rare but severe side effects, such as hypoglycemia, retina and cardiac toxicity. to further uncover the toxicity profile of cq and hcq in different tissues, we evaluated the cytotoxicity of them in cell lines, and further adopted the physiologically-based pharmacokinetic models (pbpk) to predict the tissue risk respectively. retina, myocardium, lung, liver, kidney, vascular endothelium and intestinal epithelium originated cells were included in the toxicity evaluation of cq and hcq respectively. the proliferation pattern was monitored in - hours by incucyte s , which could perform long-term continuous image and video of cells upon cq or hcq treatment. cc and the ratio of tissue trough concentrations to cc (rttcc) were brought into predicted toxicity profiles. the cc at h, h, h of cq and hcq decreased in the time-dependent manner, which indicates the accumulative cytotoxic effect. hcq was found to be less toxic in cell types except cardiomyocytes h c cells (cc - h= . μm; cc - h= . μm). in addition, rttcc is significant higher in cq treatment group compared to hcq group, which indicates that relative safety of hcq. both cq and hcq have certain cytotoxicity in time dependent manner which indicates the necessity of short period administration clinically. hcq has the less impact in cell lines proliferation and less toxicity compared to cq in heart, liver, kidney and lung. the severe acute respiratory syndrome coronavirus (sars-cov- ), was first emerged in china and has spread globally due to its high transmissibility and infectivity, resulting in an unprecedented global public health challenge ( , ). as of april , , more than , , cases have been confirmed around the world, according to data supplied by johns hopkins university, and at least , people have died from the disease ( ). judging from current status, most patients have a good prognosis, nevertheless approximately % of the patients with covid- experienced critical complications, including arrhythmia, acute kidney injury, pulmonary edema, septic shock, and acute respiratory distress syndrome (ards) ( - ). apart from primarily inflammation in the lungs, it is also suggested that other vital organs like kidneys, heart, gut, as well as liver, were also suffered severe damage according to the autopsies, suggesting that individuals or older with chronic underlying diseases appear to have a higher risk for developing severe outcomes. such huge numbers of infected people call for an urgent demand of effective and available drugs to manage the pandemic. unfortunately, at present, there are still no specific antiviral drugs for prevention or treatment of covid- patients. recent publications have demonstrated that chloroquine (cq) and hydroxychloroquine (hcq) efficiently inhibited sars-cov- infection in vitro assay ( - ). cq, together with its derivate hcq, has been commercialized as antimalarial drugs in the clinic for several decades. hcq has also been broadly used in autoimmune diseases treatment, such as systemic lupus erythematosus (sle) and rheumatoid arthtitis ( - ). several clinical trials have confirmed that both cq and hcq were superior to the control group in inhibiting the exacerbation of pneumonia, improving lung imaging findings, as well as promoting the virus negative conversion and shorten the disease course. moreover, the u.s. food and drug administration (fda) also approved cq and hcq for emergency use to treat hospitalized patients for covid- . although exhibiting apparent efficacy and acceptable safety profile for covid- treatment, cq and hcq still have some potential concerns with prolonged usage, including heart rhythm disturbances, gastrointestinal upset, retinal toxicity, in particular for retinopathy ( , ( ) ( ) ( ) ( ) . additionally, risambaf et al. found that cq/hcq may increase the risk of liver and renal impairment when it used to treat . toxicity tolerability in key tissues about drug effectiveness and side effect were critical to understand their mechanism and to optimize dosing regimen by integrating calculated at the given target organ, respectively. the data suggest that hcq was demonstrated to be much less toxic than cq, at least at certain key tissues (heart, liver, kidney, and lung). taken together, this study provides the information regarding cytotoxicity in a wide spectrum and will be beneficial for both pharmacologists and the effect of cq on cell proliferation to gain the more comprehensive cytotoxic information upon cq and hcq treatment, both cq and hcq show strong and immediate toxicity on all cell lines upon treatment more than μm of cq or hcq. as shown in figure and , when the concentration of cq or hcq is higher than μm, the proliferation shows a sudden decline or brake compared with lower dosing regimens. h c (heart) 、 hek ( kidney), and iec- (intestine), are the more sensitive cells to cq compared with other cell lines, as their cc value at h are less than μm ( . with that of h in vero, which may be due to special drug metabolism or stability in it. as the selection index (si) is the safe range to evaluation the drug effect. table ) ( ). therefore, we can preliminarily conclude that the selectivity index (si) of hcq is higher than that of cq in most cell types. using our pbpk models, we simulated the tissues concentrations of hcq ( mg bid for day, mg bid for day to ) and cq ( mg bid for days) ( , ). the cmax of tissue concentrations were summarized in table . results of simulated tissue concentration showed that tissue trough concentration of cq in liver and lung reached the highest level of drug accumulation ( . μg/ml), which is times more than that in heart ( . μg /ml). however, the tissue trough concentration of hcq in lung is the highest level ( . μg/ml) compared with liver, kidney and heart (table and figure ). in order to better predict the toxicity risk of cq and hcq in different tissues, we used the ratio of simulated tissue trough concentration to cc (r ttcc ) to predict the risk of tissue toxicity for the safety profile of these two drugs in the given tissues. as shown in figure , we systematically compared the toxicity between cq and hcq, the r ttcc value of cq is - times more than that of hcq in lung, heart, kidney and liver, which suggests that the toxicity risk of hcq in the above tissues is much lower than that of cq. were obtained as previously reported. the lung to blood concentration ratio for cq and hcq (obtained from animal studies) was used to predict the drug concentration in the lungs, heart, liver, and kidney. to better investigate the potential toxicity in vivo and in vitro, we proposed r ttcc (ratio of tissue concentration and cc ) derived from pbpk model to predict the risk of toxic profiles in different tissues. we compared the r ttcc data collected from heart, liver, kidney, lung, and revealed hcq has shown significantly safe profiles than that of upon cq treatment ( ). however, recent publication reported that cq was safer than hcq according to si ( , ) . we speculate that the safety difference might be due to their complex pharmacokinetic characteristics in vivo, which possessed specific distribution and long half-life of around days. in short, based on our just published study, we further developed the novel parameters to predict the potential toxicity besides the traditional selectivity index (si), (the ratio of the cc to ec ), which is a commonly accepted to measure the window between cytotoxicity and antiviral capacity ( ). as a result, our data shows that kidney, lung and heart are prone to the toxicity of cq, otherwise lung and kidney are relative vulnerable upon hcq treatment ( figure ). in the meantime, considering the un-negligible effect on cardiocytes and retina cells, of which the most patients with the severe symptoms are more likely suffered the dysfunction in heart and eye sight with aging simultaneously. therefore, ecg monitoring should be necessary during clinical usage, even for the patients only infected with covid- but without the underlying diseases. in addition, the more attention should be paid to the patients in the changes of their eye sight when using hcq. in this study, we perform dynamic imaging system to accurately and precisely monitor the whole proliferation process other than conventional cck assay. health organization declares global emergency: a review of the novel coronavirus (covid- ) transmission of -ncov infection from an asymptomatic contact in germany the origin, transmission and clinical therapies on coronavirus disease (covid- ) outbreak -an update on the status population movement, city closure in wuhan and geographical expansion of the -ncov pneumonia infection in china in covid- : a novel coronavirus and a novel challenge for critical care hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro lupus erythematosus-an ongoing need for international consensus and collaborations from pathogenesis, epidemiology, and genetics to definitions, diagnosis, and treatments of cutaneous lupus erythematosus and dermatomyositis: a report from the rd international conference on cutaneous lupus erythematosus (iccle) hydroxychloroquine-induced hyperpigmentation in systemic diseases: prevalence, clinical features and risk factors: a cross-sectional study of cases antimalarials and ophthalmologic safety updated recommendations on the use of hydroxychloroquine in dermatologic practice effects of chloroquine on viral infections: an old drug against today's diseases? liver and kidney injuries in covid- and their effects on drug therapy; a letter to editor kinetics of the distribution and elimination of chloroquine in the rat simultaneous quantitation of hydroxychloroquine and its metabolites in mouse blood and tissues using lc-esi-ms/ms: an application for pharmacokinetic studies drug treatment options for the -new coronavirus ( -ncov) chloroquine and hydroxychloroquine as available weapons to fight covid- response to recent commentaries regarding the involvement of angiotensin-converting enzyme (ace ) and renin-angiotensin system blockers sars-cov- infections what is the role of covid- infection in hypertensive patients with diabetes? new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid- ? mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology chloroquine is a potent inhibitor of sars coronavirus infection and spread key: cord- -vdrix cp authors: d’acquarica, ilaria; agranat, israel title: chiral switches of chloroquine and hydroxychloroquine: potential drugs to treat covid- date: - - journal: drug discov today doi: . /j.drudis. . . sha: doc_id: cord_uid: vdrix cp nan publishing product-specific guidances (psgs) to support generic drug development for these drugs (docket no. fda- -d- ). cq and hcq through the looking glass: the importance of chirality cq and hcq are chiral drugs administered as racemates [i.e., each as a : mixture of two paired enantiomers, namely (s)-(+) and (r)-(-)] (figure ). the chirality of drugs has become a major theme in the design, discovery, development, patenting and marketing of new drugs [ ] . for many years, including at the time of the approvals of cq and hcq, the pharmacopoeias were dominated by racemates. this trend was inverted in the mid- s: most of the chiral new molecular entity (nme) drugs were developed and marketed as single enantiomers [ ] . the strategy of chiral switches has emergedthe development of a single enantiomer from a chiral drug that has previously been developed (and often approved and marketed) as a racemate or as a mixture of diastereomers. nevertheless, the development and approval of racemic drugs has continued to be viable and the continuing approval of racemic nmes could have implications for the persistence of the chiral-switch strategy [ ] . according to the ema guidelines for the development of a new single enantiomer from an approved racemate [ ] , suitable 'bridging studies' should be carried out to link the complete racemate data to the incomplete data on the selected enantiomer. the extent of bridging studies should be defined on a case-by-case basis. the chiral switch of hcq was initiated in the early swith method-of-use patents us , , (priority date - - , assignee sterling winthrop, new york) and ep b claiming the enantiomer (s)-(+)-hydroxychloroquine [(s)-(+)-hcq] for treatments of malaria, ra and le. however, these initiatives and earlier and subsequent studies on hcq and cq enantiomers have not led to regulatory single-enantiomer drug approval(s) for any indication. the sterling winthrop portfolios of cq and hcq, including the pharmacological studies of the two enantiomers, were probably transferred to sanofi (proprietor of ep b ) in june . we aim preferentially at (s)-(+)-hydroxychloroquine [(s)-(+)-hcq], the more-promising enantiomer (patents: us , , and ep b , proprietor sanofi, priority date - - , now expired), followed by (s)-(+)-chloroquine [(s)-(+)-cq]. the rationale on which our call is based is driven by the following considerations. covid- is a pandemic without any approved drug or vaccine. cq and hcq could potentially display therapeutic efficacy for the treatment of covid- [ , , ] . the toxicity profiles of cq and hcq have been well known for many years; their administration is safe, although both can have serious side effects, especially at high doses or when combined with other medicines. advantages of (s)-(+)-hcq (the eutomer) vis-à-vis (r)-(-)-hcq and the racemate (r,s)-(±)-hcq have been recorded in the above-mentioned patents, especially with regard to retinopathy, a severe side effect of hcq which is caused by an enantioselective accumulation of the (r)-(-)-hcq enantiomer in the ocular tissue, r/s ratio = . ± . (in rabbits, ep b ). furthermore, studies of enantioselectivity in the pharmacokinetics of hcq reported that there was no (s)-(+)-hcq ⇌ (r)-(-)-hcq interconversion between the enantiomers [ ] . the pointed clinical implications of using the (s)-(+)-hcq 'substantially free' of the (r)-(-)-hcq as the active ingredient were lower adverse effects and the possibility of higher dose levels and/or longer periods of administration. various syntheses of the enantiomers of hcq and cq have been reported, including a simple method of synthesis for large-scale production of the cq enantiomers (patent cn b, priority date - - ). urgent guidance for navigating and circumventing the qtc interval prolongation and torsadogenic potential side effects of hcq and cq potential therapies for covid- are noted [ ] . according to the ema guidelines [ ] (vide supra), it would be productive in the present case [namely, (s)-(+)-hcq] to use data on the corresponding racemate (i.e., hcq) as far as is applicable to the enantiomer. under the highly demanding, urgent circumstances, relaxations of the regulations are needed. the fda has just created the coronavirus treatment acceleration program (ctap) to speed up coronavirus therapies and move new treatments to patients as quickly as possible. the ema indicated that it will be flexible and pragmatic during the assessment of affected clinical trial data submitted to the agency as part of marketing authorization applications. hopefully, the bridging studies (vide supra) will be reduced, in consultation between the sponsor and the regulator, to shorten the development and approval periods. on march , the italian medicines agency (aifa) expressed a favorable opinion on including the off-label use of cq and hcq for the treatment of covid- . on march , the fda issued an emergency use authorization (eua) to allow hcq sulfate and cq phosphate products donated pro bono publico by leading pharmaceutical companies to the us strategic national stockpile (sns) to be distributed and used for certain hospitalized patients with covid- . emergency drug approvals of (s)-(+)-hcq and/or (s)-(+)-cq should be considered. government agencies in major jurisdictions could also take up the challenge. it has not escaped our minds that the incentives of regulatory and secondary patent exclusivities could be diminished in the current crisis. however, a successful chiral switch of hcq could be rewarded. sanofi, the owner of the portfolios of cq and hcq, is in a preferred position to pursue the chiral switch. philanthropic foundations might also be recruited for the cause of overcoming the covid- pandemic. our call for repurposing hcq and/or cq by urgently developing the chiral switches of these racemates to their (s)-(+)-enantiomers for the treatment of covid- is based on the expectations of safer pharmacological profiles of the selected enantiomers, favorable risk:benefit profiles and shortened development and approval processes. demand for hcq has grown dramatically in recent weeks as a result of the attention raised by the ctap program. the further step that we propose here, taking into account the necessary vigilance and risk management, is the switch to (s)-(+)-hcqthe more-promising single enantiomer of a known drug that proved safe and well tolerated in most patients. therapeutic options for the novel coronavirus ( -ncov) breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro chloroquine and hydroxychloroquine as available weapons to fight covid- the market of chiral drugs: chiral switches versus de novo enantiomerically pure compounds the predicated demise of racemic new molecular entities is an exaggeration investigation of chiral active substances cc a, previously eu iii/ / chloroquine for the novel coronavirus sars-cov- enantioselective disposition of hydroxychloroquine after a single oral dose of the racemate to healthy subjects urgent guidance for navigating and circumventing the qtc prolonging and torsadogenic potential of possible pharmacotherapies for covid- the authors declare no conflicts of interest.j o u r n a l p r e -p r o o f key: cord- - g rw authors: simonis, alexander; theobald, sebastian j; fätkenheuer, gerd; rybniker, jan; malin, jakob j title: a comparative analysis of remdesivir and other repurposed antivirals against sars‐cov‐ date: - - journal: embo mol med doi: . /emmm. sha: doc_id: cord_uid: g rw the ongoing sars‐cov‐ pandemic stresses the need for effective antiviral drugs that can quickly be applied in order to reduce morbidity, mortality, and ideally viral transmission. by repurposing of broadly active antiviral drugs and compounds that are known to inhibit viral replication of related viruses, several advances could be made in the development of treatment strategies against covid‐ . the nucleoside analog remdesivir, which is known for its potent in vitro activity against ebolavirus and other rna viruses, was recently shown to reduce the time to recovery in patients with severe covid‐ . it is to date the only approved antiviral for treating covid‐ . here, we provide a mechanism and evidence‐based comparative review of remdesivir and other repurposed drugs with proven in vitro activity against sars‐cov‐ . coronaviruses (cov) are known to cause respiratory tract infections in humans and animals. since the emergence and subsequent characterization of the severe acute respiratory syndrome coronavirus (sars-cov) in (drosten et al, ; ksiazek et al, ; peiris et al, ) and middle east respiratory syndrome coronavirus (mers-cov) in (corman et al, ) , coronaviruses have increasingly been recognized as potential source of epidemic diseases. both pathogens seem to cause zoonotic infections that originate from viral reservoirs in bats li et al, ; mohd et al, ) . in , a novel coronavirus (sars-cov- ) emerged in china (zhu et al, ) and spread globally in a very short period of time. the rapid geographical extension of sars-cov- in comparison to previous outbreaks with sars-cov and mers-cov may be caused by an increased infectivity of the pathogen (sigrist et al, ; wrapp et al, ) . as of september , the ongoing coronavirus disease (covid- ) pandemic caused over million detected sars-cov- infections and more than , deaths (johns hopkins university, ). the dramatic global implications of this pandemic stressed the urgent need for therapeutic agents that can quickly be applied in the clinic without a long-lasting preclinical development phase. several therapeutic strategies were therefore investigated by repurposing of known antimicrobial or immunomodulatory substances that might be beneficial for patients with covid- . these agents can roughly be divided into compounds with a direct antiviral effect that impairs viral replication and host-directed drugs that may support recovery from covid- by attenuating an excessive host immune response. in this article, we focus on repurposed drugs against covid- with proven antiviral effects against sars-cov- in cell-based studies. the most advanced developed antiviral of this type is the nucleoside analog remdesivir that was previously unsuccessfully tested against ebolavirus disease in clinical trials (mulangu et al, ) . based on recent clinical and preclinical data on its efficacy against covid- , remdesivir received emergency use authorizations (ema) in the united states and japan and was recently approved by the european medicines agency (ema) for the treatment of adult patients with severe covid- that require supplemental oxygen. although approval of this drug is a very encouraging signal, its clinical efficacy seems to be relatively modest based on available evidence (beigel et al, ; goldman et al, ; grein et al, ; wang et al, c) . we will review preclinical and clinical outcomes of repurposed antivirals and their molecular mechanism of action (moa) to provide a comparative analysis of remdesivir with the ultimate aim to support a rational appraisal of its efficacy. mostly via droplet transmission, the life cycle of sars-cov- is initiated by the attachment of the virion to the host cell by the spike glycoprotein (s-protein) and its receptor. several studies could show that entry, as shown for sars-cov before, depends on binding of the receptor-binding domain (rbd) (subunit s ) of the s-protein to the human angiotensin converting enzyme receptor (ace ; hoffmann et al, a; walls et al, ) . notably, the rbd of sars-cov- shows a -to -fold higher affinity to ace than sars-cov, which may explain its increased transmissibility (wrapp et al, ) . furthermore, single-cell rna-sequencing data revealed a high expression level of the ace receptor in human nasal epithelial cells, which may also enhance the efficiency of sars-cov- transmission (sungnak et al, ) . after initial binding of the s subunit to ace , entry into the host cell required proteolytic cleavage of the s-protein at the s /s and s ' site, which leads to fusion of the viral and cellular membrane mediated by the s subunit. proteolytic cleavage of the s-protein is induced by the membranous serine protease tmprss of the host cell (hoffmann et al, a) . interestingly, a new furin cleavage site at the s /s boundary could be found in sars-cov- . the exact role of this site in pathogenesis is controversially discussed (walls et al, ; xia et al, a) . cleavage of s-protein exposes the s subunit which contains an internal fusion peptide and two hydrophobic (heptad) repeat regions (hr and hr ). hr and hr self-assemble into a stable helical bundle that brings viral and cellular membranes in close proximity for fusion. several bundles can form a fusion pore and finally release the viral genome into the cytoplasm (bosch et al, ; xia et al, b) . moreover, several studies could show that virus entry is not only ensued by direct fusion with the plasma membrane, but rather by endosomal/lysosomal uptake and intra-lysosomal activation of the spike protein by cathepsin l followed by membrane fusion and glossary antiviral drugs drugs that directly interfere with the ability of a virus to replicate in vivo or in cell-based models. most antiviral drugs interfere with the host celldependent life cycle of the virus. thus, mode of action of most antivirals is the inhibition of the viral entry into the host cell, blockage of viral proteases, or inhibition of viral rna replicase. used to describe the fraction of a drug or its active metabolite that reaches the systemic circulation and organ tissue after administration. the term cell-based assay is commonly used to refer to any assay, where living cells are used as model to study physiologic or pathophysiologic processes under various conditions (e.g., exposure to an antiviral agent). due to their cost efficiency and high standardization/reproducibility, cellbased assays are essential tools in preclinical drug discovery. the infectious disease caused by sars-cov- in humans. coronaviruses are a group of rna viruses that cause diseases in mammals and birds. coronavirus-associated diseases in humans include severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), and coronavirus disease (covid- ). in addition, there are endemic human covs that cause mild respiratory infections. drug repositioning or repurposing a term that describes a drug discovery strategy based on the identification of new therapeutic approaches by using already known substances that may be at a preclinical or clinical development stage. this strategy offers a time-and cost-saving method to develop therapeutics against newly emerged or neglected diseases. the concentration of a substance which is required to obtain % of its maximal effect. it is used to determine potency of a drug. for some analyses (for example antibacterial activity), the % inhibitory concentration (ic ) is used in analogy. besides the half-maximal concentration, the % maximal effective concentration (ec ) can be determined. middle east respiratory syndrome-related coronavirus causes the middle east respiratory syndrome (mers) in humans which is associated with severe respiratory symptoms and high mortality. the first confirmed case of mers was reported in . nucleoside/nucleotide analogs nucleosides are endogenous compounds composed of a nucleobase and a five-carbon sugar (ribose or '-deoxyribose), while nucleotides contain one more phosphate group. nucleosides/nucleotides are essential for the synthesis of dna and rna but are also involved in other cellular processes like signaling and metabolism. nucleoside/nucleotide analogs are synthetic, chemically modified nucleosides/nucleotides that are able to mimic their physiological counterparts. assembly of nucleoside/nucleotide analogs into the rna/dna leads to premature termination of the strand synthesis and inhibition of, e.g., viral replication. pseudovirions/pseudotyped particles pseudovirions are synthetic viral particles with modified genomes and/or envelope proteins in order to facilitate specific investigations. the particles usually lack genes essential for pathogenicity and cannot replicate. this is an advantage for experiments on otherwise highly pathogenic viruses like sars-cov- . pseudotyping is the combination of viral particles with foreign viral envelope proteins. pseudotyping can be used to study the function of viral envelope proteins and mechanisms of viral entry. the severe acute respiratory syndrome (sars) coronavirus was first described in . it causes a respiratory disease that accompanies a high rate of complications and mortality. after the epidemic outbreak in asia in - , sporadic cases have been observed in several countries until . severe acute respiratory syndrome coronavirus , initially described as -ncov, causes respiratory infections that can progress to viral pneumonia in covid- . it emerged in december in wuhan, china, and rapidly developed to a pandemic which is still ongoing. intracellular release of genomic rna burkard et al, ; ou et al, ) . after release of viral rna into the cytosol viral replication is initiated by the translation of the replicase gene encoded by two large orfs (rep a and rep b), which express the two polyproteins pp a and pp ab. the polyproteins contain several non-structural proteins (nsp) (pp a = nsp - ; pp ab = - ) also including a rna-dependent rna polymerase (rdrp) domain (nsp ) and proteases that cleave the polyproteins (initiated by the enzyme's own autolytic cleavage from pp a and pp ab) (anand et al, ; pertusati et al, ) . most of the nsp forms the replicase-transcriptase complex (rtc): the rtc replicates the genomic rna and subgenomic rna, which encodes the structural proteins and other accessory proteins. while the nucleocapsid (n) protein remains in the cytosol and forms complexes with the genomic rna, the viral structure proteins m, e, and s are translated, inserted into the membrane of the rough endoplasmatic reticulum (er) and subsequently transported to the er-to-golgi intermediate compartment (ergic) (fehr & perlman, ) . here, the genomic rna-nucleocapsid complexes get enveloped by the virion precursors, are transported to the cell surface in vesicles, and are released by exocytosis. an overview of the life cycle of sars-cov- including targets that might be exploited for inhibition of viral replication is illustrated in figure . based on its moa, repurposed drugs with anti-sars-cov- activity can be divided into substances that prevent viral entry into host cells ( - ) and inhibit viral proteases ( ) and inhibitors of viral replicase ( ). other compounds elicit multiple effects, or its specific moa in sars-cov- is unknown. viral entry is initiated by the s subunit, which requires prior sprotein priming by proteolytic cleavage of the s subunit. as shown for other coronaviruses, viral entry in cell lines depends on the serine protease tmprss and the endosomal cysteine proteases cathepsin b and l (kawase et al, ; hoffmann et al, a) . however, several studies indicate that cell entry is driven preferentially via the cell surface or early endosomes by tmprss and that proteolytic cleavage of the s-protein by tmprss is crucial for infection of the host (shirato et al, ; iwata-yoshikawa et al, ) . thus, inhibition of the tmprss and/or cathepsin b and l seems a promising target to prevent virus entry. camostat/nafamostat tmprss is a cell membrane-anchored serine protease and belongs to the family of type ii transmembrane serine proteases. these proteases share a common catalytic mechanism involving a triad of three amino acids, serine, aspartate, and histidine present in highly conserved sequence motifs (antalis et al, ) . serine proteases underlie a strict regulation by endogenous inhibitors (e.g., a /a antitrypsin, and antithrombin iii) and need a prior activation leading to hemostasis under physiological conditions. thus, imbalance can cause several pathophysiological processes like thrombosis (rau et al, ) . however, the exact physiological functions of tmprss are still unknown. synthetic protease inhibitors like camostat mesilate or nafamostat mesilate have been clinically tested in patients with acute or chronic pancreatitis, which is pathophysiologically related to an inappropriate activation of digestive enzymes inside the pancreas, including the serine protease trypsin (chang et al, ; ramsey et al, ) . due to their capability to inhibit tmprss , serine protease inhibitors have been tested for their antiviral effects on sars-cov- and other coronaviruses. camostat partially blocked the entry of vesicular stomatitis virus (vsv) pseudotyped particles harboring the sars-cov- spike protein (pseudovirions) into the human epithelial colorectal adenocarcinoma cell line caco- , vero-tmprss + cells, and human airway epithelial (hae). a complete inhibition of viral entry could only be reached when camostat was used in combination with e- d, an inhibitor of cathepsin b/l, suggesting that sars-cov- can exploit both pathways for entry into the host cell (hoffmann et al, a) . however, tmprss is essential for viral transmission and pathogenesis while catb/l activity is dispensable so that inhibition of tmprss displays a rational antiviral strategy (iwata-yoshikawa et al, ). wang et al demonstrated inhibition of sars-cov- by nafamostat with a % effective inhibitory concentration (ec ) of . lm in vero e cells . a comparative assessment of the serine protease inhibitors gabexate mesilate, camostat mesilate, and nafamostat mesilate and their ability to inhibit viral entry was done by hoffmann et al efficiency of entry inhibition was determined h post-inoculation by using calu- cells infected with sars-cov- pseudovirions. nafamostat demonstrated an almost -fold higher efficiency (ec nm) compared with camostat ( nm), both superior to gabexate (ec . m). nafamostat also showed to inhibit sars-cov- infection of lung-derived human calu- cells in vitro even at a low dose of nm (hoffmann et al, b) . although antiviral efficacy of tmprss inhibitors seems to be inferior to other strategies (table ) , entry inhibitors may be developed that are beneficial in covid- when given alone or in combination with other antivirals. three randomized controlled trials (rct) are currently listed that evaluate nafamostat in patients with covid- (nct , nct , nct ), but currently no clinical data can be reported. umifenovir is a broad-spectrum antiviral approved in russia and china for the prophylaxis and treatment of human influenza a and b infections (boriskin et al, ) . its antiviral mechanism of action is thought to be related to an impaired virus-mediated membrane fusion that is essential for viral entry. umifenovir seems to modify the physicochemical properties of the host cell membrane by influencing the negatively charged phospholipids (villalaín, ) . furthermore, it has been shown that umifenovir interacts with hemagglutinin (ha) of the influenza virus by preventing the ph-induced transition of ha into its functional state (leneva et al, ). in a recent study, the efficacy of six currently available and licensed anti-influenza drugs (umifenovir, baloxavir, laninamivir, oseltamivir, peramivir, and zanamivir) were tested against sars-cov- in vero e cells. among tested drugs, only umifenovir inhibited sars-cov- replication efficiently with an ec of . lm . these results could be reproduced by another in vitro study with an ec of . lm . although umifenovir demonstrated anti-sars-cov- activity in vitro, a therapeutic role of umifenovir in covid- is uncertain and results of qualitative clinical trials are lacking. retrospective analyses currently indicate no significant impact on clinical outcomes (huang et al, ). ª the authors. embo molecular medicine e | a crucial step in sars-cov- replication is the proteolytic cleavage and release of functional polypeptides from the polyproteins pp a/pp ab by viral proteases. subsequently, released non-structural proteins form the replicase-transcriptase complex, which initiates the viral rna synthesis machinery. translated viral structure proteins and replicated genomic rna originate new infectious virus particles, which are released from the infected host cell. in coronaviruses, the main protease (mpro) also known as c-like protease ( cl pro ) cleaves the polyprotein at conserved sites between leu-gln and ser-ala-gly. this well- attachment of sars-cov- to its host cell is mediated by binding of the viral spike protein to the ace receptor. after proteolytic cleavage of the s domain by the membrane-anchored serine protease tmprss , fusion of the viral and host cell membrane is initiated by the exposed s subunit. alternatively, sars-cov- can invade the host cell upon endosomal uptake and activation of the spike protein by cathepsin l. released viral rna is translated by ribosomes of the host cell. polyproteins pp a/pp ab are cleaved mainly by the viral main protease ( c-like proteinase). released non-structural proteins form the replicase-transcriptase complex, which initiates the viral rna synthesis machinery. viral structure proteins and genomic rna form new particles, which are released by exocytosis. the replication cycle of sars-cov- can be inhibited at various stadiums: viral entry ( - ); protease inhibition ( ), and rna replication ( ). characterized enzyme represents an ideal antiviral target as its function is critical for viral replication (anand et al, ; zhang et al, b) . due to its intrinsic proteolytic activity and the absence of homologous enzymes in humans, toxicity of specific inhibitors is expected to be limited. of known protease inhibitors that were repurposed for sars-cov- , the combination of lopinavir and ritonavir has been in focus of interest as other protease inhibitors (e.g., darunavir) showed no in vitro activity at applicable concentrations (de meyer et al, ) . lopinavir/ritonavir is used as combination regimen in the treatment of infections with human immune deficiency virus (hiv- ). both lopinavir and ritonavir are inhibitors of hiv- protease, an enzyme that cleaves the hiv polyproteins gag and gag-pol by bond hydrolysis. since ritonavir also acts as inhibitor of cytochrome p - a (cyp a ), an enzyme that normally metabolizes protease inhibitors, ritonavir is added to enhance the bioavailability of lopinavir (sham et al, ). lopinavir has been tested in vitro against sars-cov, mers-cov, and human coronavirus e (de wilde et al, ) . here, the mean ec of lopinavir ranged from . µm (ae . ) µm (hcov- e) and . µm (ae . ) mers-cov to . µm (ae . ) (sars-cov). recent analysis demonstrated that lopinavir is also active against sars-cov- with an ec of . - . µm (choy et al, ; pizzorno et al, ) while ritonavir alone was not effective (choy et al, ) . in vivo efficacy of lopinavir/ritonavir has been assessed in mice and common marmosets for mers-cov with ambiguous results: in a study published in , lopinavir/ritonavir-treated marmosets had improved clinical findings and reduced viral loads associated with a better outcome. animals were treated with mg/kg/day of lopinavir plus mg/ kg/day of ritonavir given orally once daily at , , and h postinfection (chan et al, ) . however, treatment of infected mice with lopinavir/ritonavir ( / mg + interferon beta) improved pulmonary function but did not reduce virus replication or occurrence of severe lung damage (sheahan et al, ) . clinical effects in patients with severe covid- were evaluated in a randomized controlled clinical trial including patients. patients were randomized in a : ratio to receive either lopinavir/ritonavir (standard dose of / mg) for days or the standard care. the primary end point of the study was clinical improvement or discharge from the hospital. unfortunately, treatment did not improve clinical symptoms and mortality, or decreased viral loads in pharyngeal swabs (cao et al, ) . the disappointing clinical results might be related to sub-therapeutic levels for inhibition of sars-cov- because application of / mg of lopinavir/ritonavir twice daily was shown to yield median serum concentrations of . mg/l ( . µm) in patients with hiv (van der lugt et al, ), which is significantly lower than the observed ec in the in vitro studies. however, summarizing the relatively low efficacy against sars-cov- in vitro in comparison with other repurposed drugs and available in vivo data it is unlikely that lopinavir/ritonavir will play a significant therapeutic role in covid- . besides lopinavir and ritonavir, other protease inhibitors with activity against sars-cov and mers-cov were identified that might be repurposed to target sars-cov- (anand et al, ; he et al, a) . once functional, non-structural proteins are released by proteolytic cleavage of the polyproteins, the replicase-transcriptase complex, which catalyzes the synthesis of the viral rna, can be formed. synthesis is initiated by binding of the rdrp at or near the ' end of the rna strand. subsequently, the complementary rna strand is generated in the elongation phase by repetitive nucleotidyl transfer reactions. several drugs are able to interfere with the rna synthesis machinery. mainly, nucleoside/nucleotide analogs have been repurposed and tested against sars-cov- . these drugs disrupt viral replication by competing with endogenous nucleosides during the elongation phase. after their insertion nucleoside analogs cause a chain termination followed by an abrogation of rna synthesis, which is crucial to produce new viral particles. remdesivir (gs- ) is a prodrug of a monophosphoramidate nucleoside that is designed to easily pass the cell membrane and efficiently deliver its active metabolite (jordheim et al, ) . upon entering the target cells, remdesivir monophosphate (rdv-mp) is rapidly converted into its active triphosphate form due to its ability to bypass an inefficient and rate-limiting first phosphorylation step (murakami et al, , a) , sars-cov, and sars-cov- (gordon et al, b) . in sars-cov- , incorporation of rdv-tp causes termination of rna synthesis after three additional nucleoside/nucleotide positions downstream (gordon et al, b) . although related analogs of rdv have been under investigation and pharmacological modification for many years (cho et al, ; seley-radtke & yates, ; yates & seley-radtke, ) , the current molecule as a candidate for the treatment of viral diseases was first described in based on preclinical data from cell-based assays and a macaque model of fatal evd (warren et al, ) . in fact, rdv has a very broad antiviral activity spectrum among rna viruses. along with efficacy against ebov and marburg virus that belong to the filoviridae family, it was shown that rdv effectively inhibits rna viruses of the paramyxoviridae, pneumoviridae, and coronaviridae families with ec in the sub-micromolar range (warren et al, ; lo et al, ; sheahan et al, ) . efficacy against sars-cov and mers-cov was mainly tested in human airway cells (hae or calu- ). using rt-pcr or reporter gene-based assays, rdv yielded ec of . - . µm (mers-cov) and . - . µm (sars-cov) (sheahan et al, ; agostini et al, ; sheahan et al, ) . in addition, rdv inhibits zoonotic and epidemic human covs (brown et al, ) . inhibitory effects on sars-cov- were evaluated in the african green monkey kidney cell line (vero e ) that supports entry and replication of sars-cov- by a high expression of ace (banerjee et al, ; hoffmann et al, a) . a clinical virus isolate from wuhan (wiv / ) rdv was inhibited with an ec of . µm in a rt-pcr-based assay . another group assessed the reduction of cytopathology effects (cpe) by rdv using an australian isolate (vic / ). here, the ec was significantly higher ( . µm) which might reflect methodological differences as this increased level was in the same order to a similarly tested sars-cov isolate (ogando et al, ) . recently, another preclinical evaluation was done using a sars-cov- isolate from hong kong ( / ). the investigators found ec between . µm and µm in different assay formats. however, these result cannot be readily compared with previous findings because a logarithmic fitted calculation model was used (choy et al, ) . rdv demonstrated beneficial therapeutic effects in several animal models of cov infections including mouse models of sarsand mers-cov infection and in mers-cov-infected non-human primates (sheahan et al, ; de wit et al, ; sheahan et al, ) . here, it also had prophylactic properties when mg/kg was administered h before inoculation of rhesus macaques with mers-cov. recently, rdv was evaluated in a macaque model of sars-cov- infection. animals were treated h post-infection with mg/kg (day ) followed by mg/kg daily (day - ) which is an equivalent dose of that recommended for humans (gilead_sciences, ) . in contrast to animals treated with placebo (n = ), rdv diminished clinical signs of disease and reduced lung virus titers and tissue damage in all six animals treated (williamson et al, ) . clinically, rdv was evaluated in two randomized controlled clinical trials of which results have been published. the first trial conducted in china was unfortunately underpowered (n rdv = ; n placebo = ) due to insufficient recruitment of patients and therefore remained inconclusive. however, in a subgroup of patients that were treated with rdv within days of symptom onset there was a numerical reduction of five days in time to clinical improvement of however, an overall assessment of rdv efficacy is not possible based on these data as no control group was included in this trial. another gilead sciences sponsored phase randomized controlled trial evaluating rdv in moderate covid- (nct ) found a significant better clinical status by day (primary outcome) in patients treated with a -day regimen of rdv compared with placebo (odds ratio . [ % ci: . - . , p = . ]). however, the clinical significance of this finding remains unclear because a -day course had no influence on this outcome and the effect was inconsistent with another evaluation on day . of all results from clinical trials on rdv, only one publication reports on its impact on sars-cov- viral load. wang et al found similar decreases in virus rna of upper and lower respiratory tract specimen of patients treated with either rdv or placebo. this finding may be of limited significance as the study was generally underpowered and only a limited number of patients was eligible for this evaluation ( % had a pcr-positive upper respiratory specimen at baseline and expectorated sputa were obtained from % of enrolled patients) (wang et al, c) . safety data of rdv are available from healthy volunteers (phase i) and more than , patients treated within phase iii trials on covid- or compassionate use programs (fda, ). in general, rdv was well tolerated and serious adverse events seem to be rare. rdv is known to interfere with several hepatic drug-metabolizing enzymes like cyp c , cyp d , and cyp a in vitro. in healthy individuals, rdv increased the risk of transient transaminase elevations. however, in randomized clinical trials similar elevations were observed in both rdv and placebo groups which might be explained by covid- -associated liver injury zhang et al, a) . a complete overview of safety information for rdv can be reviewed elsewhere (fda, ). favipiravir (t- ) is an oral pyrazine derivate that inhibits rdrp of several rna viruses. for influenza, it was shown that the active triphosphate form functions as a nucleotide analog that competes with atp and guanosine-triphosphate (gtp) for incorporation into the nascent rna strand, thereby causing chain termination (sangawa et al, ) . in addition to its action as competitive inhibitor of viral rdrp, favipiravir-tp triggers accumulation of random point mutations that ultimately lead to lethal mutagenesis of the virus (vanderlinden et al, ; preprint: shannon et al, ) . the drug has potent antiviral activity against influenza a and b in vitro and is currently approved in japan for the treatment of influenza infections (furuta et al, ; furuta et al, ) . furthermore, it demonstrated a broad antiviral spectrum against other rna viruses like paramyxoviruses, human metapneumovirus, respiratory syncytial virus, human parainfluenza virus and measles virus (jochmans et al, ) . however, cell-based assays that evaluated efficacy against sars-cov- showed only low activity at a high micromolar range or no activity at the highest concentration tested (choy et al, ; pizzorno et al, ) . despite its poor in vitro efficacy, favipiravir was evaluated in an open-label nonrandomized trial which compared time to viral clearance and radiological improvement after days treatment with either lopinavir/ ritonavir ( / mg twice daily; n = historical controls) or favipiravir ( mg d , mg d - , twice daily; n = ) plus the standard of care (soc) in hospitalized patients with covid- . the investigators found a significant shorter median time to viral clearance ( days, iqr: . - vs. days, iqr: - ; p < . ) and a higher rate of patients with improved chest imaging on day ( . % vs. . %; p = . ) in the group treated with favipiravir . however, these data are difficult to interpret as there was no placebo control and all patients received additional treatments with interferon (ifn)-a b. taken together, no convincing evidence for favipiravir as antiviral agent against sars-cov- can be reported. based on its poor in vivo efficacy, it seems unlikely that this drug will be assessed in another clinical trial. ribavirin is a guanosine analog with structural similarities to favipiravir. like other nucleoside or nucleotide analogs, it abrogates viral rna synthesis by incorporation into nascent rna strands. however, additional processes may also contribute to its antiviral activity. for influenza, for example it was shown that ribavirin provides a mutagenic effect on the viral genome and decreases cellular gtp pools by interfering with cellular inosinmonophosphatdehydrogenase (streeter et al, ; wray et al, ) . ribavirin is an approved drug for the treatment of chronic infections with hepatitis-c virus (hcv) in combination with other antiviral drugs. like other rdrp inhibitors, ribavirin has a broad activity among rna viruses, especially in those belonging to the flavivirus family (crance et al, ) . although virtual molecular docking studies do suggest an interaction with to sars-cov- rdrp, its efficacy against sars-cov- is very limited (choy et al, ; wang et al, a) . this is not surprising as ribavirin also lacks activity against related coronaviruses (cinatl et al, ) . therefore, ribavirin was not evaluated in vivo. pencivlovir is another guanosine analog that is an approved antiviral for topical treatment of herpes simplex virus infections or reactivations. it is closely related to acyclovir but has a very poor bioavailability. its prodrug form, famciclovir, has an optimized bioavailability and is used as systemic treatment for herpes ª the authors. embo molecular medicine e | infections including herpes zoster. virtual binding studies based on an nsp homology model suggest that pencivlovir binds to sars-cov- rdrp with an affinity even higher than that of rdv (preprint: dey et al, ) . nevertheless, it demonstrated low efficacy against sars-cov- in vitro (ec µm) . additional preclinical or clinical studies with penciclovir or its prodrug famciclovir as treatment for covid- have not been reported. chloroquine/hydroxychloroquine chloroquine (cq) is a -aminoquinoline that has been used as antimalaria drug for decades but its use steadily decreased because of emerging resistant plasmodium falciparum (wellems & plowe, ) . cq and its derivate hydroxychloroquine (hcq) however are still in clinical use to treat rheumatic diseases where it has beneficial immunomodulatory effects. hydroxychloroquine demonstrated less toxicity in animal studies that tested high doses in mice, rats, and dogs (mcchesney, ) . in the past years, cq/hcq has gained attention for its potential use as therapeutic agent in the field of bacterial and viral infectious diseases because of its ability to inhibit several intracellular bacteria, viruses, and fungi (savarino et al, ; rolain et al, ) . the moa of cq/hcq is not completely understood and varies among different pathogens to some extent. in general, non-protonated forms of cq/hcq enter the cell and subsequently become protonated according to the henderson-hasselbach law (savarino et al, ) . consequently, cq/hcq accumulates in acidic organelles, such as endosomes, lysosomes, and golgi vesicles. within these organelles, cq/hcq increases the ph because of its biochemical behavior (o'neill et al, ) . two main antiviral mechanisms have been identified: i) low-ph-depended inhibition of viral conformational changes that are essential for responsible for viral fusion, penetration, and uncoating; and ii) inhibition/modification of posttranslational processing of viral glycoprotein's in the trans-golgi compartment and within endoplasmic vesicles (randolph et al, ; sieczkarski & whittaker, ; rolain et al, ) . a third mechanism has been proposed, which is based on the immunomodulatory and anti-inflammatory properties. known effects related to this category include inhibition of intracellular toll-like receptors (such as tlr ), inhibitory effects on the cyclic-amp synthase pathway, and interference with major histocompatibility complex presentation (rolain et al, ; pal et al, ; schrezenmeier & dörner, ) . antiviral activity of cq/hcq has been shown for viruses from several families and seems to cover a relatively broad spectrum (rolain et al, ) . its antiviral mechanism is best explored in hiv where cq induces modifications of the glycosylation pattern and amino acid charges from gp viral envelope protein, which may affect the immune escape mechanism of hiv (savarino et al, ; naarding et al, ) . although its clinical efficacy in hiv is not comparable to current antiretroviral drugs, several clinical studies have proven anti-hiv effects of hcq in vivo (paton et al, ; paton & aboulhab, ) . in covs different antiviral mechanisms of cq/hcq have been proposed including modifications to the viral spike glycoprotein (gallagher et al, ; vincent et al, ) and terminal post-translational modification of ace -receptor glycosylation, which might interfere with virus binding and consequent fusion vincent et al, ) . however, cq/hcq seems to elicit multiple effects on virus and host cell that ultimately inhibit viral replication. in a time-of-addition experiment, liu et al confirmed that cq/hcq affects the viral life cycle both at cell entry and post-entry stages. intracellularly, cq/hcq showed to impair endosome maturation at intermediate stages of endocytosis, a crucial function for the transport of virions to its releasing site . activity against sars-cov was demonstrated in vero e cells with an ec of . µm (ae . ) which approximates the plasma concentrations reached during treatment of acute malaria (keyaerts et al, ) . in sars-cov- , cq yielded ec of . - . µm and hcq yielded . - . µm in rt-pcr-based assays (maisonnasse et al, ; pizzorno et al, ; wang et al, a) . moreover, liu et al directly compared in vitro efficacy of cq with that of hcq. by using vero e cells that were exposed to sars-cov- with increasing multiplicities of infection (moi), they found ec of . - . µm (cq) and . - . µm (hcq), respectively, in rt-pcr-based assays . the authors concluded that hcq is less potent compared with cq. in contrast, yao et al found lower ec for hcq ( . µm) compared with cq ( . µm) when using an moi of . (the lowest moi used by liu et al). they also included a physiologically based pharmacokinetic (pbpk) model of hydroxychloroquine concentrations in lung fluid. based on this model, they predicted that an hcq dose of mg twice daily on day one followed by mg twice daily seems to yield appropriate drug levels for treating covid- . these simulations are in line with an early pharmacokinetic study in children with rheumatic disease where - . mg/kg hcq per day yielded serum levels of . - . µm in humans (laaksonen et al, ) . chloroquine yielded plasma concentrations of - µm when applied with . mg/kg in another study (wollheim et al, ) . tissue levels of both cq and hcq in animals were found to be - times higher than those in the plasma, including lung tissue (popert, ) . this suggests that sufficient drug concentrations may be reached at the site of infection in humans when using recommended doses but final evidence is lacking and pharmacokinetics can differ significantly in humans. however, based on these models, clinical trials have been conducted with maintenance doses of - hcq mg daily. numerous clinical trials evaluating cq/hcq alone or in combination with additional drugs for the treatment of covid- were conducted or are still ongoing. at the time of writing, clinicaltrials.gov has registered trials of cq/hcq in association with covid- treatment. however, most of the published results originate from observational studies or had a low enrollment size. meanwhile, other trials have been halted due to emerging reports of hcqinduced cardiovascular events (kalra et al, ; kamp et al, ) . consequently, there has been discussion in the media and scientific community (colafrancesco et al, ; lenzer, ; sharma, ) . some debate leading clinical finding will be mentioned briefly. early reports from china suggested a breakthrough in covid- treatment as results from more than patients treated with cq it could be concluded that it has a positive impact on disease course, viral clearance, lung images in contrast to control treatments (gao et al, ) . although no clinical data were reported to support this hypothesis, hcq was subsequently introduced in chinese clinical guidelines. in a small chinese randomized trial of patients, hcq seemed to reduce body temperature recovery time and the cough of embo molecular medicine e | ª the authors remission time (preprint: chen et al, b ) and another small analysis of patients treated with hcq suggested effects on viral load reduction without any clinical implication (gautret et al, ) . nevertheless, other studies could not identify any significant beneficial effect of cq/hcq (mahévas et al, ; chen et al, a) including one observational study with patients enrolled (geleris et al, ) . one placebo-controlled rct sponsored by the nih (nct ) was recently halted after the forth interim analysis that included enrolled patients suggested no beneficial effects of hcq in covid- (nih, ) . meanwhile, hcq with or without azithromycin was tested in a non-human primate model of sars-cov- infection where it showed no effects on viral load or clinical endpoints. in additional in vitro analyses published along with this animal study, anti-sars-cov- activity of hcq evident in vero e cells could not be reproduced in human airway epithelial (hae) cells which might explain diminished effects of cq/hcq in vivo (maisonnasse et al, ) . in conclusion, cq/hcq seems to be a broadly active antimicrobial agent that elicits multiple antiviral mechanisms and has potent in vitro efficacy against sars-cov- when tested in a vero e cell model. however, clinical studies that demonstrate beneficial effects are lacking and available data mainly point toward a neglectable role in the clinical management of covid- . myriads of clinical approved drugs have been tested regarding their activity against sars-cov- in vitro. although other potent inhibitors could be identified, the clinical significance of those compounds is currently uncertain. moreover, in vivo efficacy and specific moa are largely unknown. thus, we do not provide a detailed review of those compounds. choy et al reported on antiviral effects of homoharringtonine (omacetaxine mepesuccinate), a natural plant alkaloid used as a treatment of patients with chronic myeloid leukemia, and emetine, an antiprotozoal agent used in the treatment of amoebiasis. both drugs block protein synthesis in eukaryotic cells (gupta & siminovitch, ; gandhi et al, ) . in a sars-cov- infection model with vero e cells, the ec was . - . lm for homoharringtonine and . À . lm for emetine depending on antiviral assay (choy et al, ) . wang et al found that nitazoxanide, a drug with broad-spectrum antiparasitic and broad-spectrum antiviral effects, inhibits sars-cov- at low-micromolar concentration (ec = . lm) . recently, pizzorno et al evaluated cyclosporine a, a calcineurin inhibitor used as an immunosuppressant medication, and berberine, an alkaloid found in several plants for anti-sars-cov- activity. cyclosporine has previously demonstrated antiviral activity against human coronavirus e (hcov- e) and mouse hepatitis virus (mhv) but not sars-cov (de wilde et al, ) . for berberine, inhibitory effects were shown against influenza, chikungunya, and enterovirus (varghese et al, ) . both drugs were found to inhibit sars-cov- replication in vero e cells significantly (ec : cyclosporine a . lm; berberine . lm). further studies have to clarify their potential role in covid- treatments. in this comparative review, we focus on repurposed drugs with antiviral effects against sars-cov- in cell-based assays as those substances offer great opportunities for a treatment early in the course of covid- by inhibition of viral replication and might be even suitable for preventive strategies as shown for neuraminidase inhibitors in case of influenza (jefferson et al, ) . in contrast, immunomodulatory drugs may be more beneficial in a later phase of infection, when the peak of viral replication has been reached and inflammatory processes dominate the pathophysiological process. this hypothesis is supported by the fact that repurposed immunomodulatory drugs like glucocorticoids seems to be beneficial in severe or critical covid- when used in a later phase after several days of symptomatic disease (preprint: corral et al, ; horby et al, ; ramiro et al, ) but probably not within the first week after symptom onset (horby et al, ) . many substances were tested in vitro for their direct antiviral effects on sars-cov- replication or their ability to reduce cytopathologic effects in vero e cells. however, to date only thirteen of them demonstrated any activity against sars-cov- (table ) . of repurposed entry and viral protease inhibitors, to date none has shown convincing evidence that support a clinical development as single agent against covid- . besides remdesivir which inhibits viral replication with an ec of . - . µm (depending on assay type, virus strain, and procedure of calculating), other nucleoside/nucleotide analogs that target the viral rdrp like favipiravir, penciclovir, or ribavirin were assessed but showed no or only weak activity against sars-cov- . inhibitors of viral protease were also investigated but only lopinavir had mentionable antiviral activity (ec . - . µm). unfortunately, the combination of lopinavir and ritonavir did not show any clinical effects in a randomized controlled trial (cao et al, ) . the anti-parasite drug cq/hcq was one of the most promising candidates against covid- based on preclinical studies but a clinical benefit could not be proven and a recently published in vivo study demonstrated no beneficial effects in a non-human primate model of sars-cov- infection (maisonnasse et al, ) . recent studies in vitro showed strong anti-sars-cov- properties of compounds with different and partly unknown modes of antiviral action like nitazoxanide, cyclosporine a, emetine, and homoharringtonine. however, of those agents none have been readily assessed in animal models or clinical trials (table ) . therefore, remdesivir is the only antiviral drug that demonstrated efficacy in the preclinical and clinical setting. in the latter situation, it reduces time to recovery and may reduce mortality. a meta-analysis which is available as preprint identified a statistically significant reduction in mortality (relative risk . ; [ % ci . - . ]) when pooling data of the two available rcts (preprint: alexander et al, ) . final results of the actt- trial will provide more data to evaluate effects of rdv on mortality and virologic outcomes. in addition, a phase b/ a trial evaluating effects of rdv on viral load when administered by inhalation of an aerosolized solution is being planned (nct ). the relatively modest effect of the drug may be explainable by its virostatic mechanism of action and the fact that effects were studied after median days of symptomatic disease (beigel et al, ) while viral replication is dominating in the first week of infection wölfel et al, ; zhou et al, ; he et al, b) . early treatment with rdv was shown to be very effective in a rhesus macaque model of sars-cov- infection where it reduced clinical signs of infection, lung damage, and virus replication in lower respiratory tract specimen (williamson et al, ) . based on ª the authors. embo molecular medicine e | these considerations, we hypothesize that treatment with rdv should therefore start early after symptom onset in the patient population with treatment indication. in contrast to other antiviral drugs, rdv is not available as oral formulation because of its poor bioavailability that is inherent to its phosphonate-containing pronucleoside design (murakami et al, ; pertusati et al, ) . this is a major disadvantage as it precludes an early treatment initiation out of hospital. however, a clinical study that aims to evaluate multiple intravenous doses of rdv in an outpatient setting (nct ) may increase our knowledge on its efficacy in early stages of covid- . the most successful antiviral therapies consist of combinations of antiviral drugs with different moa's as shown for hiv and hcv-therapy. here, this approach is necessary to prevent development of antiviral resistance during long-term treatments. nevertheless, combining of rdv with other antiviral or immunomodulatory agents may be a successful strategy to improve treatment outcomes. for more information i https://www.who.int/emergencies/diseases/novel-coronavirus- /globa l-research-on-novel-coronavirus- -ncov. ii https://clinicaltrials.gov/ct /who_table. iii https://www.covid treatmentguidelines.nih.gov/. iv https://www.bmj.com/content/ /bmj.m . i effects of rdv on mortality and virologic outcomes need to be addressed in the final publication of data from the actt- trial and in subsequent meta-analyses to substantiate the full potential of this antiviral drug. ii safety and efficacy studies of rdv in combination with other antivirals or immunomodulatory drugs (including systemic corticosteroids) are needed. hereby, drug-drug interactions must be taken into account as rdv interferes with several hepatic drug-metabolizing enzymes. iii the optimal timing of rdv treatment is still unknown. following studies should focus on patients in an early stage of covid- . iv the poor oral bioavailability of rdv has many disadvantages and precludes a timely use out of hospital or in remote areas. additional pharmacological efforts should be put into the development of an antiviral against sars-cov- , which is 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absence of trypsin fusion mechanism of -ncov and fusion inhibitors targeting hr domain in spike protein in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) the evolution of antiviral nucleoside analogues: a review for chemists and non-chemists. part ii: complex modifications to the nucleoside scaffold liver injury in covid- : management and challenges crystal structure of sars-cov- main protease provides a basis for design of improved a-ketoamide inhibitors viral dynamics in asymptomatic patients with covid- key: cord- -t yvy s authors: pothen, lucie; yildiz, halil; de greef, julien; penaloza, andrea; beauloye, christophe; belkhir, leila; yombi, jean cyr title: safety use of hydroxychloroquine and its combination with azithromycin in the context of sars-cov- outbreak: clinical experience in a belgian tertiary center date: - - journal: travel med infect dis doi: . /j.tmaid. . sha: doc_id: cord_uid: t yvy s nan despite some encouraging preliminary clinical data ( , ), major concerns have been raised about the use of hcq to treat covid- ( ), particularly regarding potential cardiac toxicity (i.e. qtc increase and risk of torsade de pointe). because hcq has been safely used for many years for various indications (e.a. connective tissue diseases) ( ), we decided to follow interim belgian guidance for all eligible patients hospitalized in our covid- wards. moreover, we were treated by supplementation if present. repeat ecg was not systematically performed during hcq treatment, except in case of drug-drug interaction which could potentially increase qtc (see foot note of table ). the main drug-drug interaction was driven by addition of azm (n= ). in this group, qtc was controlled at day of combination therapy (n= ). we observed a significant increase in mean qtc, from to msec (p< , with paired t-test), but none of the patients showed a qtc over msec. furthermore, in our entire cohort there were no sudden deaths nor syncope requiring resuscitation or icu admission. all icu admissions (n= ) were linked to respiratory failure resulting from covid- pneumonia. one patient on hcq presented av nodal reentry tachycardia in parallel with respiratory failure, and was successfully treated with adenosine. all deaths in our cohort (n= ) were attributed to covid- infection. in conclusion, based on our clinical experience, no safety issues were encountered with the use of hcq for the treatment of covid- . in coherence with recent data published here ( ), its association with azm also seems to be safe, despite a significant increase of qtc that should be carefully monitored. the efficacy of hcq and its combination with azithromycin on covid- infection needs, of course, to be strengthened with further evidence from large randomized clinical trials. however, at this point of the covid- pandemic, we find it relevant to share our clinical experience with this well-known, readily available compound (hcq) which has limited contraindications and may help in the fight against this outbreak. abdominal pain, diarrhea); "moderate" as clinical (fever and cough) and radiological pneumonia (infiltrates) without hypoxemia; "severe" as clinical and radiological pneumonia with hypoxemia (o saturation < %). **other consisted in escitalopram, citalopram, fluconazole, valproate, mirtazapine and olanzapine. in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) efficacy of hydroxychloroquine in patients with covid- : results of a randomized clinical trial. medrxiv ( ) published online clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in covid- patients with at least a six-day follow up: a pilot observational study online ahead of print chloroquine and hydroxychloroquine in covid- electrocardiogram abnormalities related to anti-malarials in systemic lupus erythematosus early treatment of covid- patients with hydroxychloroquine and azithromycin: a retrospective analysis of cases in marseille, france key: cord- -y ebt jm authors: monari, caterina; gentile, valeria; camaioni, clarissa; marino, giulia; coppola, nicola title: a focus on the nowadays potential antiviral strategies in early phase of coronavirus disease (covid- ): a narrative review date: - - journal: life (basel) doi: . /life sha: doc_id: cord_uid: y ebt jm background: the outbreak of the severe acute respiratory syndrome coronavirus (sars-cov- ) infection and the related disease (covid- ) has rapidly spread to a pandemic proportion, increasing the demands on health systems for the containment and management of covid- . nowadays, one of the critical issues still to be pointed out regards covid- treatment regimens and timing: which drug, in which phase, for how long? methods: our narrative review, developed using medline and embase, summarizes the main evidences in favor or against the current proposed treatment regimens for covid- , with a particular focus on antiviral agents. results: although many agents have been proposed as possible treatment, to date, any of the potential drugs against sars-cov- has shown to be safe and effective for treating covid- . despite the lack of definitive evidence, remdesivir remains the only antiviral with encouraging effects in hospitalized patients with covid- . conclusions: in such a complex moment of global health emergency, it is hard to demand scientific evidence. nevertheless, randomized clinical trials aiming to identify effective and safe drugs against sars-cov- infection are urgently needed in order to confirm or reject the currently available evidence. the new coronavirus disease (covid- ) caused by severe acute respiratory syndrome coronavirus- (sars-cov- ) began in wuhan, china, in december [ ] . since then, it has rapidly spread worldwide, so far that in march , the world health organization (who) has declared it a global pandemic and public health emergency [ ] . by st august , the who has reported a total of , , confirmed cases and , confirmed deaths due to covid- globally [ ] . the novel coronavirus is structurally similar to severe acute respiratory syndrome coronavirus- (sars-cov- ) and middle east respiratory syndrome coronavirus (mers), which were responsible a narrative review was performed using medline, google scholar, and embase from january up to the beginning of july , in order to identify the main evidences about covid- treatment in the first phase of the infection. the last research was made on the july . we included the following search terms: "covid- " and "sars-cov- " in combination with "treatment" and "therapy". the reference lists of all the included studies were analyzed, in order to identify any other studies that might have deserved inclusion. we excluded the non-englishlanguage articles. moreover, we have identified ongoing clinical trials (rcts) using the search term "covid- " and "treatment" or "therapy" on clinicaltrials.gov. the above-mentioned viral life-cycle steps identify potential targets for antiviral drugs, whereas the clinical disease staging may help clinicians to understand the best timing of the different treatment approaches. the first phase of the disease, i.e., the "early infection", is characterized by viral entry and viral replication; thus, antivirals may play a key role in the treatment of the sars-cov- infection. according to this, many drug targets have been identified. several agents with apparent in vitro and in vivo activity against sars-cov- and mers-cov have been suggested as potential candidates for sars-cov- , even though the clinical benefits of any of this regimen were not demonstrated. in this paragraph, we will review the current evidence regarding the main proposed antiviral drugs for covid- in the first phase of infection. figure summarizes the viral cycle steps and possible therapeutic targets. antiviral agents proposed against sars-cov- , their mechanism of action and their main adverse reactions are summarized in table . antiviral agents proposed against sars-cov- , their mechanism of action and their main adverse reactions are summarized in table . possible inhibition of sars-cov- -chymotrisyn-like ( cl)-protease and papain-like protease lopinavir is excreted in the gastrointestinal (gi) tract, and thus coronavirus-infected enterocytes might be exposed to higher concentrations of the drug lpv/r tab / mg: tab bid lpv/r oral sol / mg: ml bid drv/cobi tab / mg: tab qd gastrointestinal: diarrhea, nausea, vomiting, increased amylase, lipase, total cholesterol and triglycerides (risk factor for pancreatitis) hepatotoxicity: increasing in ggt, ast, alt, total bilirubin, hepatitis cardiological: qt-and pr-interval prolongation, hypertension, bradyarrhytmias; torsade de pointes have been reported in patients treated with lpv/r metabolical: hyperglycemia and diabetes mellitus, increased uric acid adenosine nucleotide analog prodrug, which inhibits viral rna-dependent rna polymerase (rdrp gastrointestinal: nausea, vomiting hepatotoxicity: transient elevation of alt and ast (grade or ), typically after multiple days of treatment [ ] hematological: mild, reversible prolonged prothrombin time (pt) without inr change (gilead ) renal: potential toxicity due to accumulation of sulfobutyl ether β-cyclodextrin sodium (sbecd) in moderate to severe renal impairment * there are no approved doses for the treatment of covid- . the doses listed here are for approved indications or from reported experiences or clinical trials for covid- . g pd: glucose- -phospate-dehydrogenase; ld: loading dose; qd: quaque die (once a day); bid: bis in die; ifn: interferon; ggt: gamma glutamyl transpeptidase; ast: aspartate aminotransferase, alt: alanine aminotransferase. chloroquine (cq) and its analogue hydroxychloroquine (hcq) are drugs that have been used in the past years to treat malaria and, more recently, chronic inflammatory diseases, such as rheumatoid arthritis (ra) and systemic lupus erythematosus (sle). cq and hcq have similar pharmacokinetic characteristics, with rapid gastrointestinal absorption and renal elimination. the current dose of hcq sulfate used against covid- consists in a loading dose of mg twice daily for day followed by a maintenance dose of mg twice daily for days, while the one of chloroquine phosphate is mg twice daily orally [ ] . the rationale in the use of both chloroquine and hydroxychloroquine in sars-cov- infection is based on the fact that they seem to hinder viral entry into host cells through the inhibition of glycosylation of host receptors, of proteolytic processing, and of endosomal acidification. moreover, they have an immunomodulatory effect, secondary to the reduction in cytokine production and an inhibition of autophagy and lysosomal activity in host cells [ , , ] . however, there are no high-quality evidence supporting the efficacy of hcq or cq therapy against sars or mers infections [ , ] . maissonasse et at. evaluated the antiviral activity of hcq both in vitro and in sars-cov- -infected macaques. in vitro, post-infection treatment of vero e cells with hcq resulted in a dose-dependent antiviral effect, with % inhibitory concentration (ic ) values of . µm ( . µg/ml) and . µm ( . µg/ml) at and h post infection. hcq tested in vivo in macaques did not show a significant effect on the viral load levels neither alone nor in combination with azithromicin, regardless of the timing of treatment initiation, either before infection, early after infection (before viral load peak), or late after infection (after viral load peak). this study shows a discrepancy from in vitro classic assays and in vivo experiments [ ] . nevertheless, the activity of cq and hcq has been further investigated in vitro against sars-cov- , showing that both these drugs decrease the viral replication in a concentration-dependent manner [ ] . moreover, chloroquine shows effectiveness at an entry and post-entry level, suggesting the possible prophylactic and therapeutic activity of this molecule against sars-cov- [ ] . given these promising in vitro results and the scenario of global emergency, several clinical trials have been launched in order to gather clinical evidence to support the use of these two drugs in covid treatment. an open-label non-randomized french study reported a better virologic clearance in patients treated with hcq: the virologic clearance by nasopharyngeal swab for sars-cov- at day was % in patients treated with hcq compared to . % in the control group ( subjects). moreover, the combination of azithromycin and hcq yielded a higher viral clearance compared to hcq alone (p < . ) [ ] . in a following study, the same authors confirmed that the use of hcq plus azithromycin improved clinical outcome in patients, although in absence of a control group of patients [ ] . another study carried out in china on more than covid- patients has shown that cq was more effective than the control group in enhancing viral clearance, improving imaging findings, and shortening the duration of symptoms [ ] . in a randomized trial conducted in wuhan, china, patients were randomly treated with a -day course of hcq or standard of care: a faster mean time to clinical recovery, i.e., resolution of fever and cough, and an improvement on chest radiography was observed in the experimental group; only patients, all in the control group, showed a progression to severe infection [ ] . a recent observational study conducted in new york city retrospectively compared hospitalized patients who received hcq ( mg twice on day , then mg daily for a median of days) with patients who did not: no significant association between hcq and lowered risk of intubation or death (hr . ; % ci . - . ) was found [ ] . however, it should be underlined that patients receiving hcq were more severely ill at baseline than those in the control group arshad et al., taking as the primary outcome the in-hospital mortality, conducted a comparative retrospective cohort study of hospitalized patients with covid- treated with different therapeutical strategies. overall in-hospital mortality was . %, . % [ % ci: . %- . %] in the hydroxychloroquine only group, . % [ % ci: . %- . %] among those with hydroxychloroquine + azithromycin, . % [ % ci: . %- . %] among the azithromycin-only group, and . % with neither drug (p < . ). treatment with hydroxychloroquine alone and in combination with azithromycin seems associated with a reduction in covid- -associated mortality, although the study has many limitations, including its design [ ] . a recent randomized double-blind placebo-controlled trial was conducted using oral hcq or masked placebo in an outpatient who had early, mild covid- or probable covid- and high-risk exposure within days of symptom onset. among outpatients at days, % ( of ) of participants receiving hydroxychloroquine had ongoing symptoms compared with % ( of ) receiving placebo (p = . ), proving that hcq did not reduce symptom severity over days [ ] . lastly, a multinational registry analysis has shown a higher mortality rate and an increased risk of ex novo ventricular arrhythmia appearance among patients affected by covid- treated with cq and hcq, alone or in combination with a macrolide [ ] . however, since these results have raised several concerns, the paper has been retracted by the auhors [ ] . furthermore, in two large randomized control trials, the solidarity trial by the who [ ] and recovery trial by the oxford university in uk [ ], the hcq arm has been recently ceased because of a lack of its efficacy in a cohort of hospitalized patients with covid- . the rationale of this decision is explained in the chapter "ongoing clinical trials". as well the discovery trial, a multicenter, adaptive, randomized open clinical trial, aiming to evaluate the clinical efficacy and safety of treatment arms (remdesivir, lopinavir/ritonavir (lpv/r), interferon-beta a, hcq) in addition to the usual standard of care, has temporarily stopped the hcq arm since may [ ] . nevertheless, hydroxychloroquine and chloroquine are considered relatively safe and well tolerated. the most common side effects include gastrointestinal symptoms, such as nausea and diarrhea, pruritus, and dermatological alterations. however, both drugs can cause severe side effects (< %), such as cardiotoxicity, proximal muscles neuromyopathy, hypoglycemia, and retinopathy. in particular, cardiotoxicity can include qt prolongation and arrhythmias, especially in patients with previous renal or hepatic problems [ ] . therefore, an electrocardiography (ecg) is deemed necessary prior to the initiation and during the treatment, especially in those patients taking concomitant qt-interval prolonging drugs, such as azithromycin. in conclusion, since there is a low level of evidence of cq and hcq efficacy against sars-cov- , other clinical trials are needed to clarify the efficacy of hcq and cq in covid- treatment and their safety profile. lopinavir/ritonavir (lpv/r) is an oral combination agent approved for the treatment of hiv infection. lopinavir is a st generation protease inhibitor, whereas ritonavir acts as a booster of lpv by inhibiting cytochrome p and p-glycoprotein. studies in vitro have demonstrated an antiviral activity of lpv against sars-cov- , mers-cov, and other coronaviruses through the inhibition of -chymotrypsin-like protease [ ] [ ] [ ] [ ] . moreover, a recent study in vitro has demonstrated the antiviral effect of lpv against sars-cov- [ ] clinical studies regarding lpv/r activity against human coronaviruses are few and have been conducted mostly on sars-cov- infection, with promising results although with retrospective and observational designs [ , ] . for example, a multicenter retrospective matched cohort study including sars-infected patients, treated with lpv/r and ribavirin and controls, suggested that the combination therapy was effective against sars-cov, in particular in the early phase of infection [ ] . data regarding lpv/r activity against sars-cov- mostly derive from case reports or small non-randomized, retrospective studies, with controversial results [ ] [ ] [ ] ; therefore, they do not allow asserting the direct efficacy of lpv/r against sars-cov- [ ] . recently, a randomized, controlled, open-label trial comparing the efficacy of lpv/r versus standard of care was conducted in hospitalized adult patients with severe covid- : no significant difference between the two groups neither in the time of clinical improvement (hazard ratio [hr] . ; % ci . - . ; p . ), nor in the -day mortality rate ( . % versus . %; % ci − . to . ) was observed [ ] . it is worthy of note that in both groups, lpv/r was started late in the course of disease, at a median time of days from the onset of symptoms (interquantile range, . thus, the timing of administration of antiviral agents seems crucial: the initiation of lpv/r beyond the peak viral replication phase (initial - days) had no effect on clinical outcomes [ , ] . rcts are underway in order to better describe the role of lpv/r in sars-cov- infection, especially in the early phase. in particular, the discovery trial, a multicenter, adaptive, randomized open clinical trial, aiming to evaluate the clinical efficacy and safety of treatment arms (remdesivir, lpv/r, interferon-beta a, hcq) in addition to the usual standard of care is still ongoing [ ] . however, the recovery trial by the oxford university in the uk has recently described no clinical benefit from the use of lpv/r in hospitalized patients with covid- [ ] . as a matter of fact, colleagues found no significant difference in the -day mortality between patients treated with lpv/r and patients randomized to usual care alone ( . % lpv/r versus . % usual care) nor in the risk of progression to mechanical ventilation or length of hospital stay [ ] . however, they were unable to study a large number of patients on mechanical ventilation; therefore, these results may not be applied to severe patients with covid- requiring invasive ventilation. other protease inhibitors (pi), such as darunavir/cobicistat (drv/c) or darunavir/ritonavir (drv/r), have been identified as potential agents with activity against sars-cov- infection, thanks to its structural similarity to lpv/r [ ] . in fact, in vitro cell models have demonstrated a significant activity of drv/c against sars-cov- [ ] . however, currently, there are very few data regarding the efficacy and safety profile of drv/c in covid- patients. interestingly, a recent case report has provided preliminary evidence that darunavir did not prevent sars-cov- infection in three hiv-positive subjects who were assuming drv/c as part of the antiretroviral regimen [ ] . anyway, clinicians should consider the possible adverse events related to the use of pis in the treatment for covid- . the rct of cao et al. showed that adverse events were observed in % of patients, thus leading to a drug discontinuation in % of them [ ] . the most commonly reported adverse effects of lpv/r include gastrointestinal symptoms (up to %), such as diarrhea, nausea, and vomiting, hepatotoxicity ( - %), hypertriglyceridemia, and hypercholesterolemia. serious adverse reactions have been described as well, including pancreatitis, qt interval prolongation, pr interval prolongation, diabetes mellitus, and/or hyperglycaemia [ ] . thus, an ecg that aimed to study qt intervals should be done prior to and during the treatment, especially in those patients taking concomitant qt-interval prolonging drugs, such as hydroxycholoroquine, chloroquine, or azithromycin. lastly, it is important to rule out drug-drug interactions, considering that these agents are cyp a inhibitors [ ] . in conclusion, further studies regarding the efficacy of pis against sars-cov- are needed, especially to evaluate the efficacy in the early phase of infection. remdesivir, known as gs- , is a novel nucleotide analogue that is the inhibitor of the rna polymerase. it is a monophosphoramide pro-drug that mimics adenosine, causing the premature termination of viral rna replication by the inhibition of rdrp. it was originally developed against ebola virus and it has been proved to have activity against mers-cov and sars-cov- both in vitro and in human cells [ ] . in a murine lung infection model, remdesivir showed both prophylactic and therapeutic efficacy against sars-cov- , resulting in a significantly reduced lung viral load and improved clinical signs of disease as well as lung function [ ] . similarly, in a mouse model of mers-cov pathogenesis, it improved pulmonary function and reduced lung viral load, both in prophylactic and therapeutic administration, compared to lopinavir/ritonavir and interferon beta [ ] . recently, in vitro data demonstrated that remdesivir had potent antiviral activity against sars-cov- in vero cells [ ] . moreover, it exerted significant antiviral and clinical effects in a non-lethal rhesus macaque model [ ] and was a potent inhibitor of viral replication in human nasal and bronchial airway epithelial cells [ ] . at this time, it is unknown how the observed efficacy of remdesivir against sars-cov- infection in animal models will translate into clinical efficacy in patients in clinical practice. however, remdesivir has been globally used in hundreds of patients infected with sars-cov- under a compassionate use protocol or expanded access. in several case series of covid- , the use of remdesivir was associated with an improvement in clinical condition [ , ] . in one multicenter, multinational series, patients with severe covid- received remdesivir for up to days: patients ( %) showed a clinical improvement (decreased requirement for oxygen support or hospital discharge) and of the patients who were mechanically ventilated at baseline ( %) were extubated [ ] . a randomized, double-blind, placebo-controlled multicenter trial enrolled patients with moderate covid- , of whom received remdesivir: although remdesivir was associated with a faster time to clinical improvement (but not significant at statistical analysis), the time to clinical improvement was days in remdesivir group and days in the placebo one (hr . ; % ci . - . ), and no difference in -days mortality was observed between the two groups [ ] . however, the preliminary report of another double-blind, randomized, placebo-controlled trial has been recently published showing encouraging effects of remdesivir in hospitalized adults affected by covid- with involvement of the lower respiratory tract [ ] . of the total patients, were assigned to the remdesivir group, and were assigned to placebo. the study arm showed a lower median recovery time ( versus days, p < . ) and a trend toward lower mortality, although it was not significant ( . versus . %). another ongoing rct evaluating the efficacy and safety of remdesivir is the discovery trial [ ] . on may , the u.s. food and drug administration (fda) has authorized the emergency use of remdesivir, stating that it can be used to treat "in-hospital adults and children with suspected or laboratory confirmed covid- and severe disease defined as spo ≤ % on room air, requiring supplemental oxygen, mechanical ventilation or extracorporeal membrane oxygenation (ecmo)" [ ] . the current dose under investigation is an intravenous (i.v.) mg-loading dose on day , followed by i.v. mg once daily for a total duration of to days. a recent randomized, open-label, phase trial did not showed a significant difference between a -day or a -day-course of remdesivir in patients with severe covid- [ ] . the safety profile has not been currently established. the most common adverse reactions reported are gastrointestinal (nausea, vomiting, and transient elevation of serum alanine aminotransferase and aspartate aminotransferase), prolonged mild and reversible prothrombin time, and renal toxicity due to the accumulation of sulfobutyl ether β-cyclodextrin sodium (sbecd). in fact, remdesivir is not recommended in case of renal impairment, with an estimated glomerular filtration rate less than ml/min [ ] . umifenovir (also known as arbidol) is a drug approved in russia and in china for oral treatment and prophylaxis of influenza a and b viruses. it prevents viral entry in the host cell by inhibiting the membrane fusion of the viral envelope and the host cell cytoplasmic membrane [ ] . studies in vitro demonstrated a broad-spectrum antiviral activity against hepatitis b and c viruses, ebola virus, lassa virus, human herpes virus , and poliovirus [ ] . since some data in vitro suggested an antiviral activity against sars virus, this agent has gained increasing interest as a potential drug for covid- . in a single-center retrospective cohort study, patients with covid- receiving umifenovir mg every h plus lpv/r were compared to a control group of patients receiving only lpv/r: compared to the control group, the experimental group showed a higher rate of viral clearance and a more significant improvement in chest imaging [ ] . a higher rate of viral clearance due to umifenovir administration was suggested by zhu et al. as well [ ] . an improvement in discharging and the mortality rate was also described in a retrospective study on patients in wuhan [ ] . on the other hand, a chinese retrospective study, comparing patients in the umifenovir group and in the control group, showed that umifenovir did not improve the prognosis of the patient nor accelerate sars-cov- clearance [ ] . since these contrasting data, rcts are underway in china. favipiravir (t- ) is an antiviral agent that selectively and potently inhibits the rna-dependent rna polymerase (rdrp) of rna viruses. it is effective against several rna viruses, including influenza virus, flavi-, alpha-, filo-, arena-and noro-viruses, as well as west nile virus, yellow fever virus, ebola virus, and lassa virus [ , ] . favipiravir is one of the potential candidates for covid- treatment, although in vitro studies showed controversial results [ , , ] . a open-label non-randomized study compared the effect of favipiravir to lpv/r in a cohort of patients with covid- : favipiravir was associated with a shorter viral clearance time ( versus days, p < . ) and significant improvements in chest imaging ( . % versus . %, p . ) [ ] . clinical trials aiming to describe the efficacy of favipiravir against covid- are underway. camostat mesylate is an agent approved in japan for the treatment of pancreatitis. it is a serine protease inhibitor, which seems to prevent sars-cov- entry in human lung cells through inhibition of the host serine protease tmprss [ ] . gabexate mesylate and nafamostat mesylate are similar agents and have been studied against covid- as well. in vitro studies demonstrated a promising effect of all these agents in inhibiting sars-cov- , in particular nafamostat mesylate [ ] . nevertheless, further evaluations in clinical trials are needed. ribavirin is a guanine analogue that inhibits viral rna-dependent rna polymerase. it is used to treat several virus infections, such as respiratory syncytial virus (rsv), hepatitis c virus, and some viral hemorrhagic fevers. moreover, it demonstrated activity against other novel coronaviruses, making it a candidate for covid- treatment. although promising results were previously obtained with ribavirin and ifn-alfa b in a mers-cov rhesus macaque model [ ] , in human mers infection, data have been discordant [ ] . in sars-cov- , a systematic review showed inconclusive results in of studies, with of them showing possible harms due to adverse reactions [ ] . interferon (ifn) both interferes with viral replication and regulates the immune system. it is an antiviral drug used in both hepatitis b and c virus infection, and it has been seen to have efficacy in reducing viral replication and disease severity in animal models of mers infection, particularly when used in combination with other drugs [ ] [ ] [ ] . in contrast, a mers-cov infection model of humanized transgenic mouse demonstrated that the combination of ifn-beta and lpv/r improved pulmonary function but did not reduce virus replication or severe lung pathology [ ] . currently, as a result of the conflicting in vitro and animal data and the lack of clinical trials, the use of interferon cannot be recommended in sars-cov- infections. the "solidarity" trial by the who is underway and aims to better describe the efficacy of different covid- treatment approaches, including ifn-beta [ ] . another rct evaluating ifn-beta efficacy and safety profile is the discovery trial [ ] ivermectin is an fda-approved anti-parasitic agent that showed a broad spectrum antiviral activity in vitro [ ] , which was probably thanks to the inhibition of nuclear import of viral and host proteins, in particular through the inhibition of importin (imp) α/β [ ] . recently, caly et al. reported that ivermectin potently inhibited sars-cov- in vero/hslam cells with a -fold reduction of viral rna at h [ ] . however, its mechanism of action against sars-cov- is still unclear, and its safety profile has not been established. moreover, a recent study analyzing its pharmacokinetic profile stated that ivermectin is unlikely to reach the ic in lungs after a single standard dose ( µg/kg), even for a dose × higher than the standard one [ ] . therefore, further studies are needed to clarify the efficacy, tolerability, and safety profile of this agent. however, a recent paper by stauffer et al. has highlighted the relevant role that ivermectine may play in covid- patients in preventing strongyloides hyperinfection, which is a potential fatal complication secondary to the administration of dexamethasone [ ] . since there is a high mortality related to this sydrome, the authors propose a test-and-treat strategy and, when it is not possible, they suggest to consider a presumptive treatment with ivermectine in moderate to high-risk patients for strongyloides [ ] . lastly, the combination of nitazoxanide/azithromycin has been proposed as a potential treatment in the early phase of covid- [ ] , since the in vitro antiviral activity of nitazoxanide against mers-cov and other coronaviruses [ ] . however, further studies are deemed necessary to better understand these preliminary results. nowadays, evidence from rcts regarding the safety profile and efficacy of the proposed therapies are still lacking. currently, there are clinical trials in progress, of which are not recruiting yet. ongoing clinical trials regarding covid- are available on the website: https://clinicaltrials.gov/ct /results?cond=covid- . it is worthy of note that the who has launched an international multicenter randomized clinical trial, "the solidarity trial", comparing four treatment options (hydroxychloroquine, lpv/r, lpv/r + ifn-β, remdesivir) to standard of care, in order to identify whether any of these drugs are effective in slowing down the disease or improving survival [ ] . however, after a review of the interim analysis of the solidarity trial, including the discovery trial data [ ] , and thanks to the results of other randomized evidences (recovery trial [ ]), the executive group has recently decided to withdraw the hcq arm because of its ineffectiveness in reducing mortality among hospitalized covid- patients [ ] . nevertheless, these results are not applicable to the use of hcq in non-hospitalized patients, where evidences about effectiveness or ineffectiveness of this drug are still lacking. the recovery trial has ceased its hcq arm for the same reason. moreover, the chief investigator of the recovery trial has recently decided to close also the randomization to the lpv/r arm because of the lack of beneficial effects in hospitalized patients [ ] . nevertheless, colleagues could not study this drug in a large number of severe patients on mechanical ventilation; therefore, they cannot make conclusions about the efficacy of lpv/r in mechanical ventilated patients. table shows the main international scientific societies (who, international society of infectious diseases (idsa), centers for disease control and prevention (cdc), and national institute of health (nih)) recommendations regarding antiviral treatment regimens for covid- . as described above, since there are no evidences from rcts that any therapy improves the outcome of patients affected by covid- , all recommendations highlight that patients should be treated in the context of a formal clinical trial in order to establish drugs safety, efficacy, risks, and benefits. as a matter of fact, antiviral drugs are not free of adverse events, which should be considered and monitored. the most important and frequent adverse events of antiviral drugs used against sars-cov- are reported in table . table . world health organization (who), international society of infectious diseases (idsa), and national institute of health (nih) recommendations regarding covid- treatment regimens [ ] [ ] [ ] [ ] . hcq + azithromicyn lpv/r or others pis remdesivir none of the drugs proposed as potential therapies against covid- have been shown to be safe and effective. many agents are now being or will soon be studied in clinical trials (including the solidarity trial). "we recommend that the following drugs not be administered as treatment or prophylaxis for covid- , outside of the context of clinical trials. . . . if it is not possible to give the treatment as part of a clinical trial, appropriate records of the use of the medicine must be kept, in compliance with national law, and outcomes for patients should be monitored and recorded. if early results from an unproven or experimental treatment are promising, the treatment should be studied in the context of a formal clinical trial to establish its safety, efficacy, risks, and benefits." awaiting the results of the ongoing trials, at the moment, it is only possible to hypothesize a different treatment strategy according to the different phases of covid- . in this view, the clinical classification proposed by siddiqi et al. may guide clinicians in the treatment decision process and identify which is the best therapy for each stage of the disease (table ). in the early phase of infection, the treatment is mainly targeted toward symptomatic relief, while antiviral agents may reduce the duration and progression of symptoms severity. drugs with antiviral activity seem important in the second stage as well, together with supportive measures, such as early o therapy (in case of hypoxia) and anti-inflammatory therapy. lastly, in the third stage, immunomodulatory agents, such as corticosteroids or cytokine inhibitors (il -receptor or il -receptor blockers), seem pivotal in order to reduce systemic hyper-inflammation and avoid the progression to a more severe disease. therefore, it seems that the sooner antiviral therapy is started, the better its efficacy; whereas its usefulness is uncertain in late stages [ , ] . on the contrary, the use of immunosuppressive agents may not be necessary in early stages, whereas it becomes essential when hyper-inflammation appears. in conclusion, the rapid identification of the critical situation and the early administration of the above-mentioned therapies are the mainstay for patients outcome [ ] . the covid- pandemic represents the greatest global public health threat of the last decades, with devastating effects not only on public health but also on economic and financial sectors [ , [ ] [ ] [ ] . in this scenario, identifying treatments against sars-cov- infection effective in the first phase of infection is crucial. in fact, in this phase, an effective antiviral drug may stop the spread of covid- and its progression toward severe forms of disease and may reduce morbidity and mortality. many antiviral drugs have been proposed as potential covid- treatment in this first phase. however, at the moment, there is no high-quality evidence to support any of the currently proposed treatment in improving clinical outcomes. as a result of the lack of scientific evidences, covid- treatment remains an issue for clinicians and scientific communities. in this situation, randomized clinical trials aiming to identify effective and safe 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hyperinfection nitazoxanide/azithromycin combination for covid- : a suggested new protocol for covid- early management nitazoxanide, a new drug candidate for the treatment of middle east respiratory syndrome coronavirus infectious diseases society of america guidelines on the treatment and management of patients with covid- treatment guidelines off-label use of medicines for covid- clinical management of covid- -interim guidance outcomes in patients with hyperglycemia affected by covid- : can we do more on glycemic control? diabetes care clinical presentation of covid- : case series and review of the literature a structural equation model to examine the clinical features of mild-to-moderate covid- : a multicenter italian study this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -la njxc authors: garcía-fernández, amaya; ramos-ruiz, pablo; ibáñez-criado, alicia; moreno-pérez, Óscar; cambra-poveda, cristina; martínez-martínez, juan gabriel title: utilidad y seguridad de la automonitorización electrocardiográfica durante el tratamiento con hidroxicloroquina y azitromicina en pacientes con covid- date: - - journal: rev esp cardiol (engl ed) doi: . /j.rec. . . sha: doc_id: cord_uid: la njxc nan utilidad y seguridad de la automonitorización electrocardiográfica durante el tratamiento con hidroxicloroquina y azitromicina en pacientes con covid- [[en]] usefulness and safety of self-electrocardiographic monitoring during treatment with hydroxychloroquine and azithromycin in covid- patients amaya garcía-fernández a* ama_garcia@hotmail.com, pablo ramos-ruiz, a alicia ibáñez-criado, a Óscar moreno-pérez, b,c cristina cambra-poveda, a juan gabriel martínez-martínez, a on behalf of the alicante covid- research group a <org>unidad de arritmias, servicio de cardiología, hospital general universitario de alicante, instituto de investigación sanitaria y biomédica de alicante (isabial)</org>, <city>alicante</city>, <country>spain</country> b <org>departamento de endocrinología y nutrición, hospital general universitario de alicante, instituto de investigación sanitaria y biomédica de alicante (isabial)</org>, <city>alicante</city>, <country>spain</country> c <org>departamento de medicina clínica, universidad miguel hernández, elche</org>, <city>alicante</city>, <country>spain</country> * corresponding author: to the editor, despite the lack of solid evidence on their efficacy, hydroxychloroquine (hcq) and azithromycin (az) have been widely used as a first-line treatment for infection with sars-cov- , the causative agent of coronavirus disease . the effect of these drugs on the qt interval and their potential to cause polymorphic ventricular arrythmias has generated growing concern in the scientific community and until more robust evidence on their usefulness is available, we must employ strategies to ensure their safe use. recently, the food and drug administration recommended the use of noninvasive remote monitoring devices to facilitate the monitoring of these patients, which minimizes contact with health care professionals, reduces the burden on health care services and allows more efficient use of resources. to this end, the kardiamobile l device, from alivecor (california, usa), has been proposed, which can provide a -or -lead electrocardiogram (ecg), offering a simple and reproducible way to determine the corrected qt interval (qtc). here in spain, there are already protocols to support its use in these patients. during march and april of , a study was conducted in our hospital to analyze the effect of treatment with hcq (either alone or in combination with az) on the qtc and the incidence of ventricular arrhythmias in patients admitted with sars-cov- pneumonia who met the high-risk criteria for qtc prolongation (female, age░>░ years, history of heart disease, chronic renal disease, or diabetes, or taking both medications together). in line with the recommendations from the experts, a protocol was designed to minimize the arrhythmic complications of these drugs. this protocol included a series of precautions to be taken before and during treatment: a) review what other medications the patient is taking that could prolong the qtc; b) correct electrolyte imbalances; c) avoid bradycardia; and d) perform close electrocardiographic monitoring. a baseline -lead ecg was performed on admission. later, the qtc was monitored using a -lead recording taken with the kardiamobile l device, at hours and hours after starting the drugs (or more often if the qtc was░>░ ms, if there was an increase░>░ ms, or if the patient had possible symptoms of arrythmia). the arrhythmia unit trained the nursing staff responsible for these patients using an informational video on the use of kardiamobile l. after a brief explanation from the nursing staff, the patient performed the recording, positioning the device on the left knee or ankle (as shown in figure ). from outside the room, the nurse recorded the ecg on a tablet and transferred it to the electronic medical records. four electrophysiologists analyzed the recordings and noted in the medical records the details of the ecg, the qtc measurement, and the recommendations on its treatment if they considered it necessary. for patients who were unable to perform the recording themselves, or when the tracing did not allow accurate measurement of the qtc, a -lead ecg was performed. of patients admitted with covid- pneumonia, received hcq and met criteria to be considered high risk. of these, were included in the electrocardiographic monitoring protocol ( were taking the hcq plus az combination). the baseline qtc was ( - ) ms. of all the patients, ( . %) had qtc prolongation on subsequent ecg; ( . %) were taking hcq alone and ( . %) were taking hcq plus az. the baseline characteristics of the study population are shown in table , as well as the comparison between the groups with and without prolonged qtc. no significant differences were found between the patients with and without prolonged qtc. the median qtc prolongation was . ( . - . ) ms, with no significant differences between the groups who were taking hcq plus az and hcq alone (p░=░ . ). the median qtc duration on monitoring was ( - ) ms. ten patients ( . %) had a significant prolongation of the qtc (increase ≥ ░ms or qtc ≥ ░ms): ( . %) were taking hcq alone and ( . %) were taking hcq plus az. five ( . %) of these patients stopped the medication for this reason. we did not observe sustained ventricular arrhythmia or death due to arrhythmia in any of our patients during the monitoring period. twelve patients ( . %) required a conventional ecg due to difficulty interpreting the recording or not being able to use the device due to limited mobility. according to our experience, hcq and az treatment in patients admitted with covid- is safe as long as measures are taken to minimize the risk of arrhythmia, including close electrocardiographic monitoring. therefore, given the high burden on the healthcare system caused by this disease, and given its high rate of transmissibility, we think that ecg recording by the patients themselves with devices such as the kardiamobile l can be a simple, useful strategy to avoid unwanted proarrhythmic effects of this treatment. the supplementary material for this article can be found in its electronic version available at http://dx.doi.org/ . /j.recesp. . . supplementary material mmc references <bibl> <bib> s jankelson f l. s karam f g. s becker f m.l. s chinitz f l.a. s tsai f m.c. at qt prolongation, torsades de pointes and sudden death with short courses of chloroquine or hydroxychloroquine as used in covid- : a systematic review jt heart rhythm. d doi . /j.hrthm. . . </bib> <bib> food and drug administration. enforcement policy for non-invasive remote monitoring devices used to support patient monitoring during the coronavirus disease (covid- ) public health emergency. available at: https://www.fda.gov/regulatory-information/search-fda-guidancedocuments/enforcement-policy-non-invasive-remote-monitoring-devices-used-support-patientmonitoring-during. hs-tnt, high-sensitivity troponin t; nt-probnp, n-terminal pro-brain natriuretic peptide pcr, polymerase chain reaction. values are expressed as n/n (%) or median this study would not have been possible without the valuable contribution of all the health care professionals involved in the care of our patients with covid- . the authors would like to give special thanks to josé luis ibáñez criado, thomas brouzet, esperanza merino de lucas, isabel lillo ródenas, vicente arrarte esteban, as well as the nurses involved in obtaining the recordings: eunice gonzález ríos, cristina sogorb garri, regina s. cardoso monteiro and norma pérez carpio. key: cord- - gmn fcj authors: almazrou, saja h.; almalki, ziyad s.; alanazi, abdullah s; alqahtani, abdulhadi m.; alghamd, saleh m. title: comparing the impact of hydroxychloroquine based regimens and standard treatment on covid- patient outcomes: a retrospective cohort study date: - - journal: saudi pharm j doi: . /j.jsps. . . sha: doc_id: cord_uid: gmn fcj background pharmacological treatments including antivirals (lopinavir/ritonavir), immuno-modulatory and anti-inflammatory drugs including, tocilizumab and hydroxychloroquine (hcq) has been widely investigated as a treatment for covid- . despite the ongoing controversies, hcq was recommended for managing mild to moderate cases in saudi arabia . however, to our knowledge, no previous studies have been conducted in saudi arabia to assess its effectiveness. methods a hospital-based retrospective cohort study involving patients with covid- was conducted from march to may , . the study was conducted at prince mohammed bin abdul aziz hospital (pmah). the population included hospitalized adults (age ≥ years) with laboratory-confirmed covid- . each eligible patient was followed from the time of admission until the time of discharge. patients were classified into two groups according to treatment type: in the hcq group, patients were treated with hcq; in the sc group, patients were treated with other antiviral or antibacterial treatments according to ministry of health (moh) protocols the outcomes were hospitalization days, icu admission, and the need for mechanical ventilation. we estimated the differences in hospital length of stay and time in the icu between the hcq group and the standard care (sc) group using a multivariate generalized linear regression. the differences in icu admission and mechanical ventilation were compared via logistic regression. all models were adjusted for age and gender variables. results a total of patients fulfilled the inclusion criteria. approximately % (n= ) received hcq-based treatment, and % (n= ) received sc. length of hospital stay and time in icu in for patients who received hcq based treatment was shorter than those who received sc. similarly, there was less need for icu admission and mechanical ventilation among patients who received hcq based treatment compared with sc, ( . % vs. . and . % vs. . %). however, the regression analysis showed no significant difference between the two groups in terms of patient outcomes. conclusion hcq had a modest effect on hospital length stay and days in icu compared with sc. however, these results need to be interpreted with caution. larger observational studies and rcts that evaluate the efficacy of hcq in covid- patients in the saudi population are urgently needed. the world health organization (who) announced coronavirus disease at the beginning of , naming it a public health emergency of international concern (world health organization. who, n.d.) . as of july nd , , the who has reported a total of , , confirmed cases and , deaths (world health organization. who, n.d.) . covid- has been associated with a substantial symptomatic burden, including dyspnea that leads to death due to respiratory and heart failure (keeley et al., ) . the economic burden of such a pandemic is also troublesome. in the united states, the direct medial cost for one patient is $ , per infection course (bartsch et al., ) . if % of the u.s. population becomes infected, the total direct medical cost will be approximately $ billion. direct medical costs are mainly incurred via hospitalization, intensive care unit (icu) admissions, and ventilator use. the burden of covid- therefore extends beyond health care and affects the societal and national economies of affected countries (keni et al., ) . because covid- is an emerging disease, treatment protocols and guidelines are being developed and updated rapidly (national institutes of health, ; world health organization. who, n.d.) . several observational and interventional studies have evaluated the effectiveness of various pharmacological treatments for covid- (matera et al., ; siemieniuk et al., ) . the main therapies being used to treat covid- are antiviral drugs which include remdesivir and lopinavir/ritonavir combination which inhibits viral protease (pascarella et al., ) . other treatments such as respiratory therapy which delivers oxygen in case of hypoxia or symptoms of respiratory distress. chloroquine and hydroxychloroquine (hcq) is among the promising treatment modalities for covid- patients geleris et al., ; rosenberg et al., ; shen et al., ) . hcq is an antimalarial drug that have been used for decades to treat autoimmune diseases such as systemic lupus erythromatus (sle) and rheumatoid arthritis (ra) (ponticelli and moroni, ) . hcq work by increasing the endosomal ph and thus enhancing the fusion between the virus and host cell (pascarella et al., ) . in addition, it has some immunomodulatory effect by interfering with the ace cell receptors. the recommended regimen is to start with a loading dose of mg bid for the first day followed by mg bid (colson et al., ) . the common side effects include nausea, vomiting and diarrhea. arrhythmogenic cardiotoxicity was also associated with the use of hcq, which require qt interval monitoring. several in vitro studies demonstrated the antiviral efficacy of hcq not only have in vitro studies suggested hcq's activity against the sars-cov- virus, but observational studies have also suggested its effectiveness in covid- patients (mahévas et al., ) . several ongoing clinical trials are aimed at examining the efficacy and safety of hcq in covid- patients ("clinicaltrials.gov-searching for covid and hydroxychloroquine," ). despite the large number of studies assessing the effectiveness of hcq, evidence is still limited and inconclusive (pascarella et al., ) . on march , , the saudi ministry of health (moh) issued the first protocol for treating adults with a confirmed diagnosis of covid- ( saudi moh and cdc, ) . in this version, hcq was one of the recommended treatments for mild to moderate cases of the disease. for severe cases, the protocol still recommends hcq, but alternatives such as lopinavir/ritonavir can also be used (saudi moh and cdc, ) . despite the recommendation to use hcq in covid- patients in the moh protocol, no observational studies or rcts that evaluate the efficacy of these drugs in the saudi arabian population have been published. therefore, the objective of this observational study is to compare the effects of hcq and standard care (sc) on length of hospital stay, icu admission, and mechanical ventilation use among covid- patients. a hospital-based cohort study involving confirmed cases of patients with covid- was conducted retrospectively from march , , to may , . the strobe guideline for cohort studies was followed (von elm et al., ) . we conducted the study at prince mohammed bin abdul aziz hospital (pmah), an infectious disease center in riyadh ("prince mohammed bin abdulaziz hospital (pmah)," n.d.). in the response to the covid- pandemic, this hospital was among the leading hospitals designated as covid- centers. as such, patients with covid- symptoms were escorted to this hospital. the population included hospitalized males and females (age ≥ years) with laboratory- patients who were transferred to other facilities, had incomplete or missing data, or received supportive treatment that only included analgesics were excluded from the final dataset. data were collected from patients' medical records by trained medical personnel. collected data included patients' basic information (e.g. age, gender, nationality); medication prescribed; and information on hospitalization, cases requiring icu care, and mechanical ventilation. a well-designed, organized checklist was used to obtain and extract necessary information from patients' medical records. the primary outcomes of interest for this study were hospital length of stay (number of days from the patient's arrival at the hospital until discharge) and time in icu (calculated as the number of calendar days from the day of admission to the day of discharge). we also assessed the patients' need for icu care and mechanical ventilation. data were cleaned, edited, and entered into sas version . for analysis. descriptive data were reported for dichotomous polychotomous frequencies and percentages to examine the distribution of study variables among members of the hcq and sc groups. a chi-square test was utilized to compare categorical variables between groups. continuous variables were presented as means ± standard deviation (sd) and/or median with interquartile range (iqr). we estimated the differences in length of hospital stay and time in icu between the two groups using a multivariate generalized linear model regression. the differences in the need for icu admission and mechanical ventilation were compared via logistic regression. all models were adjusted for age and gender variables. no imputation was performed for all tests, and statistical significance was considered at a p-value of less than . . ethical clearance was obtained from the institutional review board (irb) at king fahad medical city with irb log no. - . hospital management's permission was obtained to conduct this study. the information and data collected were kept confidential. this study included no personal information or identifiers such as names or id number. a total of patients fulfilled the inclusion criteria and were included in the study. a total of regimens were prescribed for these patients. in the hcq group, hcq, azithromycin, and ceftriaxone comprised the most prescribed regimen ( %), whereas in the sc group, an azithromycin and ceftriaxone regimen accounted for % of the participants (see appendix a for additional information). approximately % of patients received hcq based treatment, and % received sc treatment. no differences were observed between the two groups with respect to age, whereas the number of male and non-saudi patients were more in the hcq group (p= . and p= . , respectively). table illustrates the demographic characteristics of the included study patients. length of hospital stay and time in icu in for patients who received hcq based treatment was shorter than those who received sc. similarly, there was less need for icu admission and mechanical ventilation among patients who received hcq based treatment compared with sc, ( . % vs. . and . % vs. . %), respectively; see table ). the results of the regression analyses after controlling for age and gender are shown in table . despite the shorter length of hospital stay and time in icu among patients who received hcq based treatment, as well as the smaller proportions of patients who needed icu care and mechanical ventilation in this group, the results indicated no significant differences in these outcomes between the two cohorts. in this study, we employed a multivariate linear regression with adjustment for gender and age and found that treatment with hcq was associated with shorter length of hospital stay and fewer days in icu when compared with sc treatment. however, the difference was not significant. in addition, the percentage of patients who required icu admission and mechanical ventilation was lower in the hcq group than in the sc group, but the difference was not significant. our results were consistent with those of other observational studies. for instance, a retrospective cohort study was conducted at new york-presbyterian hospital (nyp)-columbia university irving medical center (cuimc) and published in nejm (geleris et al., ) . in this study, the outcomes were intubation rate and death rate (geleris et al., ) . hcq use was not associated with a significant decrease in intubation or death (geleris et al., ) . the second study that evaluated the effectiveness of hcq in covid- patients was a systematic review that evaluated the efficacy of hcq based on peer reviewed articles and preprint studies. hcq showed controversial results among studies (das et al., ) . in conclusion, our study results are consistent with other observational studies on the effectiveness of hcq in covid- patients. generally, the effectiveness of pharmacological treatments of covid- including antivirals such as remdesivir and lopinavir/ritonavir, chloroquine and hydroxychloroquine is limited and inconclusive (cortegiani et al., ; das et al., ; siemieniuk et al., ) . this is mainly due to small sample size of most studies, lack of randomization and potential risk of selection bias (pascarella et al., ) . according to a recently published meta-analysis which aim to assess the effectiveness of pharmacological intervention in covid- (siemieniuk et al., ) . the only promising treatment that demonstrated a substantial impact on mortality, length of stay and mechanical ventilation is glucocorticoids. however, glucocorticoid are only recommended for covid- patients having severe acute respiratory distress syndrome (ards) . the potential benefits of glucocorticoid for patients with no symptoms of ards is still inconclusive (matera et al., ) . to our knowledge, this is the first study in saudi arabia that clearly describes treatment options for covid- patients. other published studies in saudi arabia mainly described patient characteristics with a minimal emphasis on treatments and outcomes (alsofayan et al., ) . the treatment options recommended by the moh protocol (saudi moh and cdc, ) were summarized in a disaggregated method to fully understand the prescribing pattern of covid- treatments. in addition, the choice of various outcomes, including hospitalization, icu admission, and mechanical ventilations targeted various levels of disease severity and facilitated comparison with other published studies that used these outcomes to assess treatment success. however, this study has some limitations. first, randomization was not feasible as this stage of study, which potentially limits the selection bias. second, the study did not have sufficient power to detect any statistical difference due to small sample size. therefore, inferential statistical analyses cannot capture the potential effect of the intervention. second, in saudi arabia, the moh provides % of health care services, whereas other governmental sectors, including teaching hospitals, the ministry of defense, and security forces provide the remainder (almalki et al., ) . this diversity in the provision of health care generates some inevitable issues, including those related to the definition of sc and generalizability. the definition of sc for covid- might vary considerably across various hospitals within the moh and other referral hospitals. moreover, this study recruited people from only one hospital; therefore, the sample might not be representative of people with covid- throughout the kingdom. covid- treatment options and guidelines continue to evolve on a daily basis. therefore, decision-makers need a dynamic source of data that captures such ongoing progress. the health electronic surveillance network (hesn) is a web based platform managed by the moh that records and analyzes infectious diseases and pandemic data (saudi moh and hesn, ) . the current use of the hesn is quite limited, as it only collects patient demographics and laboratory data (alsofayan et al., ) . therefore, decision-makers should consider expanding the scope of such platforms to include treatment regimens and patient outcomes. in addition, decision-makers should mandate that all moh and non-moh hospitals register covid- patients and record their treatments and outcomes on a daily basis. this will ultimately generate a valuable representative data source that could help clinicians, researchers, and decision-makers assess the impact of emerging treatments on patient outcomes. in this study, the choice of hydroxychloroquine and the comparators (mainly antivirals) was mainly informed by the moh guidelines for managing covd- patients ( ). the guideline gave a range of therapeutic options according to the disease severity. additionally, the guideline did not provide any preference as for st and nd line treatment. therefore, it was left to the treating physician to start either with hcq or antiviral. we believe that the selection of st line therapy was based on the availability of the medication and the potential side effect of hcq which need to be used cautiously for patients with arrythmia. future clinical practice guidelines should consider the cost, availability of medication, patient preference and potential side effect to ensure the consistency of clinical practice among different hospitals. despite that hcq based regimens reduce hospitalization and icu admission, the results were not statistically significant. this was mainly due to the small size. in addition, the study's participants were recruited from a single hospital, which limits the generalizability of our results. larger observational studies and rcts that evaluate the efficacy of hcq in covid- patients in the saudi population are urgently needed. health 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observational studies in epidemiology (strobe) statement: guidelines for reporting observational studies who director-general's remarks at the media briefing on world health organization. who, n.d. who coronavirus disease (covid- ) dashboard [www document clinical management of covid- : interim guidance [www document in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) efficacy and safety of corticosteroids in covid- based on evidence for covid- , other coronavirus infections, influenza, communityacquired pneumonia and acute respiratory distress syndrome: a systematic review and *data expressed as mean ± sd and median (iqr) abbreviations: hcq, hydroxychloroquine; sc key: cord- -zadogqiu authors: davido, benjamin; boussaid, ghilas; vaugier, isabelle; lansaman, thibaud; bouchand, frédérique; lawrence, christine; alvarez, jean-claude; moine, pierre; perronne, véronique; barbot, frédéric; saleh-mghir, azzam; perronne, christian; annane, djillali; de truchis, pierre title: nimpact of medical care including anti-infective agents use on the prognosis of covid- hospitalized patients over time date: - - journal: int j antimicrob agents doi: . /j.ijantimicag. . sha: doc_id: cord_uid: zadogqiu introduction: interest of anti-infective agents in covd- showed discrepant results. however, there is no evaluation about the impact in changes of practices on the prognosis over time. methods: single center, retrospective study, conducted from march (th) to april (th) , in adults hospitalized in a medicine ward for a covid- . patient characteristics were compared between periods (before/after march (th)) considering french guidelines issued by learned societies. aim of the study was to evaluate how medical care impacted unfavorable outcome, namely admission in intensive care unit (icu) and/or death. results: one hundred thirty-two patients were admitted, mean age was . ± . years, mean crp level was . ± . mg/l, % had a lymphocyte count< /mm( ). when prescribed, anti-infective agents were lopinavir-ritonavir (n= ), azithromycin (azi) (n= ) and azi combined with hydroxychloroquine (hcq) (n= ). between the periods we noted a significant decrease of icu admission, from % to % (p< . ). delays until transfer in icu were similar between periods (p= . ). pulmonary ct-scan were significantly more performed (from % to %, p< . ), as oxygen-dependency ( % vs %, p= . ) and prescription of azi±hcq (from % to %, p< . ) were greater over time. multivariate analyses showed a reduction of unfavorable outcome in patients receiving azi±hcq (hr= . , %ic [ . - . ], p= . ), especially among an identified category of individuals (lymphocyte≥ /mm( ) or crp≥ mg/l). conclusion: the present study revealed a significant decrease of admission in icu over time probably related to multiple factors, including a better indication of pulmonary ct-scan, of oxygen therapy, and a suitable prescription of anti-infective agents. impact of medical care including anti-infective agents use on the prognosis of covid- hospitalized patients over time introduction: interest of anti-infective agents in covd- showed discrepant results. however, there is no evaluation about the impact in changes of practices on the prognosis over time. methods: single center, retrospective study, conducted from march th to april th , in adults hospitalized in a medicine ward for a covid- . patient characteristics were compared between periods (before/after march th ) considering french guidelines issued by learned societies. aim of the study was to evaluate how medical care impacted unfavorable outcome, namely admission in intensive care unit (icu) and/or death. results: one hundred thirty-two patients were admitted, mean age was . ± . years, mean crp level was . ± . mg/l, % had a lymphocyte count< /mm . when prescribed, anti-infective agents were lopinavir-ritonavir (n= ), azithromycin (azi) (n= ) and azi combined with hydroxychloroquine (hcq) (n= ). between the periods we noted a significant decrease of icu admission, from % to % (p< . ). delays until transfer in icu were similar between periods (p= . ). pulmonary ct-scan were significantly more performed (from % to %, p< . ), as oxygen-dependency ( % vs %, p= . ) and prescription of azihcq (from % to %, p< . ) were greater over time. multivariate analyses showed a reduction of unfavorable outcome in patients receiving azihcq (hr= . , %ic [ . - . ], p= . ), especially among an identified category of individuals (lymphocyte≥ /mm or crp≥ mg/l). conclusion: the present study revealed a significant decrease of admission in icu over time probably related to multiple factors, including a better indication of pulmonary ct-scan, of oxygen therapy, and a suitable prescription of anti-infective agents. management and medical care of covid- pneumonia in hospitalized patients is currently still debated, especially because data regarding an emerging pathogen are constantly evolving over time and across countries. numerous therapies including oxygen, anti-infective agents and corticosteroids have been proposed. historically, gautret et al. [ , ] and million et al. [ ] observed in marseille (france) that a combination therapy using hydroxychloroquine (hcq) and azithromycin (azi) could potentially reduce viral shedding and the incidence of covid- pneumonia. concomitantly, an observational study conducted by mahevas et al. [ ] evaluating hcq alone prescribed in an in-hospital setting, showed no impact of hcq on the transfer rate in intensive care unit (icu) and/or death. this study is concordant with a publication issued in the united states by geleris et al. [ ] who concluded that hcq administration was not associated with a greatly lowered risk of intubation or death. interestingly, although corticosteroids were considered potentially harmful in the early care of covid- infected patients [ ] , the recovery trial (nct ) stated that dexamethasone could reduce mortality rate up to % in severely-ill patients admitted for a covid- pneumonia and revealed no interest of hcq (data not published), meanwhile the azithromycin arm is still being investigated. very recently a multicenter study in the united states reopened the debate concerning the efficacy of hcq with or without azi [ ] . furthermore antiviral therapies, notably lopinavirritonavir, revealed no benefit in comparison to standard of care in a large randomized trial [ ] , whereas remdesivir showed a reduction in time to clinical improvement in trials but no significant impact on mortality [ , ] . overall those reports have raised concerns about the true interest of anti-infective agents in covid- pneumonia in a context where medical practices between these different studies are heterogeneous and have evolved over time. indeed, in the absence of a clear recommendation for treatment initiation, it is difficult to assume or to invalidate the effect of anti-infective agents on the prognosis of covid- patients. to our knowledge, there is no evaluation over time about changes of practices, including anti-infective agents, and their impact on the prognosis of patients admitted in a medical ward for a covid- pneumonia. considering controversies, we retrospectively evaluated the potential factors associated with an unfavorable outcome, namely admission in icu and/or death, during this first wave of the epidemic. we conducted a single center and retrospective study, from march th to april th , regarding adults admitted in our medicine wards in a tertiary university hospital namely hôpital raymond poincaré (ap-hp), garches, france. we included all the adults admitted in medicine for a covid- infection confirmed by sars-cov- rt-pcr and/or a compatible pulmonary ct-scan. exclusion criteria were: i) patients directly admitted in icu; ii) patients discharged from icu to a medicine ward; iii) opposition to collect data expressed by the patient. the following data were collected from patient's medical charts: -patient characteristics: age, sex, diabetes, cardiovascular risk factors, smoking habits, obesity, chronic pulmonary disease, charlson comorbidity index (cci) [ ] , -infection characteristics: delay between onset of symptoms and admission, presence of super-infection, c-reactive protein (crp) and white blood cell count (wbc) at admission, percentage of lung injuries on ct-scan if applicable, positive pcr amplifying the betacoronavirus e gene and the sars-cov- rdrp gene on nasopharyngeal swab or sputum, -treatment characteristics: requiring icu support with invasive ventilation and associated therapeutic strategies (e.g. oxygen, anti-infective agents), -endpoint was defined as unfavorable outcome assessed by the requirement of a transfer in icu for invasive ventilation and/or death within days, -patients were followed-up until hospital discharge. after discharged, patients were monitored during days by the telemedicine through the french covidom platform [ ] , -derived variables: moderate lymphocytopenia was based on a lymphocyte count with a threshold at /mm and high systemic inflammation was defined as a crp threshold ≥ mg/l. all patients who required oxygen received systematically a beta-lactam for at least days, using preferentially ceftriaxone or cefotaxime to treat a potential superinfection. patients were eligible to a supposed effective anti-infective agent against covid- (hcq, azi, lopinavir-ritonavir), independently of biological abnormalities and considering the following indications: i) patient presenting a clinical pneumonia confirmed by sars-cov- pcr, requiring oxygen therapy (independently of the ct scan findings); ii) high suspicion of covid- pneumonia considering the clinical presentation and/or pulmonary ct-scan showing ground-glass opacity affecting ≥ % of the whole parenchyma. patients were categorized as receiving an anti-infective agent once they received at least one dose. patients who received lopinavir-ritonavir were categorized in no treatment group, considering this antiviral drug did not show any benefit for the treatment of covid- [ ] . before hcq or azi initiation, patients had systematically an electrocardiogram (ecg) to evaluate the corrected qt interval using the framingham formula, and monitored times per week during the whole treatment, as well as serum potassium levels. a loading dose at day with mg/day was administered followed by a maintenance dose of mg/day up to mg/day in case of obesity (body mass index (bmi) > ) for a total days. in addition, mg of azithromycin was prescribed the first day, followed by mg for days. patients were informed that hcq and lopinavirritonavir were currently off-label for the treatment of covid- pneumonia until the th of march in france, where the ministerial decree # - authorized the in-hospital prescription of hcq in this particular indication. in case they refused the prescription of hcq or the latter was contraindicated (by ecg or drug interactions), it was noted into their medical chart and patients did not receive hcq. aim of the study was to describe the medical care over time (oxygen therapy, antiinfective agents, pulmonary ct-scan) and to determine whether potential factors were related to an unfavorable outcome (transfer in icu and/or death). descriptive statistics are presented as counts and percentages, or means and standard deviations, with skewed continuous data summarized as medians and interquartile ranges. interactions between treatment and lymphocyte count or crp level were tested and kaplan-meier curves were plotted to assess unfavorable outcome from admission depending on these biological parameters. statistical significance was set at . (two-tailed test). all statistical calculations were performed using r software version . . . all procedures performed in studies involving human participants were in accordance with the ethical standards and with the helsinki declaration and its later amendments or comparable ethical standards. this study has passed the cesrees/health data hub regarding ethics committee approval (mr ) and is registered on clinicaltrials.gov (nct ). as part of an anonymous and retrospective study, a non-opposition and information letter was sent to participants afterwards. greater than mg/l. seventy-two percent of patients were oxygen-dependent at admission, with % of patients with an oxygen flow therapy greater than l/min. among the patients who underwent a pulmonary ct scan, % had lung injuries compatible with covid- greater than % of the whole parenchyma. sars-cov- rt-pcr was positive in . % (n= ) of cases. overall, ( %) patients received one anti-infective agent. among them, ( %) received lopinavir-ritonavir, ( %) azithromycin (azi) and ( %) azi combined with hcq ( during the first period, ( %) patients were hospitalized whereas ( %) were admitted thereafter. there were significantly more oxygen-dependent patients hospitalized during the second period than the first one ( % vs %, p= . ). also, a significant higher number of pulmonary ct scan performed was observed over time between periods of hospitalization from % to % (p< . ), independently of ct-scan severity (table ) . concomitantly, prescription of azi whether or not combined with hcq increased over time, from % to % between the periods (p< . ) (figure ). of note, among patients who did not receive hcq, had cardiac contraindication and refused to be treated with this molecule. during the course of treatment using azi in combination with hcq, we report only patient that presented an adverse event (a prolonged qt interval on ecg without clinical event) that led to discontinuation of hcq within h, and was switched to azithromycin alone. a total of ( %) patients had an unfavorable outcome, among them ( %) were transferred to icu and ( %) died without being transferred in icu. mean delay between hospitalization and admission in icu was . ± . days ( . ± . days during the first period vs . ± . days during the second one, p= . ). a trend towards a lower frequency of admission to icu was observed, from % in the first period to % in the second period (p< . ) (figure ). overall, the risk of death or admission to icu was significantly related to the oxygen flow (p< . ) and to lymphocyte count in a first model (i.e. lymphocyte there was a significant interaction between treatment and crp level (p= . ) and at the limit of statistical significance for the lymphocyte count (p= . ) supporting a subgroup analysis. in univariate analysis, patients who benefited from azi whether or not combined with hcq with a lymphocyte count ≥ /mm , were less likely to have an unfavorable outcome compared to patients without any treatment (p= . ) (fig .a) . concomitantly, patients who benefited from azi whether or not combined with hcq with a crp ≥ mg/l, were less likely to have an unfavorable outcome compared to patients without any treatment (p= . ) (fig .b) . however, these results are not reproducible in patients with a lymphocyte count < /mm (p= . ) and similarly in patients with a crp level < mg/l (p= . ) ( figure s .a, s .b in supplementary data). our study highlights that unfavorable outcome (transfer to icu and/or death) because of lockdown, it looks like patients were admitted later in the second period than during the first period of the epidemic and it might explain why they required more oxygen therapy at baseline. we suggest that in case of a second wave, it could be relevant to introduce telemedicine monitoring of vital signs including pulse oximetry at home. indeed, oxygen therapy at home, as proposed by the french covidom platform in patients discharged from the hospital during the first wave of the epidemic was of interest [ ] . in multivariate analyses, our models adjusted on the lymphocyte count or crp, showed that patients who benefited from azi whether or not combined with hcq were . and . times less likely to have an unfavorable outcome than patients without treatment (p= . ), respectively. this finding suggests that the lymphocyte count which is already known to be closely related to covid- disease severity [ , ] could be also a predictive factor of anti-infective therapy response. indeed, patients with lymphocyte count ≥ /mm might be patients at an early stage of covid- , arguing for the earliest initiation of anti-infective agents, as previously demonstrated with oseltamivir treatment in severely-ill patients with pandemic influenza a (h n ) [ ] . however, we did not study whether there was a relationship between the lymphocyte count and the delay from first onset of symptoms to the admission, because this variable is declarative and thus not reliable. likewise, azi whether or not combined with hcq showed interest in hospitalized patients with a high systemic inflammation (crp level ≥ mg/l), known as the so called -cytokine storm‖. this is one argument pleading for a possible immune-modulator effect of the treatment as previously described by zhao et al. [ ] . interestingly, our study does focus on the potential interest of treatment with azithromycin whether or not combined depending on certain biological parameters. indeed, azithromycin's potential antiviral activity is concordant with previous in vitro studies regarding sars-cov- [ ] or h n -pdm [ ] and one clinical randomized trial in in the prevention of children respiratory infections [ ] . in addition a recent publication emphasized the role of azithromycin against covid- through the cd receptor of stem cell [ ] . moreover, one study published in the jama by rosenberg et al. [ ] highlighted a potential trend to a decreased mortality in patients receiving azithromycin versus hcq or standard of care despite being non-statistically significant (p= . ). moreover, authors discussed that the rapidity with which patients entered the icu (within hours) might have underestimated the treatment efficacy. also, as azithromycin is commonly prescribed for bronchitis and authorized in ambulatory care, a study conducted among general practitioners could be relevant to evaluate early indication of this single therapy for the treatment of covid- in fragile outpatients. in addition, our experience does not report any serious side effect of this combination therapy as long as we take the necessary caution and perform follow-up ecg using a conventional dose of hcq as proposed by borba et al. [ ] . our study has several limitations. the first limitation is the single center nature of the study, describing the experience of a unique center whose results might not be generalizable. however, it was carried out in a hospital specialized for decades in the moreover, considering inherent limitation of a descriptive study with a limited sample size (n= ), we could not infer causality in the association between the use of azi±hcq and the ameliorated prognosis in covid- patients. besides, we also noted that some unforeseen confounders (e.g., pre-hospital medication and delay to admission) may still potentially alter the magnitude of azithromycin effects on the outcome of covid- pneumonia. also, choices in anti-infective agents have differed between the first and second period, notably because prior to march th , hcq was not authorized by the french minister of health and explained partly the common use of lopinavir-ritonavir at this period. finally, we decided to choose a multivariate model rather than a propensity score because the aim of this study was not to evaluate the effect of azi±hcq on the prognosis but to evaluate all factors which could have impacted on medical care. in conclusion, findings from this study showed that rate of admission in icu all authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. authors would like to thank pr xavier paoletti for his proofreading of the manuscript and his particular attention to the statistical analyses. ( ) < . azi ± hcq ( ) ( ) † in first period is define between / to / ; ‡in second period is define between / to / ; azi, azithromycin; hcq, hydroxychloroquine; n, number; %, percent; sd, standard deviation; m, men; obesity with body mass index ≥ kg/m²; *cci, charlson comorbidity index; pmn, polymorphonuclear leukocyte; crp, c-reactive protein; ct : computerized tomography; pulmonary ct scan category normal [ %], limited < %, mild % - %, moderate % - %, severe > %; a student test (equal variance) or a welche-satterthwaite t test (unqual variance) was used to analyze the quantitative variables, a mantel-haenszel chi-square test was used to analyze the qualitative variables and the exact test of fisher was used when the sample sizes were small (< ). test significant (p< . ) infection & inflammation-u- centre d'evaluation et de traitement de la douleur-u- handicap neuromusculaire-u- centre d'investigation clinique clinique de la muette clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in covid- patients with at least a six-day follow up: a pilot observational study hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial early treatment of covid- patients with hydroxychloroquine and azithromycin: a retrospective analysis of cases in marseille, france clinical efficacy of hydroxychloroquine in patients with covid- pneumonia who require oxygen: observational comparative study using routine care data observational study of hydroxychloroquine in hospitalized patients with covid- clinical evidence does not support corticosteroid treatment for -ncov lung injury treatment with hydroxychloroquine, azithromycin, and combination in patients hospitalized with covid- a trial of lopinavir-ritonavir in adults hospitalized with severe covid- remdesivir in adults with severe covid- : a randomised, double-blind, placebo-controlled, multicentre trial remdesivir for the treatment of covid- -preliminary report a new method of classifying prognostic comorbidity in longitudinal studies: development and validation assistance publique-hôpitaux de paris' response to the covid- pandemic recommandations d'experts portant sur la prise en charge en réanimation des patients en période d'épidémie à sars-cov : hematological findings and complications of covid- lymphopenia predicts disease severity of covid- : a descriptive and predictive study early versus late oseltamivir treatment in severely ill patients with pandemic influenza a (h n ): speed is life n.d cytokine storm and immunomodulatory therapy in covid- : role of chloroquine and anti-il- monoclonal antibodies outcomes of , covid- patients treated with hydroxychloroquine/azithromycin and other regimens in marseille, france: a retrospective analysis in vitro screening of a fda approved chemical library reveals potential inhibitors of sars-cov- replication azithromycin, a -membered macrolide antibiotic, inhibits influenza a(h n )pdm virus infection by interfering with virus internalization process early administration of azithromycin and prevention of severe lower respiratory tract illnesses in preschool children with a history of such illnesses: a randomized clinical trial cd as a target for covid- treatment: suggested effects of azithromycin and stem cell engagement association of treatment with hydroxychloroquine or azithromycin with in-hospital mortality in patients with covid- in new york state effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus (sars-cov- ) infection /n number/total; * indicates the reference category no treatment defined as patients who have had no treatment or lopinavir-ritonavir multivariate cox model regression was used to identify the potential factors associated with unfavorable outcome (icu admission or death after icu), adjusted on cci (including age), obesity, oxygen and treatment strategies groups according to crp key: cord- -temt b f authors: brown, ronald title: hydroxychloroquine and “off-label” utilization in the treatment of oral conditions date: - - journal: oral surg oral med oral pathol oral radiol doi: . /j.oooo. . . sha: doc_id: cord_uid: temt b f nan hydroxychloroquine and "off-label" utilization in the treatment of oral conditions to the editor: in response to president trump's remarks made on march , , concerning the potential of chloroquine and hydroxychloroquine (hcq) as treatment for the novel coronavirus- infections: "the u.s. food and drug administration (fda) swiftly issued a statement to clarify that, no, these drugs are not approved as treatments for covid- , the disease caused by the coronavirus sars-cov- . both drugs are approved to treat malaria, lupus, and rheumatoid arthritis but must still be assessed in clinical trials before being declared a safe and effective covid- treatment. doctors in the united states have wide latitude to prescribe drugs "off-label," meaning for conditions beyond their initial fda approval." hcq may or may not pan out to be a successful therapeutic agent in the treatment for covid- infections. however, it appears likely that many health care providers may begin using this drug without knowledge of accepted dosage regimens and toxicity. hcq is a drug specifically approved for the prevention and treatment of malaria. however, it is utilized extensively by both physicians and dentists (oral medicine clinicians) in the treatment of rheumatologic conditions, such as systemic lupus erythematosus, sjogren syndrome, rheumatoid arthritis, chronic ulcerative stomatitis, immune thrombocytopenia purpura, lichen planopilaris, and oral lichen planus. in the realm of treatment of autoimmune secretory and oral mucosal conditions, hcq has been deemed safe and effective for such oral conditions as sjogren syndrome, chronic ulcerative stomatitis, and oral lichen planus. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] a noted possible negative effect of hcq is druginduced conjunctivitis. this toxicity is typically addressed by advising the patient to see his or her ophthalmologist at least once yearly. recently, it has been noted that there is a rare complication related to hcq use, that is, sudden death resulting from a particular cardiac arrhythmia. torsade de pointes arrhythmia is associated with prolonged qt duration secondary to high-dose hcq administration. , however, as reported by o'laughlin et al., hcq-related qt interval prolongation and secondary arrhythmia are extremely rare and may be related to higher dosage regimens. danielsson et al. reported that the results of their recent study on sudden death in older patients indicated an increased risk of torsade de pointes arrhythmia with the use of the selective serotonin reuptake inhibitor citalopram. therefore, there appears to be the possibility of additive drug interactions when prescribing hcq to patients already taking citalopram and other drugs that significantly prolong the qt duration and increase the risk of a torsade de pointes arrhythmia. over years ago, it was noted that the antihistamine h blocker terfenadine was cardiotoxic in higher doses and that particular drugs used in dentistry, such as ketoconazole and erythromycin, and even grapefruit juice could result in a drugÀdrug interaction and potentially lethal serum values, with the possible result of cardiotoxicity (specifically the torsade de pointes arrhythmia) and death. the danger of this sudden death condition resulted in the eventual removal of terfenadine as a clinical therapeutic agent worldwide. [ ] [ ] [ ] [ ] at rheumatologic therapeutic dosage levels, hcq has been regarded as a reasonably safe therapeutic agent. however, oral medicine clinicians and other health care providers should be advised of the potential issues with the use of hcq, such as drugÀdrug interactions, the additive toxicity of qt duration prolongation, and the association with sudden death, in the treatment of older patients. could the anti-malarial drug chloroquine treat covid- ? available at bhattacharyya i. chronic ulcerative stomatitis: diagnostic and management challenges-four new cases and review of literature. oral surg oral med oral pathol oral radiol endod metabolic and cardiovascular benefits of hydroxychloroquine in patients with rheumatoid arthritis: a systematic review and meta-analysis chronic ulcerative stomatitis: a distinct clinical entity effects of hydroxychlorquine on salivary flow rates and oral complaints of sjogren patients: a prospective Ó hydroxychloroquine: a multifaceted treatment in lupus the role of hydroxychloroquine as a steroid-sparing agent in the treatment of immune thrombocytopenia: a review of the literature hydroxychloroquine and lichen planopilaris: efficacy and introduction of lichen planopilaris activity index scoring system hydroxychloroquine sulphate therapy of erosive oral lichen planus. australas ocular adverse effects associated with systemic medications: recognition and management chronic hydroxychloroquine use associated with qt prolongation and refractory ventricular arrhythmia cardiac complications attributed to chloroquine and hydroxychloroquine: a systematic review of the literature life threatening severe qtc prolongation in patient with systemic lupus erythematosus due to hydroxychloroquine drug use and torsades de pointes cardiac arrhythmias in sweden: a nationwide registerbased cohort study the role of adenosine receptors and endogenous adenosine in citalopram-induced cardiovascular toxicity grapefruit juice alters terfenadine pharmacokinetics, resulting in prolongation of repolarization on the electrocardiogram cardiotoxicity of histamine and the possible role of histamine in the arrhythmogenesis produced by certain antihistamines cardiac k+ channels and drug-acquired long qt syndrome next generation antihistamines: therapeutic rationale, accomplishments and advances key: cord- -djv syy authors: ullah, waqas; m. abdullah, hafez; roomi, sohaib; sattar, yasar; almas, talal; narayana gowda, smitha; saeed, rehan; mukhtar, maryam; ahmad, ammar; oliver, tony; alraies, m. chadi; haas, donald c.; fischman, david l. title: safety and efficacy of hydroxychloroquine in covid- : a systematic review and meta-analysis date: - - journal: j clin med res doi: . /jocmr sha: doc_id: cord_uid: djv syy background: during the initial phases of the coronavirus disease (covid- ) epidemic, there was an unfounded fervor surrounding the use of hydroxychloroquine (hcq); however, recently, the centers for disease control and prevention (cdc) has recommended against routine use of hcq outside of study protocols citing possible adverse outcomes. methods: multiple databases were searched to identify articles on covid- . an unadjusted odds ratio (or) was used to calculate the safety and efficacy of hcq on a random effect model. results: twelve studies comprising , patients (hcq , and control ) were included. the odds of all-cause mortality (or: . , % confidence interval (ci): . - . , p value < . ) were significantly higher in patients on hcq compared to patients on control agent. the response to therapy assessed by negative repeat polymerase chain reaction (pcr) (or: . , % ci: . - . , p = . ), radiological resolution (or: . , % ci: . - . , p value = . ) and the need for invasive mechanical ventilation (imv) (or: . , % ci: . - . , p value = . ) were identical between the two groups. overall, four times higher odds of net adverse events (naes) were observed in the hcq group (or: . , % ci . - . , p value = . ). the measures for individual safety endpoints were also numerically lower in the control arm; however, none of these values reached the level of statistical significance. conclusions: hcq might offer no benefits in terms of decreasing the viral load and radiological improvement in patients with covid- . hcq appears to be associated with higher odds of all-cause mortality and naes. on march , , the world health organization (who) officially declared coronavirus disease (covid- ) a global pandemic. emerging as a handful of pneumonia case clusters in wuhan, china, in late december , covid- has now reached all corners of the world. the epicenter of the outbreak has shifted over the past months from china to italy and now to new york. as of april , , more than million cases from over countries and more than , deaths have been documented worldwide, with the usa accounting for nearly a quarter of all fatalities. the projected number of deaths in the usa is between , and , , with an estimated total burden of over million covid- cases [ ] . in response to this extraordinary public health challenge and the urgent need for medications to treat this disease, the food and drug administration (fda) established a coronavirus treatment acceleration program (ctap) and issued emergency use authorization (eua) for chloroquine (cq) and its derivative hydroxychloroquine (hcq). cq and hcq (an analog of cq) are primarily approved for the treatment of malaria and chronic inflammatory disorders (rheumatoid arthritis), respectively [ ] . scientific literature pertaining to the immunopathology of the severe acute respiratory syndrome (sars) and middle eastern respiratory syndrome virus (mers) has previously shown some effect of these medications against those viruses [ ] . this lends some credence to the use of these medications against severe acute respiratory syndrome coronavirus (sars-cov- ). both drugs are thought to prevent the ingress of virions into the host cell, halt post-entry cascades and replication of the virus. the former is achieved by inhibition of the downstream glycosylation of angiotensin-converting enzyme (ace ) receptors, while en-hydroxychloroquine and covid- j clin med res. ; ( ): - dosomal acidification helps in reducing the overall virus burden [ ] [ ] [ ] . although hcq was widely adopted for the treatment of covid- , this appears to have been based on little concrete evidence, but rather on anecdotal data emerging from china and france. while a few small-scale studies have demonstrated some favorable outcomes, the safety and efficacy of these regimens remain to be proven. this, along with considerable concern for hcq-related adverse outcomes, has prompted us to conduct this meta-analysis as we await the results of randomized control trials (rcts). the medline (pubmed, ovid), embase, clinicaltrials. org and cochrane databases were queried with various combinations of medical subject headings (mesh) to identify relevant articles. there were no language or time restrictions placed. backward snowballing was performed to retrieve unidentified studies that were missed on the initial search. the mesh used included two subsets: one for the covid- and another for hcq. the two subsets of mesh were combined in a : combination using boolean operators. results from all possible combinations were downloaded into an endnote library. all rcts and observational cohort studies (ocss) until april , , were evaluated. studies comparing the safety and efficacy of the hcq in covid- were included. the efficacy endpoints were radiological resolution or decreased virological load on repeat polymerase chain reaction (pcr). safety outcomes included net adverse events (naes), a composite of gastrointestinal, respiratory, neurological and dermatological outcomes. secondary outcomes included components of nae. review articles, conference papers, and studies with no control arm or insufficient data were excluded. the statistical analysis was performed using the dersimonian and laird test on a random-effects model to calculate an unadjusted odds ratio (or). the probability value of p < . was considered statistically significant. the "test for overall effect" was reported as a z-value corroborating the inference from the % confidence interval (ci). if the eligibility of a study was dubious or unduly influencing results due to its large sample size, a sensitivity analysis was performed. subgroup analysis based on the type of control group (placebo vs. antiviral) was also performed. higgins i-squared (i ) statistical model was used to assess variations in outcomes of the included studies. i less than % corresponded to low heterogeneity. depending upon the strength of evidence for heterogeneity (p value from the chi-square χ analysis), i of - % indicated moderate (p ≥ . ) or moderate to severe (p ≤ . ), and i of % or higher suggested substantial heterogeneity. publication bias was il-lustrated graphically using a funnel plot. the methodological quality assessment of the included rcts was performed using the cochrane collaboration tool for the systematic review and meta-analysis, where each study was screened for five different types of bias (selection, performance, detection, attrition, and reporting bias). all statistical analysis was performed using the digitize and the cochrane review manager (revman) version . . the overall quality of the included rcts was high (fig. ). due to adequate randomization and allocation concealment, the risk of selection bias in rcts was low. however, three retrospective cohort studies introduced a % overall risk of selection bias. the risks of performance and detection bias were high because of inadequate blinding of participants and outcomes, respectively. reporting bias across all studies was reduced due to an adequate description of the study results. the fact that most rcts used an intention to treat model or had a minimal loss to follow-up of its participants, the risk of attrition bias was minimal. an approval was obtained from the institutional review board (irb). the initial search revealed articles. after removal of irrelevant and duplicate items, articles were deemed relevant for full-text review. of these, articles were excluded based on our selection criteria. fourteen articles (seven rcts, seven ocss) qualified for quantitative analysis [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the preferred reporting items for systematic reviews and meta-analyses (prisma) flow diagram is shown in figure . a total of , patients, , in hcq and , in the control arm were included. the mean age was years, comprising % male patients. eight studies compared hcq therapy to standard of care therapy (sct) on outcomes including mortality, viral clearance and radiological resolution. sct is defined as a supportive therapy with no specific treatment for covid- . the study of borba et al compared high dose hcq ( mg twice daily) with low dose hcq ( mg twice daily) [ ] . huang et al compared hcq against lopinavir/ritonavir therapy [ ] . carlucci et al compared hcq + azithromycin + zinc with hcq + azithromycin in the control group [ ] . the baseline characteristics were comparable between the hcq and the control group with a proportion of hypertension ( % vs. . %), diabetes mellitus ( % vs. %), smoking ( . % vs. . %) and chronic kidney disease ( . % vs. %), respectively. the follow-up period varied from days to days with a median follow-up duration being days. the detailed account of the inclusion criteria of studies and the baseline characteristics of included patients are given in supplementary material (www.jocmr.org). a comprehensive pooled analysis of studies did not show any benefit with the use of hcq in any of the efficacy endpoints. virological clearance assessed by negative repeat pcr (or: . , % ci: . - . , p = . ) and radiological resolution of concerning findings for covid- (or: . , % ci: . - . , p = . ) were similar between the two groups. the in-hospital requirement for an invasive mechani- was also identical between patients on hcq compared to those not on hcq. there was no significant difference between the disease progression between the two groups (or: . , % ci: . - . , p = . ), while, hcq was found to be associated with significantly higher odds of all-cause mortality (or: . , % ci: . - . , p value < . ) compared to patients in the control group. the heterogeneity in the outcomes of the included studies was moderate to high (i = % - %) (figs. , a) . the incidence of nae, a composite of all systemic complications, was used to gauge the safety of hcq. the nae with hcq was significantly higher in the hcq group as compared to control groups (or: . , % ci: . - . , p value = . ) (fig. b) . interestingly, the odds of all individual adverse events were consistently higher but statistically nonsignificant in patients receiving hcq for covid- ; respiratory complications (fig. ) . in the study of borba et al [ ] , a high dose of hcq was associated with a higher rate of qt prolongation, and ventricular arrhythmias. subgroup sensitivity analysis based on the type of medications used in combination with hcq in the experimental group mirrored the overall findings of all efficacy endpoints except that mechanical ventilation was lower in the study by carlucci et al [ ] , and control agent was favored in the study by gautret et al [ ] in terms of virological seroconversion. the safety profile was in concordance with the pooled results for all subgroups (supplementary materials - , www.jocmr.org). on visual assessment of the funnel plots, publication bias was minimal for overall outcomes (fig. ) . the vertical axis of the plot used the standard error to estimate the sample size of the study; it is evident that the majority of the included studies fall at the bottom, indicating a small sample size. the horizontal spread reflected that most studies were underpowered due to a wide ci of the effect size. our meta-analysis of studies comprising , patients showed no significant difference in the efficacy of hcq compared to the control arm, while the overall naes were five times higher in in the former group [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the individual safety measures including the incidence of gastrointestinal, respiratory, cardiac, dermatological and neurological complications were numerically higher but statistically non-significant between the two groups. efficacy was gauged by a difference in the mortality rate, decrease in the viral load and radiological resolution of covid- -related findings. while there was no significant difference in most of the efficacy endpoints, patients on hcq had a two-fold higher rate of in-hospital allcause mortality compared to patients on sct. of the included studies, the major efficacy endpoint referred to the attainment of a negative pcr result, indicating total viral clearance usually at - days of treatment. a pooled analysis of five studies comprising patients revealed that a virologic cure was achieved in % of patients treated with hcq compared to % in the control group. gautret et al presented the results of a -day trial with patients [ ] . this was an outlier as the only study that had a statistically significant improvement in virologic cure rate. it is unclear if these findings could be attributed to the substantial amount of attrition and performance bias seen in this study [ ] . six patients on hcq were prematurely excluded before completion of the study, and there were inconsistencies in the methods and centers of pcr testing between the two groups. nevertheless, this study triggered massive interest and prompted other researchers to further evaluate the efficacy of hcq. these subsequent studies have, however, shown an identical rate of virological clearance among both the hcq-treated and sct groups, mirroring the findings of our pooled analysis. it should be noted that the wide ci in our results indicates that it is difficult to draw definitive conclusions from the limited data currently available. three studies included in our analysis reported resolution of suggestive radiological findings on computerized tomography (ct) as an efficacy outcome [ ] [ ] [ ] . a pooled analysis of these studies that comprised (hct) and patients (sct group) showed a trend favoring resolution in the hcq group ( % vs. %), though statistical significance was not achieved. huang et al was the only study that provided a breakdown of radiologic features. they reported a higher percentage of patients in the control group with the involvement of more than two lobes and also bilateral distribution of patchy shadows ( % vs. % and % vs. %, respectively) [ ] . the most debilitating complication of severe acute respiratory syndrome coronavirus (sars-cov- ) infection is acute respiratory failure necessitating the use of imv and other concurrent resource-intensive needs in critical care units [ ] . the need for artificial ventilation was reported by four studies, comprising , patients in the hcq and patients in the control group, showing % vs. % utilization, respectively, with a pooled or of . [ , , , ] . while this favors the hcq group, it was statistically not significant with p value of . . among these four studies, we found that the study of magagnoli et al was not only subject to the bias of nonrandomized data but also to ascertainment issues, i.e., sicker patients were more likely to receive hcq on compassionate grounds and hence were more prone to have adverse outcomes [ ] . our mortality analysis of , (hcq group: , ; control: , ) showed a significantly higher rate of all-cause mortality in patients in hcq group compared to those on sct. it should be noted, however, that this difference is principally driven by three out of studies with higher population [ ] [ ] [ ] . magagnoli et al reported a mortality rate three times higher in the hcq group than the sct group [ ] . this finding is likely reflective of the inclusion of a sicker population in the treatment group, as evidenced by the higher rate of lymphopenia and azithromycin use. the study by borba et al differed in terms of its inclusion criteria, comparing a high dose of hcq against a low dose of hcq [ ] . similarly, carlucci et al studied the efficacy of hcq, azithromycin, and zinc against hcq and azithromycin, demonstrating increased frequency of discharges (or: . , % ci: . - . ) and low mortality (or: . , % ci: . - . ) in the zinc regimen arm [ ] . on the contrary, rosenberg et al compared sct with hcq and azithromycin, or hcq alone, favoring the control arm due to a significantly lower need for imv and mortality [ ] . these findings were validated by geleris et al [ ] . overall, the inconsistencies in the results of the included studies indicate that currently there is not enough data available to confirm or refute the efficacy of hcq in this patient population, and we would have to wait for further studies to be reported before a more accurate assessment can be made [ ] . while the therapeutic benefits of hcq have now been widely touted, the adverse effects have not been adequately highlighted. in the current context, hcq is generally thought to be safe due to a typically short course of therapy employed [ ] . in light of these trends and with previous studies demonstrating major side effects of hcq (cardiomyopathy, qt prolongation, and retinopathy), a considered risk to benefit determination should be made prior to hcq use. in summary, the available data regarding the effect of hcq on safety and efficacy are limited and fraught with inconsistencies. a better analysis may be possible once more data become available from the numerous trials that are currently underway. however, it is important to keep in mind that the virologic cure does not necessarily correlate with the resolution of symptoms and patient outcomes. at present, our analysis shows that hcq use is not associated with any benefit and could potentially be harmful. our study is constrained by the limitations of the included studies. all studies were vastly underpowered due to the small sample size. there was significant heterogeneity in the inclusion criteria limiting our ability to perform subgroup analysis. some therapeutic decisions were impacted by the prescriber's discretion, prioritizing treatment of the sicker population, potentially skewing results. variability in the doses of hcq, addition of antiviral agents to control arms, differing pcr testing methods and paucity of major rcts call for caution while interpreting the results of this meta-analysis. hcq does not appear to offer significant benefits in terms of reducing the viral load, resolution of radiological findings, or progression of the covid- . hcq might be associated with a higher mortality. though there are trends in the data, pointing towards a higher complication rate with hcq, greater sample size and thus more statistically significant results, are needed to better inform the current debate. this meta-analysis supports the recommendation that hcq should not be used outside of study protocols. author contributions conceptualization: waqas ullah. data curation and formal analysis: hafez m. abdullah and sohaib roomi. investigation and methodology: ammar ahmad. project administration: m writing of original draft: yasar sattar and talal almas. writing review and editing jhu. covid- dashboard by the center for systems science and engineering (csse) at johns hopkins university (jhu) breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of cov-id- associated pneumonia in clinical studies regulatory review of novel therapeutics-comparison of three regulatory agencies fda approval and regulation of pharmaceuticals hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial efficacy of hydroxychloroquine in patients with cov-id- : results of a randomized clinical trial zhejiang da xue xue bao yi xue ban treating covid- with chloroquine chloroquine diphosphate in two different dosages as adjunctive therapy of hospitalized patients with severe respiratory syndrome in the context of coronavirus (sars-cov- ) infection: preliminary safety results of a randomized, double-blinded, phase iib clinical trial (cloro-covid- study) outcomes of hydroxychloroquine usage in united states veterans hospitalized with covid- . medrxiv no evidence of clinical efficacy of hydroxychloroquine in patients hospitalized for covid- infection with oxygen requirement: results of a study using routinely collected data to emulate a target trial hydroxychloroquine in patients mainly with mild to moderate covid- : an open-label, randomized, controlled trial potential therapeutic agents against covid- : what we know so far hydroxychloroquine and azithromycin plus zinc vs hydroxychloroquine and azithromycin alone: outcomes in hospitalized covid- patients association of treatment with hydroxychloroquine or azithromycin with in-hospital mortality in patients with covid- in new york state observational study of hydroxychloroquine in hospitalized patients with covid- hydroxychloroquine is associated with slower viral clearance in clinical covid- patients with mild to moderate disease: a retrospective study efficacy of hydroxychloroquine in patients with covid- : results of a randomized clinical trial none to declare. none to declare. none to declare. not applicable. the data supporting the findings of this study are available from the corresponding author upon reasonable request. key: cord- - trkti authors: abd-elsalam, sherief; esmail, eslam saber; khalaf, mai; abdo, ehab fawzy; medhat, mohammed a.; abd el ghafar, mohamed samir; ahmed, ossama ashraf; soliman, shaimaa; serangawy, ghada n.; alboraie, mohamed title: hydroxychloroquine in the treatment of covid- : a multicenter randomized controlled study date: - - journal: am j trop med hyg doi: . /ajtmh. - sha: doc_id: cord_uid: trkti the covid- pandemic is showing an exponential growth, mandating an urgent need to develop an effective treatment. indeed, to date, a well-established therapy is still lacking. we aimed to evaluate the safety and efficacy of hydroxychloroquine (hcq) added to standard care in patients with covid- . this was a multicenter, randomized controlled trial conducted at three major university hospitals in egypt. one hundred ninety-four patients with confirmed diagnosis of covid- were included in the study after signing informed consent. they were equally randomized into two arms: patients administrated hcq plus standard care (hcq group) and patients administered only standard care as a control arm (control group). the primary endpoints were recovery within days, need for mechanical ventilation, or death. the two groups were matched for age and gender. there was no significant difference between them regarding any of the baseline characteristics or laboratory parameters. four patients ( . %) in the hcq group and ( . %) patients in the control group needed mechanical ventilation (p = . ). the overall mortality did not differ between the two groups, as six patients ( . %) died in the hcq group and ( . %) died in the control group (p = . ). univariate logistic regression analysis showed that hcq treatment was not significantly associated with decreased mortality in covid- patients. so, adding hcq to standard care did not add significant benefit, did not decrease the need for ventilation, and did not reduce mortality rates in covid- patients. coronaviruses are a large family, which may cause illness in animals or humans. in humans, several coronaviruses are known to cause respiratory infections, ranging from common cold to more severe diseases such as middle east respiratory syndrome and sars. [ ] [ ] [ ] [ ] [ ] [ ] the most recently discovered coronavirus is sars-cov- which causes covid- . as cases of covid- continue to rise in different countries, health systems are facing enormous pressure to manage covid- patients. by august , , covid- has been confirmed in about , , million individuals worldwide and has resulted in more than , deaths. these numbers are still increasing. more than countries have reported laboratoryconfirmed cases of covid- on all continents, except antarctica. [ ] [ ] [ ] [ ] in egypt, the official number of infected patients was , , with , deaths as of august , . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] although many vaccines are in development, effective therapy is needed to treat currently infected patients and prevent mortality. chloroquine (cq) and hydroxychloroquine (hcq) have been used for decades in the treatment and prophylaxis of a number of conditions including malaria. the ability of these drugs to inhibit other coronaviruses, such as sars-cov- , has been explored. although generally considered safe, there are potential risks associated with taking these medications, including cardiac arrhythmia. [ ] [ ] [ ] [ ] [ ] although an initial study in france found encouraging results for the treatment of covid- with hcq, the study was later criticized for its methodological problems, leading to skepticism about the validity of its results. other similar results were not represented in any further subsequent studies, but even reported deleterious clinical outcomes especially cardiac adverse events like prolongation of qt interval. on march , , the food and drug administration (fda) granted an emergency use authorization for use of oral formulations of cq and hcq in the treatment of covid- . - based on emerging data showing cq and hcq as unlikely to be effective in the treatment of covid- , , the fda revoked its previous emergency use authorization for both drugs on june , . in this study, we aimed to evaluate the safety and efficacy of hcq added to the standard of care versus the standard of care alone in patients with covid- . patients admitted to three tertiary referral centers (n = ) managing patients with suspected and confirmed covid- in egypt in the period between march and june were enrolled. the patients were clinically stratified into mild, moderate, and severe disease according to the who interim guidelines published on march , . mild cases represented patients with uncomplicated upper respiratory tract viral infection, moderate cases represented patients with pneumonia but without need for supplemental oxygen, whereas severe disease represented cases with fever or suspected respiratory infection, plus one of the following: respiratory rate > breaths/min, severe respiratory distress, or spo £ % on room air. the egyptian ministry of health (moh) adopted a standard of care treatment protocol for covid- patients. it included paracetamol, oxygen, fluids (according to assessment), empiric antibiotic (cephalosporins), oseltamivir if needed ( mg/ hours for days), and invasive mechanical ventilation with hydrocortisone for severe cases if pao < mmhg, o saturation < % despite oxygen or noninvasive ventilation, progressive hypercapnia, respiratory acidosis (ph < . ), and progressive or refractory septic shock. patients were randomized into two groups using a computerized random number generator using simple randomization with an equal allocation ratio. during randomization, the proportional allocation of each clinical stratum was equalized in both groups. study groups. . hydroxychloroquine group: this group included patients who received hcq mg twice daily (in day ) followed by mg tablets twice daily added to the standard of care treatment adopted by the egyptian moh for days. . control group: this group included patients who received only the standard of care treatment adopted by the national moh for days. all the patients were followed up for weeks. the study included all patients admitted with sars-cov- infection and enrolled both genders. patient who had allergy or contraindication to hcq, pregnant and lactating females, and patients with cardiac problem (chronic heart failure or prolonged qt interval on electrocardiogram [ecg]) were excluded from the study. informed written consent was obtained from each participant, and the study was approved by the ethics committee of the faculty of medicine, tanta university. privacy of the participants and confidentiality of the data were assured. risks and benefits were explained to the patients. the study was registered on clinicaltrials.gov with registration number nct . all the participants were subjected to thorough history taking and full clinical examination including age, gender, weight and height measurements, and calculation of body mass index (bmi); medication history; and investigations in the form of complete blood picture, liver function tests, computed tomography of the chest (ct chest), and sars-cov- detection in nasopharyngeal swabs using pcr and ecg. assessment of the studied medication side effects was performed using a questionnaire. statistical analysis. data were analyzed using statistical package for social sciences v. and were expressed in number, percentage (%), mean (x̅ ) and sd. the variables were tested for normality by the shapiro-wilks test. student's t-test was used for normally distributed quantitative variables and mann whitney's test for not normally distributed ones. chi-square test (χ ) was used to study association between qualitative variables, and whenever any of the expected cells were less than five, fischer's exact test was used. binary logistic regression was used to ascertain the effect of the potential risk factors on the patients' mortality. a two-sided p-value of < . was considered statistically significant. post hoc power analysis. considering the percentage of recovery as a primary endpoint and by using g*power program, post hoc power analysis revealed a sample power of . % with the following input parameters: two-tailed α error . , . % recovery rate in the hcq group, . % recovery in the control group, and sample size in each group. at the time of presentation, interrupted fever was present in . %, continuous fever in . %, headache in . %, sore throat in . %, anorexia in . %, anosmia in . %, pallor in . %, cyanosis in . %, fatigue in . %, vomiting in . %, diarrhea in . %, abdominal pain in . %, cough in . %, and dyspnea in . % of the included patients. oxygen saturation between and was present in . %, - in . %, and less than in . % of all the participants. the computed tomography chest scans were normal in . %, ground-glass opacities in . %, confluent opacities in . %, consolidation in . %, extensive consolidation in . %, and emphysema in only . %. the two groups were matched for age and gender, with no significant difference between them. they had no significant difference regarding bmi, residence, smoking, pregnant females, or the presence of comorbidities. the patients were randomized equally between the two groups regarding the disease severity (table ) . there was no significant difference between the two groups regarding laboratory parameters (table ) . mechanical ventilation was needed in four patients ( . %) in the hcq group and ( . %) in the control group, with no significant difference between the two groups (p = . ). six patients ( . %) died in the hcq group, and five patients ( . %) died in the control group without any significant difference between the two groups either (p = . ). eleven patients ( . %) in the hcq group needed intensive care unit (icu) admission, and patients ( . %) in the control group needed the same (p = . ). the mean duration to negative pcr was ± days in the hcq group and ± in the control group (p = . ). the hcq group had a mean of ± days to show clinical improvement and ± days to hospital discharge, whereas the control group had a mean of ± to clinical improvement and ± to hospital discharge (p = . and . , respectively) ( table ) . after days, there was no significant difference between the two groups regarding the clinical outcome (p = . ). complete recovery was achieved in cases ( . %) of the hcq group, whereas cases ( . %) were in mild, ( . %) were in moderate, ( . %) in severe disease status, and six patients ( . %) died. among the control group, patients ( . %) recovered completely, ( . %) were in mild, ( . %) were in moderate, ( . %) were in severe disease status, and five patients ( . %) died. by logistic regression, the overall mortality was not significantly associated with hcq therapy; however, it was significantly related to the patient's age, alanine aminotransferase, serum creatinine, serum ferritin, c-reactive protein, oxygen saturation, and the presence of diabetes mellitus (table ) . chloroquine and hcq are well-known drugs and have been used for decades as antiparasitic and anti-inflammatory drugs to treat malaria and rheumatological disorders. chloroquine was shown to be effective against sars-cov in invitro studies. this may be because of disruption of viral replication, changing immune system activity in addition to its inflammatory effect. the two drugs have been tried earlier for the treatment of sars infection and showed promising efficacy. with the emergence of sars-cov- pandemic, they have been suggested as potential treatment for the new coronavirus based on the previous evidence from different coronavirus strains. although cardiac toxicity is a known adverse event requiring monitoring during treatment, hcq showed promise in treating sars-cov- -infected patients with multiple comorbidities including coronary artery disease. a large trial from india showed that hcq can decrease time to recovery both in symptomatic and in asymptomatic patients with no effect on mortality. at the beginning of the pandemic in europe, a small series of covid- patients treated in france with hcq showed improved decline in sars-cov- viral load compared with controls, which was augmented by the addition of azithromycin. however, this study had serious methodological flaws and could not be considered as a good evidence in the favor of hcq use. [ ] [ ] [ ] [ ] many other conflicting trials have been published in the past few months leading initially to emergency use authorization for hcq use in the treatment of covid- and later on withdrawal of this authorization by the fda. initial observational trials of hcq use in hospitalized patients showed that there were no increased risks of mortality or intubation in groups receiving hcq or the control group who received only standard of care although patients who received hcq were more critically ill. however, many published trials had some methodological flaws and missed important patient outcomes urging the need for properly designed, adequately powered trials to support clinical decisions of hcq use in treating covid- patients. administration of hcq did not result in a significantly higher probability of conversion from positive to negative pcr than standard care alone in patients admitted to hospital with nonresponsive mild-to-moderate covid- in china. adverse events were more frequent in hcq recipients than in non-recipients. a meta-analysis included seven studies with a large number of patients showing that treatment with hcq was associated with faster improvement of fever, cough, and less radiological progression of lung lesions. however, there was no difference in the virological cure, clinical improvement, or mortality. many subsequent trials did not show benefit for hcq use in covid- , with some of them suggesting more adverse events associated with its use. [ ] [ ] [ ] a recent clinical trial by skipper et al. studied the change in symptom severity over days in nonhospitalized patients between hcq and control groups and did not find any significant difference (p = . ). another trial by cavalcanti et al. compared three groups; standard care group, standard care plus hcq, and standard care plus hcq and azithromycin. the clinical status at days assessed by a seven-level ordinal scale did not show any significant difference among the three groups. moreover, elevated liver enzymes and prolonged qt intervals were more frequent among patients who used hcq. in our study, adding hcq to standard care did not add an extra benefit for the patients. hydroxychloroquine arm was similar in all outcomes. moreover, hcq was not effective as postexposure prophylaxis against covid- when administered within days after exposure. [ ] [ ] [ ] [ ] [ ] limitations of the study include small sample size which was not adequately powered for survival endpoint. the number of the included patients was limited because in egypt, tertiary care hospitals were assigned lately to deal with covid- patients and had many regulations by the egyptian moh. the study lacks long-term follow-up which could be addressed in a prospective trial. the utility of hcq should be evaluated in larger multicenter trials either alone or in combination with other drugs/lines of treatment. the role of hcq as a prophylaxis against sars-cov- infection should be among the future trials also. in conclusion, our trial adds extra evidence from egypt that hcq may not be beneficial as a treatment for covid- . a novel coronavirus from patients with pneumonia in china mers, sars and other coronaviruses as causes of pneumonia a pneumonia outbreak associated with a new coronavirus of probable bat origin lopinavir/ritonavir combination therapy amongst symptomatic coronavirus disease patients in india: protocol for restricted public health emergency use case of the index patient who caused tertiary transmission of covid- infection in korea: the application of lopinavir/ritonavir for the treatment of covid- infected pneumonia monitored by quantitative rt-pcr a trial of lopinavir-ritonavir in adults hospitalized with severe covid- hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label nonrandomized clinical trial no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe covid- infection identification of falsified chloroquine tablets in africa at the time of the covid- pandemic insight into novel coronavirus -an updated interim review and lessons from sars-cov and mers-cov chloroquine and hydroxychloroquine for the prevention or treatment of covid- in africa: caution for inappropriate off-label use in healthcare settings hydroxychloroquine in nonhospitalized adults with early covid- : a randomized trial hydroxychloroquine with or without azithromycin in mildto-moderate covid- clinical management of severe acute respiratory infection (sari) when covid- disease is suspected interim guidance egyptian ministry of health and population (moh), . diagnosis and treatment protocol for covid- g*power : a flexible statistical power analysis program for the social, behavioral, and biomedical sciences chloroquine or hydroxychloroquine for prophylaxis of covid- hydroxychloroquine for covid- : what is our current state of knowledge? 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key: cord- -m igkcxv authors: asli, rosmonaliza; abdullah, muhammad syafiq; chong, pui lin; metussin, dhiya; momin, riamiza natalie; mani, babu ivan; chong, vui heng title: case report: right bundle brunch block and qtc prolongation in a patient with novel coronavirus disease (covid- ) treated with hydroxychloroquine date: - - journal: the american journal of tropical medicine and hygiene doi: . /ajtmh. - sha: doc_id: cord_uid: m igkcxv novel coronavirus disease (covid- ) is a highly contagious disease caused by severe adult respiratory syndrome-coronavirus- that has resulted in the current global pandemic. currently, there is no available treatment proven to be effective against covid- , but multiple medications, including hydroxychloroquine (hcq), are used off label. we report the case of a -year-old woman without any cardiac history who developed right bundle brunch block and critically prolonged corrected electrocardiographic qt interval (qtc ms) after treatment for days with hcq, which resolved on discontinuation of the medication. this case highlights a significant and potentially life-threatening complication of hcq use. novel coronavirus disease (covid- ) is a highly contagious disease caused by severe adult respiratory syndromecoronavirus- (sars-cov- ) that was identified in december in china and is now a global pandemic. currently, there is no proven effective treatment, and medications proposed to inhibit the virus life cycle such as hydroxychloroquine (hcq), chloroquine, lopinavir/ritonavir, and remdesivir are used off label. [ ] [ ] [ ] [ ] these medications are widely used despite the lack of evidence for their efficacy and safety, and are often used in combination. a -year-old woman was admitted to the national isolation centre in brunei after her nasopharyngeal and throat swabs tested positive (reverse transcriptase [rt]-pcr) for adult respiratory syndrome coronavirus- (sars-cov- ). she was among a group of infected travelers and was linked to a confirmed covid- case through contact tracing. she had just returned from indonesia days before and developed symptoms (fever, dry cough, weakness, and dyspepsia) on returning. these symptoms had already improved when she was called for testing. her comorbid conditions included hypertension, hyperlipidemia, and being overweight ( . kg/m ), but she had no known heart disease. admission chest radiograph (cxr) was normal, and laboratory investigations revealed mildly elevated c-reactive protein, without lymphopenia (table ) . she was empirically started on intravenous amoxicillin-clavulanic acid ( . g three times daily) and oseltamivir ( mg twice daily). a repeat cxr on the second day of hospitalization showed bilateral lower zone opacities. as a result, she was transferred to the intensive care unit for close monitoring and was started on lopinavir mg/ritonavir mg (twice daily). as her condition did not improve, hcq ( mg stat dose followed by mg twice daily) was initiated on the fourth day of hospitalization. an electrocardiograph (ecg) on hospital day (before initiation of hcq) was normal, with a corrected qt interval (qtc) of ms. repeat ecgs the following day remained normal (qtc ms). the patient's condition deteriorated, requiring intubation and ventilatory support on the fifth day of admission. blood and urine cultures were negative. sputum culture isolated pseudomonas aeruginosa and serratia marcescens, both sensitive to meropenem. amoxicillin-clavulanic acid was discontinued, and meropenem ( , mg three times daily) was initiated. she was also started on inotropic support. the timeline of events and medications prescribed are shown in figure and laboratory investigations in table . on the fifth day, serum troponin i was noted to be mildly elevated. transthoracic echocardiogram (tte) showed normal ejection fraction and no regional wall motion abnormalities. myocarditis secondary to sars-cov- was considered. serial monitoring twice daily showed fluctuation of troponin i. on the seventh day of hospitalization, repeat ecg before the morning dose of hcq showed a new right bundle branch block (rbbb) and critically prolonged qtc ( ms) ( figure ). hydroxychloroquine was discontinued after a cumulative dose of , mg. blood investigations on that day showed normal serum mg + and k + but slightly low corrected ca + (table ) . this was corrected with calcium replacement. a repeat ecg performed hours after the last dose of hcq showed normalization of the qtc ( ms). on the tenth day of hospitalization, a repeat tte showed normal cardiac function. she was weaned off the inotropes on that day and extubated on the th day of hospitalization. investigations on the th day showed improvement of laboratory parameters apart from elevated d-dimer, and she was otherwise well and had no leg swellings. she was started on low molecular weight heparin. after three consecutive negative rt-pcr results for sars-cov- , she was transferred on the rd day of hospitalization to a tertiary hospital, where a computed tomography pulmonary angiogram showed scattered ground-glass opacities consistent with covid- and a small pulmonary embolism on the right. she was started on dabigatran mg twice daily (planned months of treatment) and remained well on follow-up. corrected qt interval prolongation is dangerous and can be associated with torsade de pointes, a life-threatening arrhythmia. our patient developed rbbb and critically prolonged qtc (qtc > ms) after days of hcq at a cumulative dose of , mg. a systematic review of chronic use of chloroquine and hcq in rheumatic conditions reported cardiac side effects to be common. among patients who were treated with hcq who experienced cardiac toxicity (n = , median duration of use years [range days to years], and cumulative dose of , g [range . g- , g]), the study reported bundle branch block ( %), atrioventricular block ( %), and firstor second-degree heart block ( %). other cardiac adverse effects of hcq included ventricular hypertrophy ( %), ventricular hypokinesia ( %), heart failure (ejection fraction < % in . %), and valvular dysfunction ( %), especially with high cumulative doses. other adverse effects of hcq include gastrointestinal, ophthalmic, neurological, musculoskeletal, psychiatric, metabolic, and dermatological abnormalities. patients with covid- who require hospitalization are at risk for complications including electrolyte derangements, which are risk factors for qtc prolongation. our patient had several risk factors for conduction abnormalities: administration of hcq, lopinavir/ritonavir, and inotropes, and hypocalcemia. lopinavir/ritonavir is also associated with the prolongation of qtc, but the ecg after starting this medication was normal. among the electrolytes associated with the prolongation of qtc, only calcium was slightly low. inotropes were started days (noradrenaline) and one (dopamine) day before the detection of conduction abnormalities. unfortunately, we did not obtain an ecg on the third day of hcq therapy. we considered the possibility of myocarditis. the elevated troponin coincided with the period leading up to conduction abnormalities and peaked several days later, before decreasing. a repeat tte was normal. we did not perform further investigations for myocarditis, as our patient remained stable. it is possible the other factors discussed contributed to the development of conduction abnormalities, but the resolution of ecgs changes after discontinuation of hcq suggested a causal relationship between hcq and these abnormalities. given the lack of proven therapies for covid- , the continued use of hcq is likely. hcq and chloroquine have also been used as prophylaxis for covid- . , with such widespread use, complications can be expected. combination therapy of chloroquine and azithromycin, both medications associated with qtc prolongation drugs, has recently been advocated. our case highlights that significant and lifethreatening conduction abnormalities can occur with the use of hcq. therefore, clinicians should exercise caution and assess cardiac risk if considering hcq treatment for covid- . this is an open-access article distributed under the terms of the creative commons attribution (cc-by) license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. world health organization, . coronavirus disease (covid- ) situation report- race to find covid- treatments accelerates a trial of lopinavir-ritonavir in adults hospitalized with severe covid- hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label nonrandomized clinical trial the epidemiology, diagnosis and treatment of covid- cardiac complications attributed to chloroquine and hydroxychloroquine: a systematic review of the literature risk factors for qtc-prolongation: systematic review of the evidence chloroquine and hydroxychloroquine for the prevention or treatment of novel coronavirus disease (covid- ) in africa: caution for inappropriate off-label use in healthcare settings hydroxychloroquine prophylaxis for covid- contacts in india key: cord- -w lap authors: cortegiani, andrea; ippolito, mariachiara; ingoglia, giulia; iozzo, pasquale; giarratano, antonino; einav, sharon title: update i. a systematic review on the efficacy and safety of chloroquine/hydroxychloroquine for covid- date: - - journal: j crit care doi: . /j.jcrc. . . sha: doc_id: cord_uid: w lap purpose: to assess efficacy and safety of chloroquine (cq)/hydroxychloroquine (hcq) for treatment or prophylaxis of covid- in adult humans. materials and methods: medline, pubmed, embase and two pre-print repositories (biorxiv, medrxiv) were searched from inception to th june for rcts and nonrandomized studies (retrospective and prospective, including single-arm, studies) addressing the use of cq/hcq in any dose or combination for covid- . results: thirty-two studies were included ( rcts, nonrandomized, , participants). two rcts had high risk, two ‘some concerns’ and two low risk of bias (rob ). among nonrandomized studies with comparators, nine had high risk and five moderate risk of bias (robins-i). data synthesis was not possible. low and moderate risk of bias studies suggest that treatment of hospitalized covid- with cq/hcq may not reduce risk of death, compared to standard care. high dose regimens or combination with macrolides may be associated with harm. postexposure prophylaxis may not reduce the rate of infection but the quality of the evidence is low. conclusions: patients with covid- should be treated with cq/hcq only if monitored and within the context of high quality rcts. high quality data about efficacy/safety are urgently needed. the spread of the coronavirus disease caused by the novel sars-cov- has reached pandemic dimensions. many drugs, both repurposed and new, have and are being investigated for preventing or treating the disease [ , ] . chloroquine (cq) and its related formulations (e.g. hydroxychloroquine -hcq) were introduced at a very early stage of the pandemic as a potential treatment for covid- . at the time, only pre-clinical rationale, in vitro findings and meager animal model data were available [ ] . the desperate need for an effective treatment has led to widespread use of the drug nonetheless. dependent on location, cq/hcq are being used in the context of clinical trials or as standard care. as the pandemic evolves, the amount of evidence accumulated regarding various treatment options is growing rapidly. however, the efficacy and safety of cq/hcq remains unclear [ ] . we therefore aimed to systematically search, assess and summarize the existing literature related to the efficacy and safety of these drugs in the clinical context of treatment and prophylaxis of covid- . we also set out to pool and meta-analyze the most updated data, if possible, in order to ascertain whether any conclusions can be reached at this time regarding the association between cq/hcq and hospital mortality in patients with covid- or disease prevention in those exposed. we prospectively registered the protocol of this review on open science framework (osf) on th may (osf.io/ yka ). journal pre-proof pico question: the current review covers studies comparing adult patients with or at risk of covid- (p) who had been administered cq or related formulations, alone or in combination with other drugs (i) to those given standard care or other regimens or drugs (c). the outcomes of interest were both efficacy (i.e. mortality, viral clearance, infection rate) and safety (i.e. adverse events, focusing on cardiac events) search strategy: the search strategy is presented in full in supplementary material . in brief, we performed a comprehensive search of medline, pubmed and embase from inception to th june for both randomized and nonrandomized studies, both retrospective and prospective [ ] addressing the pico question. we did not apply any language or quality restrictions. following full-text download (see below) the reference lists of relevant articles were also screened (i.e. snowballing method). two major pre-print servers (biorxiv and medrxiv) were also searched for relevant not peer-reviewed articles from inception to th june . tool (revised tool for risk of bias in randomized trials) was used for assessing randomized trials [ ] . the robins-i tool (risk of bias in non-randomized studies of interventions) was used for rob of nonrandomized studies with comparison between relevant study groups [ ] . the newcastle ottawa scale (nos) was used for assessing single-arm nonrandomized studies, without evaluating the -comparability‖ item [ ] . for each domain we rated the overall rob as the highest risk attributed to any criterion. we used the robvis tool (visualization tool for risk of bias assessments in a systematic review) [ ] for presenting the data as appropriate. the final rob assessments are reported as either a plot or a table as per requirement (see below). data collection and management: the primary study outcomes were all-cause mortality at the longest reported follow-up for studies evaluating cq/hcq as treatment, and infection rate in prophylactic studies. two authors (ac, mi) extracted information regarding study design, sample size, patients' characteristics, interventions and outcomes using a pre-piloted data extraction form in duplicate. if important data were missing or remained unpublished in any of the relevant studies, the authors were contacted by one of the reviewers with the aim of retrieving this data. the extracted data were used for both the description of the included studies and the qualitative analysis. borba et al. conducted a block-randomization phase iib trial, enrolling hospitalized patients with severe acute respiratory syndrome (e.g. fever, tachypnea, hypotension, altered mental status, oliguria) and suspected diagnosis of covid- in a single centre in brazil [ ] . patients were enrolled before laboratory confirmation of covid- . the study aimed to compare the efficacy and safety of two doses of cq base ( mgx /day for days vs. mgx /day on day and mgx day on j o u r n a l p r e -p r o o f days - ). placebo pills were used to mask the treatment from the participants and researchers. all patients also received azithromycin and some also received oseltamivir. the trial was terminated prematurely after enrolling patients of the intended when unplanned interim analysis was requested by the independent data and monitoring board due to concerns regarding safety. higher drug doses were found to be accompanied by higher rates of -day mortality ( % vs. %), qtc interval prolongation > milliseconds ( . % vs. . %) and ventricular tachycardia ( patients versus none). the proportion of patients with detectable viral rna levels was similar in the two groups. france. the study included patients with a positive pcr test for sars-cov- who were admitted to either day-care or wards [ ] . all patients received oral hcq mg x /day for ten days and azithromycin mg on the first day, followed by mg daily for four days. all patients also underwent pre-treatment workup including electrocardiography and serum electrolyte testing to rule out the presence of contraindications to treatment. good clinical outcomes, defined as survival, no icu or hospital admission and negative nasal viral shedding were achieved in . % of the patients by day . adverse events occurred in . % of the patients but none were cardiac. the same group of investigators published another article with similar methods and smaller sample size, without data overlap [ ] . molina et al. described a prospective case series of patients. all the patients were treated with the same regimen proposed by the authors of the series of papers described above, but % of the patients had a positive pcr assay for nasopharyngeal swab specimens at - days [ ] . pcr for sars-cov- , treated with hcq mg (+ azithromycin %; + lopinavirritonavir %; both drugs %). an ecg was recorded after days of treatment (qt corrected with bazett formula). proportion of patients with mild, moderate and severe (> ms) qt prolongation were %, % and % respectively. in % of cases who had an ecg off-therapy, the median increase of qtc was ms [ ] . treated with hcq ( with azithromycin). qt prolongation was detected in . % of the patients, normalizing after treatment completion or discontinuation. one nonagenarian died of sepsis. [ ] . boulware et al. conducted a randomized, double blind, placebo controlled trial in us and canada, enrolling asymptomatic non-hospitalized adults with selfreported high-risk or moderate risk of exposure to a positive covid- case within days of exposure. high risk was defined as household or occupational exposure at a distance of less than ft for more than minutes while wearing neither a face mask nor an eye shield. moderate risk was defined as exposure while wearing a face mask but no eye shield [ ] . the study had a power of %; the sample size was calculated based on a prior study that had actively monitored exposed cases [ ] where % of close contacts developed covid- plus an attrition rate of %. participants (recruited by social media) were assigned to hcq ( mg once, mg - hours later, then mg daily for days) or placebo and followed up through emails. data were provided by participants via a portal to an online database. the j o u r n a l p r e -p r o o f median age of the cohort was years, . % were healthcare workers (hcws) and the rate of adherence to the trial intervention was %. the incidence of new illness compatible with covid- did not differ between those taking hcq and those taking the placebo ( . % vs. . %; p= . ). participant-reported side effects were more common in those receiving hcq ( . %) than placebo ( . %). gastrointestinal side effects were the most common and no serious adverse reactions were reported. the proportion of patients hospitalized due to covid- was similar among those using cq/hcq and among those who were not ( % [ / ] ) vs. % [ / ], p=ns) [ ] . nine studies were retrieved from the search of pre-print repositories. these included one rct [ ] and eight non-randomized studies [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the studies evaluated a total of hospitalized patients with covid- . we hereby describe only the rct and the two retrospective studies that included the largest number of patients. a detailed description of all the studies is available in table . among the included studies, the six rcts [ , , , , , ] were evaluated using the rob tool [ ] . only two studies had low risk of bias [ , ] . details regarding downgrading are provided in figure , and the weighted risk of bias is presented as a plot in figure s (supplementary material ) fourteen [ , , , , , , , , , , , , , ] nonrandomized studies were evaluated using the robins-i tool [ ] . the most frequent domain causing downgrading was confounding. details regarding downgrading are provided in (table s ). we planned to perform quantitative synthesis if two or more studies were identified with a low risk of bias that also had sufficient homogeneity in study design, interventions and outcomes [ ] we identified only two studies with a low risk of bias, and these differed in participants (ill inpatients vs. exposed outpatients), intervention j o u r n a l p r e -p r o o f (treatment vs. prophylaxis) and outcomes (mortality rate vs. incidence of compatible illness with covid- ) [ , ] . therefore, particularly in light of the outcomes at stake, we decided to not perform quantitative synthesis of the data. two of these trials included only [ ] and [ ] patients. a third included a large number of comparators but only patients who actually received treatment [ ] and in a fourth study only patients were in the treatment arm [ ] . the largest number of patients who actually received treatment in the published studies with comparators j o u r n a l p r e -p r o o f comes from one retrospective study (rosenberg et al. n= ) [ ] and one prospective study (geleris et al. n= ) [ ] , both at moderate risk of bias. both provided data from the same geographical area (new york). the first (multicentre) did not collect data on other antiviral drugs co-administered in included patients [ ] and the second (single-centre) had a composite primary outcome measure [ ] . composite outcomes are often used to increase power, raising questions regarding outcome selection and interpretation [ ] . the existing data regarding efficacy as treatment is also limited by issues that are not included in standard data quality tools. two included rcts were terminated early, one for concerns regarding side effects in a high cq dose arm [ ] and another due to a decline in cases [ ] . these two studies were therefore underpowered for their primary outcomes. underpowering has also been shown to be a major issue in studies on antiviral medications in patients with covid- [ ] . from the specific aspect of critical care, at least two of the studies included patients who were mostly not critically ill. one a-priori excluded patients with organ failure [ ] and the second excluded patients who died within hours of presentation to hospital [ ] . finally, all of the published studies reported allcause mortality at various timepoints, rather than attributable mortality and none reported on the rate of withholding and withdrawal of care. this issue is particularly poignant since one of the studies reported that among the patients included almost half died without intubation [ ] . the possibility of crisis standards of care is very valid in the context of a pandemic, and patients that do worse may have been more likely to receive both compassionate medication and expectant care. prolongation of qtc is a consistent finding with cq/hcq, suggesting that patients receiving treatment with cq/hcq require in the least periodic electrocardiographic assessment or, better yet, continuous monitoring. higher doses j o u r n a l p r e -p r o o f of these drugs may be associated with higher risk of harm and side effects however this remains uncertain given that the study suggesting so was terminated very early [ ] . importantly, the association between cq/hcq and qt prolongation and arrhythmia was not adjusted for the presence of conventional risk factors for qt prolongation such as older age, electrolyte disorders, cardiac disease, genetic predisposition and other qt prolonging drugs in any of the studies. drug induced prolongation of the qt interval is a known risk factor for torsades de pointes but the exact electrographic markers of a tendency towards this arrhythmia remain unknown. treatment with azithromycin has been associated with induction of short-coupled polymorphic vt irrespective of qt prolongation [ ] . conversely -the effect (of cq/hcq) on the qtc (is) driven entirely by prolonging the repolarization and regardless of qrs, as evident by the corresponding jtc prolongation‖ [ ] . reports of qtc prolongation and arrythmias during combination treatment of cq/hcq with azithromycin have been anecdotal thus far and mostly based on case reports [ ] . the initial recommendation to combine the two drugs was based on the outcome of six patients [ ] and has not been supported by any real evidence of benefit. one included nonrandomized large study suggest an independent association between the combination hcq with azithromycin and higher risk of cardiac arrest compared to no drug (or, . , % ci . - . ) [ ] and so it is probably time to rethink the efficacy and safety of this approach. more detailed study of the coupling interval of the arrhythmia-initiating beat may perhaps contribute to differentiate between ventricular arrythmias caused by the two drugs. evidence from available data suggests that monitoring baseline and subsequent (e.g. daily) ecg during treatment especially in high risk patients with known risk. we identified only two studies with low risk of bias (one for treatment and one for post-exposure prophylaxis) and our included studies varied widely in term of patients' characteristics, outcomes definitions, interventions and design ( table ) . our early decision to proceed with quantitative synthesis only for the primary outcomes and only if the data would clearly lend itself to such analysis may seem conservative. however, given the stakes at hand such an approach may save lives as it should lead to better monitoring and research and, hopefully in the interim, table ) . finally, we identified only one trial on post-exposure prophylaxis with hcq. although this trial seems appropriately powered and has a low risk of bias, delayed initiation of treatment (usually ≥ days) and the use of self-reported outcomes make it pharmacologic treatments for coronavirus disease (covid- ): a review hydroxychloroquine in the management of patients with covid- : the need for an evidence base a systematic review on the efficacy and safety of chloroquine for the treatment of covid- chloroquine for covid- : rationale, facts, hopes including non-randomized studies on intervention effects rob : a revised tool for assessing risk of bias in randomised trials robins-i: a tool for assessing risk of bias in non-randomised studies of interventions critical evaluation of the newcastle-ottawa scale for the assessment of the quality of nonrandomized studies in meta-analyses risk-of-bias visualization (robvis): an r package and shiny web app for visualizing risk-of-bias assessments synthesizing and presenting findings using other methods | cochrane training a pilot study of hydroxychloroquine in coronavirus disease- (covid- ) observational study of hydroxychloroquine in hospitalized patients with covid- clinical efficacy of hydroxychloroquine in patients with covid- pneumonia who require oxygen: observational comparative study using routine care data association of treatment with hydroxychloroquine or azithromycin with in-hospital mortality in patients with covid- in hydroxychloroquine in patients with mainly mild to moderate coronavirus disease : open label, randomised controlled trial low dose of hydroxychloroquine reduces fatality of critically ill patients with covid- effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus (sars-cov- ) infection early j o u r n a l p r e -p r o o f treatment of covid- patients with hydroxychloroquine and azithromycin: a retrospective analysis of cases in marseille, france covid- patients with at least a six-day follow up: a pilot observational study no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe covid- infection association of hydroxychloroquine with qtc interval in patients with covid- risk of qt interval prolongation associated with use of hydroxychloroquine with or without concomitant azithromycin among hospitalized patients testing positive for coronavirus disease (covid- ) qt interval prolongation and torsade de pointes in patients with covid- treated with hydroxychloroquine/azithromycin. hear rhythm incidence and determinants of qt interval prolongation in covid- patients treated with hydroxychloroquine and azithromycin assessment of qt intervals in a case series of patients with coronavirus disease (covid- ) infection treated with hydroxychloroquine alone or in combination with azithromycin in an intensive care unit chloroquine-induced qtc prolongation in covid- patients the effect of chloroquine, hydroxychloroquine and azithromycin on the corrected qt interval in patients with sars-cov- infection experience of short-term hydroxychloroquine and azithromycin in covid- patients and effect on qtc trend a randomized trial of hydroxychloroquine as postexposure prophylaxis for covid- active monitoring of persons exposed to patients with confirmed covid- -united states hype study: hydroxychloroquine prophylaxis-related adverse events' analysis among healthcare workers during covid- pandemic: a rising public health concern continuous with sars-cov- : insights from a large healthcare database analysis baseline use of hydroxychloroquine in systemic lupus erythematosus does not preclude sars-cov- infection and severe covid- efficacy of hydroxychloroquine in patients with covid- : results of a randomized clinical trial hydroxychloroquine and azithromycin plus zinc vs hydroxychloroquine and azithromycin alone: outcomes in hospitalized covid- patients treatment response to hydroxychloroquine, lopinavir-ritonavir, and antibiotics for moderate covid- : a first report on the pharmacological outcomes from south korea outcomes of hydroxychloroquine usage in united states veterans hospitalized with covid- hydroxychloroquine is associated with slower viral clearance in clinical covid- patients with mild to moderate disease: a retrospective study experience with hydroxychloroquine and azithromycin in the covid- pandemic: implications for qt outcomes of hydroxychloroquine treatment among hospitalized covid- patients in the united states-real-world evidence from a healthcare network for design assistance to complete this project hydroxychloroquine and tocilizumab therapy in covid- patients -an observational study doxycycline and hydroxychloroquine as treatment for high-risk covid- patients: experience from case series of patients in long-term care facilities composite outcomes in randomized trials: greater precision but with greater uncertainty? remdesivir for covid- : challenges of underpowered studies azithromycin causes a novel proarrhythmic syndrome azithromycin, cardiovascular risks, qtc interval prolongation, torsade de pointes, and regulatory issues: a narrative review based on the study of case reports hydroxychloroquine and azithromycin as a treatment of covid- : results of an open j o u r n a l p r e -p r o o f key: cord- -imciixde authors: babayeva, mariana; loewy, zvi title: repurposing drugs for covid- : pharmacokinetics and pharmacogenomics of chloroquine and hydroxychloroquine date: - - journal: pharmgenomics pers med doi: . /pgpm.s sha: doc_id: cord_uid: imciixde background: a new coronavirus sars-cov- has been identified as the etiological agent of the severe acute respiratory syndrome, covid- , the source and cause of the – coronavirus pandemic. hydroxychloroquine and chloroquine have gathered extraordinary attention as therapeutic candidates against sars-cov- infections. while there is growing scientific data on the therapeutic effect, there is also concern for toxicity of the medications. the therapy of covid- by hydroxychloroquine and chloroquine is off-label. studies to analyze the personalized effect and safety are lacking. methods: a review of the literature was performed using medline/pubmed/embase database. a variety of keywords were employed in keyword/title/abstract searches. the electronic search was followed by extensive hand searching using reference lists from the identified articles. results: a total of results were obtained after screening all sources. mechanisms underlying variability in drug concentrations and therapeutic response with chloroquine and hydroxychloroquine in mediating beneficial and adverse effects of chloroquine and hydroxychloroquine were reviewed and analyzed. pharmacogenomic studies from various disease states were evaluated to elucidate the role of genetic variation in drug response and toxicity. conclusion: knowledge of the pharmacokinetics and pharmacogenomics of chloroquine and hydroxychloroquine is necessary for effective and safe dosing and to avoid treatment failure and severe complications. sars-cov- is a new coronavirus type that has not been previously identified in humans. little is known about the highly infectious virus or how to combat it. the current strategy considers two broad categories of therapies: antivirals, which may target the coronavirus directly, and host modifiers and immune-based medications, which may influence the immune response to the virus. currently, all the therapeutic agents are repurposed medications. for approximately identified medical conditions, only have approved therapies; a critical need currently exists for the availability of drug therapies. [ ] [ ] [ ] drug repurposing helps to minimize the deficiency and delivers a candidate at a shorter development time and a lesser cost. a key advantage of repurposed drugs is that safety has been established and only efficacy of the new indication needs to be assessed. many of the wellknown repurposed drugs, including sildenafil, minoxidil and aspirin emerged by chance from "unorganized" drug discovery processes. several diverse disease states have common transcriptional inflammatory and metabolic pathways, suggesting that drugs designed for treatment of one disease can potentially be used to treat other diseases. drug repurposing is being applied to finding a therapeutic approach for the covid- pandemic. thirty-one potential broad-spectrum antiviral agents (bsaas) were recently identified as having potential for treating sars-cov- /covid- . several existing bsaas have been initiated into clinical trials ( table ) . two of the drugs listed in table , hydroxychloroquine and chloroquine, have been proposed as treatments for covid- . chloroquine (aralen ® ) and hydroxychloroquine (plaquenil ® ) are -aminoquinoline medications used to treat several disease states. chloroquine (cq) was first developed for the treatment of malaria. hydroxychloroquine (hcq) is β-hydroxylated analogue of cq. both medications have been successfully used to treat extraintestinal amebiasis, several infectious (hiv, q fever, zika virus, fungal infections) and rheumatological (systemic lupus erythematosus, antiphospholipid syndrome, rheumatoid arthritis, sjögren's syndrome) diseases. [ ] [ ] [ ] in the therapy of malaria, the agents inhibit the action of heme polymerase, which causes the buildup of toxic heme in plasmodium species. , the antiviral activity is not fully understood. the drugs accumulate in human organelles, raise the endosomal ph and prevent viral activity. [ ] [ ] [ ] the elevated ph inhibits nucleic acid replication, glycosylation of viral proteins, viral fusion and entry into the cell, viral assembly and release. postmarketing cases of life-threatening and even fatal events have been reported for chloroquine and hydroxychloroquine. an overdose of cq can cause acute poisoning and death. hcq was demonstrated to be % less toxic than chloroquine, although prolonged and overdose administration can still cause poisoning. , patients may present with atrioventricular block, pulmonary hypertension, sick sinus syndrome or with cardiac complications. the most life-threatening adverse reaction is qtc prolongation with subsequent risk of ventricular arrhythmias. concomitant qtc-prolonging medications may increase the severity of the complication even more. the mechanism of qtc prolongation by chloroquine and hydroxychloroquine is unknown, largely depending on the cardio-vascular health of the patients. [ ] [ ] [ ] another complication of the two medications is retinopathy (chloroquine retinopathy), which can result in irreversible impairment of the retina. , high concentrations of the drugs in the retina, due to binding to retinal melanin, result in the damage of the tissue. hydroxychloroquine may also produce a severe cutaneous adverse effect such as a generalized pustular figurate erythema (gpfe). high concentrations of the medications in the skin and very slow cutaneous elimination (longer than months) may result in this severe cutaneous reaction. , the most common adverse effects of the drugs are nausea, vomiting, diarrhea. other complications include hypoglycemia in diabetics, hemolytic anemia in g pd deficiency patients, tinnitus and headache. , chloroquine and hydroxychloroquine for covid- therapy chloroquine and hydroxychloroquine have shown the ability to inhibit replication of multiple coronaviruses in vitro, [ ] [ ] [ ] including sars-cov- in concentration-dependent manner. , , , , the anti-sars-cov- activity of hcq seems to be less potent compared to cq. the ec for cq ( . μm) was significantly lower than that of hcq ( . μm). in contrast, hydroxychloroquine was found more potent than chloroquine against sars-cov when given post-infection and prophylactically. clinical evidence of the effectiveness of hcq or cq for the treatment of covid- is limited. some small clinical trials have shown therapeutic benefits of the drugs, while others have shown the opposite. in recent clinical trials, over people with covid- have been treated with pharmacogenomics and personalized medicine : chloroquine. these patients had less severe disease and a shorter illness duration compared to those who did not receive chloroquine. another open-label non-randomized clinical trial with covid- patients demonstrated that hydroxychloroquine treatment resulted in viral load reduction/disappearance in the patients. the effect was reinforced by azithromycin. contrasting results were reported in a small study with hospitalized patients; no difference in clinical outcomes was observed between patients treated with hcq and azithromycin and patients on standard care. in a randomized trial with hospitalized patients, patients on hcq had a more substantial proportion of clinical improvement of pneumonia ( % vs %) than patients with standard care. in another clinical trial with covidpatients, an increased overall mortality was observed in the patients treated with hydroxychloroquine. more clinical trials are going on. the fda has issued an emergency use authorization for cq and hcq to treat covid- infection, allowing the unapproved use of these medications in light of a public health emergency. , on april , the fda issued warning against hcq or cq unless the therapy is closely supervised by a healthcare professional. the caution was initiated after the agency received reports of serious adverse effects in covid- patients. these findings do not apply to the use or evaluation of hydroxychloroquine in pre-or post-exposure prophylaxis in patients exposed to covid- . these results bring forward the need for large controlled clinical trials to provide guidance on safe and effective dosing of cq/hcq for covid- therapy. furthermore, response to drugs is subject to interindividual variability. patients treated with the same dose of the same drug, may exhibit lack of efficacy, or adverse reactions. the variability, at least in part, is attributed to genetic polymorphisms. knowledge of pharmacogenomic (pgx) of the drugs is necessary to estimate effective and safe dosing and to avoid/minimize adverse reactions. no pgx studies have been conducted to investigate the interpatient variability of cq and hcq in covid- patients. the purpose of the study was to highlight the importance of large, randomized, controlled clinical trials and pharmacogenomic studies to assess the optimal dosing of cq and hcq in diverse populations as a treatment for covid- . a review of the literature was performed using medline/ pubmed/embase database resources for english language papers from up to july to identify appropriate articles that addressed the objectives of this review. a variety of keywords were employed in keyword/title/abstract searches that included: chloroquine, cq, hydroxychloroquine, hcq, pharmacokinetics, pharmacogenomics, covid- , sars-cov- . we obtained appropriate results after screening all sources; relevant and nonrelevant. the publications were reviewed independently by two investigators. the investigators extracted the data and inspected each reference identified by the search. in cases where the same studies were reported in more than one publication, the study's results were accounted for only once. limits to the search strategy were english language articles and human studies. the electronic search was followed by extensive hand searching using reference lists from the identified articles. the search method was used to strengthen existing concepts and to identify study designs for upcoming research studies. this paper presents the current knowledge on chloroquine (cq) and hydroxychloroquine (hcq) pharmacokinetics (pk) with a focus on stereoselectivity of their disposition. both drugs are racemic mixtures, consisting of equal amounts of r(-) and s(+)-enantiomers. the pharmacokinetics of these two -aminoquinolines are similar and regulate dosing of the drugs. cq is available as chloroquine phosphate, aralen ® . each -mg tablet of chloroquine phosphate contains mg of chloroquine. hcq is available as hydroxychloroquine sulfate, plaquenil ® . each -mg tablet of hydroxychloroquine sulfate contains mg of hydroxychloroquine. doses of both agents are based on ideal body weight (ibw). chloroquine doses are . - . mg/kg/day and produce plasma levels of to × − m/l. doses of hydroxychloroquine are . - . mg/kg/day and produce plasma concentrations of . to . × − m/l. an initial adult dose of chloroquine phosphate for malaria therapy is g followed by mg given at - hours, , and hours. lupus erythematosus and rheumatoid arthritis chloroquine phosphate dosage is mg daily with dose reduction after remission. initial adult dose of hydroxychloroquine sulfate is mg followed by mg given at - hours, , and pharmacogenomics and personalized medicine : submit your manuscript | www.dovepress.com dovepress hours. lupus erythematosus and rheumatoid arthritis initial treatment is - mg of hydroxychloroquine sulfate daily for several weeks or months. pediatric dosage of chloroquine and hydroxychloroquine is based on body weight. an initial pediatric dose of chloroquine phosphate for treatment of malaria is . mg/kg followed by . mg/kg given at , , and hours after initial dose. maximum total dose is . g. initial pediatric dose of hydroxychloroquine sulfate for treatment of malaria is . mg/kg followed by . mg/ kg given at , , and hours after the first dose. maximum total dose is g. cq and hcq have been used for prophylaxis and treatment of malaria in pregnant women without evidence of adverse effects on the fetus. dosing for treatment and prophylaxis of uncomplicated malaria is the same in pregnant and nonpregnant adults. due to pregnancy-induced physiologic changes, some pharmacokinetic properties of the drugs may be altered, suggesting dose adjustments may be needed. but data are not sufficient to determine an appropriate dosing during pregnancy. , small amounts of chloroquine and hydroxychloroquine excrete into breast milk. the amounts of the drugs are not sufficient to harm the infant nor to protect the child from malaria. weekly cq/hcq of / mg may be given until breastfeeding is completed. , no information is available on the effect of chloroquine and hydroxychloroquine in geriatric patients. but because cq and hcq are mostly excreted in the urine, elderly patients with age-related kidney problems may require caution and a dose adjustment for the patients. the dose adjustment should be based on the kidney function. the optimal dosing of hcq and cq for treatment of covid- is unknown. most of the published clinical studies had hcq dosage of mg/ days or mg on the first day and mg for the next days. the latest regimen was supported by pharmacokinetic modelling, where an oral hcq sulfate loading dose of mg twice daily, followed by a maintenance dose of mg twice daily for days was able to achieve treatment efficacy and a good safety profile. this regimen reached three times the potency of cq phosphate given mg twice daily for days. however, more reliable information is required before it can be widely used to treat covid- . oral absorption of chloroquine and hydroxychloroquine in humans is efficient. both drugs have oral bioavailability of . - . . , although - -fold difference in the absorbed fraction of oral doses was reported. , maximum blood concentrations (cmax) for the oral doses showed significant differences between subjects (range - ng/ml), but not within subjects. , oral bioavailability of chloroquine is - %. cq oral tablets have slightly greater bioavailability than oral solutions, - % vs - %, respectively. hydroxychloroquine has oral bioavailability of - %. , antacids may decrease the bioavailability of both drugs. oral chloroquine reaches cmax faster than hydroxychloroquine. time to reach the maximum level (tmax) for cq was estimated at minutes, , - while tmax for hcq was estimated . hours. , absorption of the r and s enantiomers was not significantly different. distribution cq and hcq have multicompartment disposition in humans with wide distribution to the body tissues. highest concentrations were found in the melanin-containing cells, the retina and the skin. high levels were observed in the liver, spleen, kidney, and lung. in the blood, concentrations in erythrocytes were up to times higher than in plasma. reported volumes of distribution were , l and , l for hcq and cq, respectively. , , [ ] [ ] [ ] plasma protein binding of the drugs ranges between % and %. , cq and hcq are mostly bound to two plasma proteins, albumins and α- -acid glycoproteins. binding of both compounds to plasma proteins is stereoselective. chloroquine is approximately % bound to plasma proteins. , extend of s(+)-chloroquine plasma protein binding is greater than binding of r(-)-chloroquine ( % vs %). binding of hydroxychloroquine to plasma proteins is around %, which is less than chloroquine binding. the s-hydroxychloroquine is % bound to plasma proteins, while the r-hydroxychloroquine is only % protein bound. following separate administration of the individual enantiomers of both drugs, r(-)-isomers reach higher and more sustained plasma and ocular concentrations than s(+)-forms. , , metabolism chloroquine and hydroxychloroquine have long half-lives and low blood clearance. cq is rapidly n-desethylated into two major metabolites: desethylchloroquine ( %) and bisdesethylchloroquine ( %). , desethylchloroquine is the pharmacologically active metabolite and further metabolizes to bidesethylchloroquine. cq is metabolized primarily by cyp c and cyp a mediating % of the total submit your manuscript | www.dovepress.com pharmacogenomics and personalized medicine : metabolism of the drug. other enzymes, cyp a and cyp d break down chloroquine to a lesser extent. , [ ] [ ] [ ] metabolism of cq is stereoselective. after administration of the individual enantiomers, the concentration of (r)-chloroquine was . -fold higher compared to concentrations of (s)-chloroquine in plasma and . -fold higher in the blood in patients with rheumatoid arthritis. blood concentrations of the active metabolite s(+)desethylchloroquine exceeded those of the r(-)forms. , , hcq has similar to chloroquine biotransformation but breaks down into more metabolites. hcq is n-dealkylated by cyp a to the two active metabolites desethylhydroxychloroquine, desethylchloroquine and an inactive metabolite bidesethylchloroquine. other cytochrome p enzymes (cyp c , d , and a ) are involved in the metabolism to a lesser extent. , biotransformation of hcq is also stereoselective. several studies have reported faster hepatic metabolism of s(+)-enantiomers compared to metabolism of r(-)-enantiomers. , , [ ] [ ] [ ] the blood and plasma concentrations of r-hydroxychloroquine exceeded those of the s-hydroxychloroquine with the mean r/s ratio of . in the blood and . in the plasma. the mean blood concentration ratio r/s for desethylhydroxychloroquine was . and for desethylchloroquine was . , indicating stereoselective metabolism of the compound. similar doses of the two drugs produced -fold variations in the blood concentrations in patients with rheumatoid arthritis , , , and in healthy volunteers, , suggesting different extend of metabolism among individuals. moreover, chloroquine and hydroxychloroquine are involved in several metabolic drug-drug interactions (ddis). a cyp a inhibitor, cimetidine increased serum concentrations of cq by %. another cyp a inhibitor, ketoconazole reduced the formation of active metabolite desethylchloroquine. , excretion urinary excretion is the main route of elimination for chloroquine and hydroxychloroquine. the % of a chloroquine dose is recovered in the urine as unchanged drug, with % of the dose recovered in the urine as its active metabolite desethylchloroquine. the % of a cq dose is recovered in feces. small amounts ( %) of the drug eliminate through the skin and up to % stored in lean tissues. elimination from the skin is very slow. cq remains in the skin longer than months, a time when the drug is no longer detectable in the plasma. chloroquine and active metabolite desethylchloroquine have elimination half-lives of to days and may be detected in urine months after a single dose. chloroquine has a total clearance of . - l/h/kg. renal clearance accounts for half of the total systemic clearance and increases by acidification of the urine. the renal excretion accounts for - % of hcq elimination, where only - % is excreted as unchanged drug. the - % of absorbed dose is excreted in the feces, which is greater than cq feces excretion. the elimination through the skin and long-term storage in lean tissues is identical to those of chloroquine, % and %, respectively. , , the total clearance of hydroxychloroquine is ml/min. iv hydroxychloroquine has a halflife of days ( . days in blood, and . days in plasma). the elimination half-life of both drugs is significantly longer in patients with chronic renal disease. , in anuretic patients, the plasma levels were % and - % higher for cq and hcq, respectively, compared to concentrations in subjects with normal kidney function. enantioselective renal elimination of the medications has been demonstrated in patients. , (s)-hydroxychloroquine had a mean renal clearance approximately twice that of (r)hydroxychloroquine. in addition, the mean renal clearance of active (s)-metabolites was also higher than that of (r)metabolites. the main mechanism of renal elimination of the medications is tubular secretion as renal excretion -fold exceeds the glomerular filtration rate. , the tubular secretion is an active process mediated by membrane proteins. it was reported that chloroquine is a substrate and potent competitive inhibitor of multidrug and toxin extrusion protein (mate ). , as substrates and/or inhibitors of active transport and metabolism, cq and hcq may be involved in several drug-drug interactions. [ ] [ ] [ ] [ ] [ ] [ ] response to drugs is subject to inter-individual variability. - % of individuals that receive a drug, exhibit lack of efficacy, or adverse drug reactions. up to % of the variability is attributed to genetic polymorphisms. cytochrome p enzymes are major determinants of drug response. they are responsible for approximately % of phase i drug metabolism, and % of drug clearance. the human cyp supergene family includes genes, of pharmacogenomics and personalized medicine : submit your manuscript | www.dovepress.com dovepress which are responsible for more than % of all drug oxidation reactions. the cyp genes are highly polymorphic composed of large numbers of single-nucleotide polymorphisms (snps) and copy number variations. the most studied are genes of cyp d , c , c , a , and a enzymes. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] however, drug pharmacokinetics also depend on the renal excretion of the medications. mate , encoded by slc a gene, has been identified as a major efflux transporter involved in the renal excretion of many drugs including chloroquine. , [ ] [ ] [ ] [ ] [ ] pharmacogenomics informing chloroquine malaria pharmacotherapy individual variation in drug response is a critical challenge in effective drug pharmacotherapy. both the nature of the drug, as well as the dose of the drug, are subjected to vary on an individual basis. genetic polymorphisms in metabolizing enzymes influence the pharmacokinetics and drug response. plasmodium vivax is the major cause of malaria disease outside africa. the world health organization (who) recommends chloroquine as a component of the treatment protocol for uncomplicated p. vivax malaria. chloroquine is metabolized by the cyp isozymes c , a , a and d . the cyp c genes are located in a cluster on chromosome q , organized as cent-cyp c -cyp c -cyp c -cyp c -tel. the cyp c gene is approximately kb in size and includes nine exons. cyp c is the most divergent with respect to its protein sequence. interindividual variability in chloroquine efficacy was previously reported in africa and asia and attributed to: p. vivax resistance to chloroquine, noncompliance, suboptimum dose and drug-drug interactions. in a study reported in , assessment of genetic polymorphisms in chloroquine metabolizing enzymes was identified as a need. to that end, the investigators focused on a cohort consisting of p. vivax malaria patients followed during malaria treatment from to . the study reported for the first time the influence of the cyp c gene on gametocyte clearance rate with patients undergoing chloroquine/primaquine malaria treatment. from baseline until the first day of treatment, wild-type cyp c homozygous individuals achieved greater reduction in gametocytes as compared to individuals without this genotype. the results suggested that cyp c , cyp c and cyp a genetic variants influenced chloroquine malaria treatment. discoid lupus erythematosus is the most common form of cutaneous lupus. patients diagnosed with systemic lupus erythematosus and rheumatoid arthritis show a positive correlation between whole blood hydroxychloroquine levels and clinical response. hcq is metabolized to n-desethylhydroxychloroquine in the liver. the reaction is mediated by cyp a , cyp c , cyp d and cyp a isoforms. , in a study reported by lee et al, systemic lupus erythematosus patients were genotyped for snps in cyp a * b, cyp d * , cyp a * . the association of the respective genotypes with blood hydroxychloroquine and n-desethyl hydroxychloroquine was the focus of the investigation. the cyp d * allelic variants were found to be significantly associated with the n-desethylhydroxychloroquine/hydroxychloroquine ratio. the study demonstrated that this ratio is related to cyp d polymorphisms in systemic lupus erythematosus patients treated with hydroxychloroquine. a multicenter observational and pharmacogenetic study with discoid lupus erythematosus patients treated with hcq was reported by wahie et al. thirty-nine percent of the patients failed to respond to hydroxychloroquine, or developed toxicity. the study showed a trend for cyp c variants to be associated with better response. the pk and pd characteristics of chloroquine and hydroxychloroquine need to be evaluated in order to provide safe and effective covid- therapy. the pharmacokinetics of chloroquine and hydroxychloroquine are similar. oral absorption of the drugs is comparable with bioavailability values of . - . . , but the two medications have significant variations in bioavailability between individuals. , genetic polymorphism of cyp enzymes involved in the presystemic metabolism can explain at least in part the individual differences in the oral absorption of the drugs and may reflect the variations in blood and tissue concentrations of the drugs in covid- patients. both cq and hcq have wide distribution to the body tissues. plasma protein binding of the drugs varies between % and %. , cq and hcq are known to be enantioselective in their dispositions. both medications have similar stereoselective patterns of protein binding. s (+)-isomers are more bound to plasma proteins than r(-)isomers, suggesting that free plasma concentrations are higher for r(-)-forms. such differences in the plasma submit your manuscript | www.dovepress.com pharmacogenomics and personalized medicine : protein binding can be responsible in part for the variations in therapeutic response and toxicity of the isomers in covid- patients treated with cq or hcq, as only free drug can interact with receptors and produce therapeutic and/or side effects. chloroquine and hydroxychloroquine have long halflives. the long half-life can be attributed to extensive tissue uptake rather than decreased elimination. chloroquine is n-desethylated into two major metabolites largely by cyp a and cyp c , while hydroxychloroquine metabolizes into three metabolites primarily by cyp a . , metabolism of both drugs is stereoselective. the higher blood and plasma concentrations of (r)-forms confirm the stereoselective metabolism of the medications, where s-enantiomers metabolize faster than r-enantiomers. , as a result, s(+)-isomers have shorter half-life than r(-)isomers. similar doses of both medications produce large ( -fold) variations in the blood concentrations in patients with rheumatoid arthritis and in healthy volunteers. , , , comparable differences in drug levels may be expected in covid- patients. the variability can be explained by the stereoselective metabolism as well as genetic polymorphism of the p- enzymes involved in the biotransformation of the medications. individual variation in drug response is a critical challenge in effective drug pharmacotherapy. up to % of individuals that receive a drug, exhibit lack of efficacy or adverse drug reactions, at least partially, due to genetic polymorphisms. gene polymorphisms influence metabolism as well as active transport. cyp d the most extensively studied cyp gene metabolizes approximately % of all drugs. genetic polymorphisms resulting in increased cyp d metabolic capabilities have been linked to decreased treatment response with tricyclic antidepressants, increased occurrences of respiratory depression and opioid toxicity. , cyp c deficiency is related to bleeding complications with warfarin and other anticoagulants treatment. fifty percent of interindividual variability in dose requirements is observed in concert with age, body surface area and polymorphisms in vkorc . cyp c is involved in the metabolism of many medications including non-steroidal anti-inflammatory drugs, thiazolidinediones, chemotherapy agents, chloroquine and hydroxychloroquine. cyp c genotyping should be considered as a viable option in africans and europeans in which . % and . % of cyp c alleles, respectively, exhibit reduced functionality. the cyp a subfamily is the most abundant of the p- enzymes. cyp a and cyp a metabolize more than half of the marketed drugs. , the most common variant of cyp a enzyme, cyp a * b has been associated with reduced cyp a activity. the cyp a * b allelic frequency varies among different ethnic groups, ranging from % in chinese to % in african americans. [ ] [ ] [ ] is polymorphically expressed in - % in caucasians, % in japanese and % in african americans. the primary variant is cyp a * , which has been associated with low cyp a protein expression and reduced metabolic activity. the cyp a * allele frequency varies from approximately % in african americans to % in caucasians. other allelic variants have been reported for both cyp a and cyp a . however, the variants occur at relatively low allelic frequencies and their functional significance has not been verified and validated. determination of cyp a, cyp c and cyp d polymorphism and, therefore, activity is important to establish safe and efficient dosing of chloroquine and hydroxychloroquine for treatment of covid- patients. a recent study reported for the first time the influence of the cyp c gene on clearance in patients with chloroquine/primaquine therapy. wild-type cyp c homozygous individuals achieved greater reduction in gametocytes as compared to individuals without this genotype. another study demonstrated cyp d polymorphisms in systemic lupus erythematosus patients treated with hydroxychloroquine. cyp d * allelic variants were found to be significantly associated with altered metabolism of hcq. a study with lupus erythematosus patients treated with hydroxychloroquine demonstrated % of the patients failed to respond to the therapy or developed toxicity. similar trend was observed in a recent clinical trial with covid-patients treated with hydroxychloroquine. additionally, the study with lupus erythematosus patients showed a trend for cyp c variants to be associated with better response. overall, the results suggest that cyp c , cyp d and cyp a genetic polymorphisms may influence chloroquine and hydroxychloroquine pharmacokinetics and covid- patients treated with the same dose of cq or hcq may exhibit lack of efficacy or adverse reactions. the variability in therapeutic response may require dose adjustment of cq/ hcq in treatment of covid- patients. urinary excretion is the primary route of elimination for chloroquine and hydroxychloroquine. the % of a cq dose is recovered in the urine as unchanged drug, while only - % of an hcq dose is renally excreted as pharmacogenomics and personalized medicine : submit your manuscript | www.dovepress.com dovepress unchanged drug. , however, a greater fraction of absorbed hcq dose excretes in the feces compared to the fraction of cq dose. the elimination half-lives of both medications are significantly longer in patients with chronic renal disease. , this finding recommends that the two drugs should be used with caution in patients with renal impairment, as kidney dysfunction may lead to greater drug retention and higher risk of adverse effects. renal elimination of both compounds is stereoselective, (s)-isomers have a mean renal clearance approximately twice that of (r)-isomers. the main mechanism of renal elimination of the medications is tubular secretion. , however, the molecular mechanisms of the renal tubular secretion remain mostly unidentified. it was reported that chloroquine is a substrate and potent inhibitor of the mate transporter. given the similarity in structure between cq and hcq, it is possible to propose that hcq is also a substrate for mate . mate is a proton-substrate antiporter expressed in the kidney and liver that facilitates the export of organic cations, such as metformin, paraquat, and oxaliplatin into urine and bile. genetic polymorphisms of mate may alter the pharmacokinetics and pharmacodynamics of the medications, as drug transporters are key determinants of elimination of the drugs. mate , encoded by the slc a gene, has been identified as a major efflux transporter involved in the renal excretion of chloroquine. functional snp of mate (rs g>a) was associated with increased glucose-lowering activity of metformin through slowing renal excretion of the anti-diabetic drug. , the allele frequency ranges from . % in african americans to % in mexican americans. other snps may also alter transport activity of mate and lead to changed elimination of the corresponding drugs. , genetic polymorphisms of mate can affect renal elimination of cq and hcq and, therefore, may require dose adjustment based on pharmacogenomic profiles of covid- patients. indeed, the active transporters and cyp enzyme polymorphisms may explain the variations in blood concentrations, therapeutic responses and severity of adverse effects of chloroquine and hydroxychloroquine. despite the evidence of the influence of genetic polymorphisms on the pharmacokinetics of chloroquine and hydroxychloroquine, no large pharmacogenomics studies have been conducted to provide guidance on the use, dosing, and duration of the therapy in covid- patients. additionally, chloroquine and hydroxychloroquine are involved in several ddis. cimetidine and ketoconazole, cyp a inhibitors increased serum concentrations of cq. , predictably, cimetidine and ketoconazole may also increase hcq blood concentrations by inhibition metabolism of the drug. co-administration of cq and hcq with moderate and strong cyp a (boceprevir, cobicistat, azole anti-fungal agents, macrolide antibiotics, etc.), cyp c (gemfibrozil, clopidogrel, deferasirox, teriflunomide) and mate inhibitors may result in increased plasma concentrations, longer half-life, exaggerated therapeutic effect and the toxicity of chloroquine and hydroxychloroquine. significant drug interactions with chloroquine and hydroxychloroquine that should be avoided or require additional monitoring include digoxin, antiepileptics, antacids, cyclosporine, amiodarone, azithromycin, moxifloxacin, insulin and other antidiabetic agents, tamoxifen, and praziquantel. , with the currently known or potential ddis, the use of chloroquine and hydroxychloroquine with other drug therapy requires consideration for patient safety in covid- patients. limited pharmacogenomic studies have been performed investigating the inter-patient variability of chloroquine and hydroxychloroquine in both malaria and lupus patient populations. moreover, data to support the use of hydroxychloroquine and chloroquine for covid- are limited and inconclusive. the off-label use of chloroquine and hydroxychloroquine to treat covid- must be used with caution given the toxicities: cardiac, retinal and cutaneous severe adverse effects. well-designed randomized trials incorporating pharmacogenomics need to be performed in a timely manner to achieve safe and effective dosing and to reduce severity of adverse effects. cq, chloroquine; ddi, drug-drug interaction; ec , % maximal effective concentration; fda, food and drug administration; hcq, hydroxychloroquine; ibw, ideal body weight; mate , multidrug and toxin extrusion protein ; pgx, pharmacogenomics; pk, pharmacokinetics; snp, single nucleotide polymorphism. the authors report no conflicts of interest in this work. omim gene map statistics diagnosing the decline in 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cyp a genetic variant and clinical presentation in african-american prostate cancer patients interaction between polymorphisms in the oct and mate transporter and metformin response slc a gene rs g>a variants enhance the glucose-lowering effect of metformin via delaying its excretion in chinese type diabetes patients genetic polymorphisms of multidrug and toxin extrusion proteins (mate and mate ) in south indian population identification of multidrug and toxin extrusion (mate and mate -k) variants with complete loss of transport activity genetic variants in multidrug and toxic compound extrusion- , hmate , alter transport function pharmacogenetics -five decades of therapeutic lessons from genetic diversity challenges for malaria elimination in brazil a . -megabase physical map spanning the cyp c gene cluster on chromosome q gene structure of cyp c and extrahepatic distribution of the human cyp cs effectiveness of antimalarial drugs the effect of snps in cyp in chloroquine/primaquine 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society's chemical abstracts service (cas). the manuscript management system is completely online and includes a very quick and fair peer-review system key: cord- -f b t ts authors: kabeerdoss, jayakanthan; danda, debashish title: understanding immunopathological fallout of human coronavirus infections including covid‐ : will they cross the path of rheumatologists? date: - - journal: int j rheum dis doi: . / - x. sha: doc_id: cord_uid: f b t ts severe acute respiratory syndrome coronavirus (sars‐cov‐ ) infection causing coronavirus disease (covid‐ ) is the biggest pandemic of our lifetime to date. no effective treatment is yet in sight for this catastrophic illness. several antiviral agents and vaccines are in clinical trials, and drug repurposings as immediate and alternative choices are also under consideration. immunomodulatory agents like hydroxychloroquine (hcq) as well as biological disease‐modifying anti‐rheumatic drugs (bdmards) such as tocilizumab and anakinra received worldwide attention for treatment of critical patients with covid‐ . this is of interest to rheumatologists, who are well versed with rational use of these agents. this brief review addresses the understandings of some of the common immunopathogenetic mechanisms in the context of autoimmune rheumatic diseases like systemic lupus erythematosus (sle) and covid‐ . apart from demographic comparisons, the role of type i interferons (ifn), presence of antiphospholipid antibodies and finally mechanism of action of hcq in both the scenarios are discussed here. high risks for fatal disease in covid‐ include older age, metabolic syndrome, male gender, and individuals who develop delayed type i ifn response. hcq acts by different mechanisms including prevention of cellular entry of sars‐cov‐ and inhibition of type i ifn signaling. recent controversies regarding efficacy of hcq in management of covid‐ warrant more studies in that direction. autoantibodies were also reported in severe acute respiratory syndrome (sars) as well as in covid‐ . rheumatologists need to wait and see whether sars‐cov‐ infection triggers development of autoimmunity in patients with covid‐ infection in the long run. there are demographic, immunological and therapeutic similarities and dissimilarities between hcov infections and autoimmunity. in general, adult women have stronger immune response and they are protected more often from infectious disease compared to men of similar age. women appears to have robust antimicrobial immune responses, especially against viral infections. x chromosomes and sex hormones are thought to be responsible for this phenomenon. in addition, negative regulators of immune response are less marked in woman as compared to men, for example lower number of circulating t-regulatory cells and lower expression of immune checkpoint inhibitors like pd-l in t-cells of women. coronavirus strains sars-cov and sars-cov- utilize angiotensin i converting enzyme (ace) as a receptor for entry into host cells. ace is differentially expressed in different organs; high levels are reported in the small intestine, colon, heart, muscle, kidney, testis and moderate levels in lungs. expression of ace is also higher in males compared to females, especially in liver and lung tissues even though its gene is present in the x chromosome. ace activity and expression is regulated by β-estradiol. messenger rna (mrna) expression of ace correlates with immune signatures in lungs and it is dependent upon age and gender. there is positive correlation between ace expression and immune signatures in the lungs of men and older individuals, whereas negative correlation is observed in women and younger individuals. this might be the reason for excessive immune response in the form of the cytokine storm observed in older-aged males that results in severe respiratory complications. however, sars-cov- infects both genders equally, although higher mortality is observed in males. , in an animal model studies, males were found to be highly susceptible to sars-cov infection and more severe lung pathology. mortality of male mice was higher than that of female mice and it was dependent on viral load. blocking estrogen receptor signaling, however, led to an increase in mortality even among sars-cov in- similar to animal studies, male population is predominantly susceptible to sars-cov- infection and accounts for nearly % of all cases of covid- with higher mortality. similar gender bias was also observed in mers-cov infection, although it was attributed to social activities and religious customs that involved more men than women in the middle east. african american women are at times higher risk and latino american as well as asian women are at times higher risk for developing sle than european american women. sle disease severity, number of clinical manifestations, prevalence of autoantibodies and nephritis as well as mortality are higher in african american, asian and hispanic populations as compared to the white populations. however, socio-economic and environmental background may also be confounding factors that influence ethnicity-based prevalence and phenotypic differences in sle. as on june , mortality rate (deaths/ total cases) of covid- infection in europe, north america, asia and africa are . %, . %, . % and . % respectively. however, mortality rate of covid in the usa is disproportionately higher among blacks ( . deaths per population) and hispanics/latino americans ( . deaths per population) than the white american population ( . deaths per ). , blacks, asians and minority ethnic (bame) groups are also found ethnicity also influences type i ifn secretion, as asians and african americans have higher type i ifn expression in peripheral blood cells as compared to caucasians. , this observation goes hand in hand with prevalence of lupus in these ethnicities, a predominantly type ifn-driven disease. type i ifn expression is also lower in male individuals compared to females, again keeping in line with high female predominance in lupus. body mass index (bmi) and smoking also increases type i ifn expression and inflammatory cytokines. these risk factors tend to adversely affect outcome of covid- disease as well as lupus and other systemic autoimmune diseases. [ ] [ ] [ ] these reports suggest that gender, ethnicity, bmi and smoking affect type i ifn secretion. hence, male gender, african ancestry, high bmi and smoking are risk factors for severe or critical covid- disease ; interestingly, these are also risk factors for severe lupus. this mechanism of type i ifn induction is strikingly similar to that of sle. early secretion of type i ifn by pdcs is important for controlling viral replication and preventing dissemination of virus to major organs. depletion of pdcs results in loss of antiviral type i ifn response and impaired survival of virus-specific natural killer (nk) or cd + t-cells. numbers of pdcs gradually decrease as age increases, whereas there is no change in conventional dc (cdc) cells. this may be the reason for reduced antiviral ifn responses in older age and increased mortality among the elderly due to covid- . there is nearly -fold higher secretion of type i ifn by pdcs upon recognition of mers-cov compared to that of sars-cov. this may be one of the factors contributing to the higher mortality rate observed in mers than sars and covid- . type iii ifn (ifn-λ) secretion is also higher by coronavirus-infected pdc and its levels were similar to that of type i ifn. signal in summary, both hcov-mediated diseases and sle have robust production of type i ifn. in both conditions, pdc is a major cellular source for type i ifn via tlr and cgas-sting signaling, as well as via the tlr pathway, a relatively lesser known mechanism. early response to sars-cov- infection is mediated by cd cells. dramatic reduction in the number of cd and cd t-cells during the acute phase of infection in patients of sars and covid- are uniformly reported. decrease in activated cd t cells and increase in antibody- during convalescence phase of covid- are other encountered observations. low levels of t-cells (cd +), both cd + t-cells and cd + t-cells, are also associated with severity and hospital death in covid- . , , neutrophil to lymphocyte ratio as a predictor for severity in covid- has also been studied. type i ifn is required for activation of cd th cells that sustain antiviral response of cd ctls. in parallel, type i ifn is also required for differentiation of tfh cells that mediate b cell differentiation and antibody production. therefore, type ifn are crucial for immediate and long-term protection against covid- . wide variability in cytokine secretion patterns that is noticed among patients with sars and covid- determines the course of disease. pre-existing comorbidities of these patients also synergistically alter cytokine levels and decide outcome. ards, disseminated intravascular coagulation and multiple-organ failure rapidly progress in severe covid- patients, leading to death within to days of intensive care unit admission. increased infiltration of neutrophils and macrophages as well as secretion of high levels of pro-inflammatory cytokines result in a condition called cytokine storm. cytokine storm could be the leading cause for respiratory complications and multi-organ failure in patients with covid- . reduced lymphocytes, increased cytokine levels and abnormal coagulation parameters are frequent in these cases. reduced ifn levels and increased viral load are higher in critical and severe cases as compared to mild to moderate patients with covid- . in view of diminished lymphocyte and dendritic cell function, neutrophils and macrophages take over antiviral defense response. interleukin (il- ) and tumor necrosis factor alpha (tnf-α) are major pro-inflammatory cytokines secreted by these cells that induce tissue damage, eventually leading to alveolar flooding and fibrosis. cytokine storm is also a relatively common complication in pa- age-dependent altered innate immune response to hcov was studied in a mice model. young mice infected with sars-cov efficiently cleared the virus. in contrast, aged mice showed exacerbated immune response to virus with increased lymphocyte infiltration in lungs. during initial infection in young mice, activation of innate immune cells, namely pdc, macrophages and nk cells were involved in viral clearance. however, this response was not effective in aged mice to contain the virus; instead, a robust cytokine storm and altered lung pathology followed the immunological war against the virus in older mice. in a macaque model of sars-cov infection too, aged macaques had more severe lung pathology, lower expression of type i ifn and higher expression of pro-inflammatory cytokines as compared to younger macaques. as mentioned earlier, sars-cov infects hosts equally across all age groups, but complications and fatality are noted much more commonly in older populations. early type i ifn response is important for preventing hcov-mediated inflammation and severe disease. both ifn secretion from innate cells like pdc and response threshold by its receptor in t-cells are impaired in older age as compared to young individuals. , , delayed ifn response causes apoptosis of t-cells and recruitment of monocytes and neutrophils, leading to cytokine storm and lung injury. , inflammation in the form of chronic subclinical systemic inflammation and immune senescence, that is reflected by blunted and impaired immune response, are other factors contributing to age-related differential covid- pathogenesis. on the other hand, sars-cov- -associated multisystem inflamma- kawasaki-like diseases have also been reported in some immunodeficiency states. it is likely that children with mis-c may have some underlying immunodeficiency state that is triggered into an auto-inflammatory syndrome by covid- infection; only future studies can reveal exact immunological mechanisms behind this unique mimic of kd. during recent times, hcq has been subjected to huge discussions in relation to its benefit and harm in covid- ; its role in autoimmune diseases like rheumatoid arthritis (ra) and lupus are already established. antimalarial agents, chloroquine (cq) and hcq are drugs of choice for various connective tissue diseases. both are immunomodulatory agents; unlike immunosuppressants, these drugs are safer for patients with chronic diseases. role and mechanism of hcq in management of autoimmune rheumatic diseases is discussed in a recent review. here we discuss its perspectives in covid- ( figure ). cq and hcq not only interfere with tlr / -mediated signaling, there is evidence that it has an effect on tlr also. cq inhibit ifn-β secretion and phosphorylation of stat in human mesangial cells treated with tlr agonist polyinosinic-polycytidylic acid (poly i:c). this may be one more effective mechanism of cq and hcq in treatment of lupus nephritis patients. as mentioned above, tlr also recognize sars-cov dsrna and activates transcription factors in a small randomized controlled trial, treatment with hcq was associated with reduced viral load and improvement in radiological progression on computed tomography images in patients with covid- . however, another clinical study involving patients showed failure of hcq to clear viral load and no clinical benefit. in an observational study, administration of hcq in severe covid- patients has shown no benefit in preventing intubation or death. in other randomized and clinical observational studies, hcq was not shown to be beneficial for patients who are transferred to the intensive care unit. these studies were different in terms of dose of medication, day of starting treatment, primary endpoint and selection bias of severity (table s ). therefore, randomized clinical trials with larger sample size is warranted for testing its efficacy. on the other hand, hcq has been recommended as prophylactic regimen specifically for healthcare workers in india. to patients with covid- that it is a mild immunomodulatory f i g u r e hydroxychloroquine (hcq) inhibits sars-cov- entry and inhibits virus-induced type i interferon (ifn) signaling and proinflammatory cytokines production. here are the various pathways: . angiotensin i converting enzyme (ace ) is an inducible gene. hcq inhibit type i ifn, thereby inhibit ace expression. also hcq may inhibit n-terminal glycosylation of ace . . hcq can also inhibit viral entry by disrupting endosomal acidification. . hcq alters endosomal ph, there by disrupts ligand binding to toll-like receptor (tlr ) and tlr . . hcq inhibit cgas-sting (stimulator of interferon genes) signal and thereby reduce type i ifn and pro-inflammatory cytokines expression agent and not an immunosuppressant. while its use in hospitalized covid- patients are not yet proven to be beneficial, several developing nations are using it in early disease and as a pre-exposure prophylaxis for frontline healthcare workers exposed to sars-cov- , as recommended by indian council of medical research (icmr) (table s ). presumable viral etiologies have been known for many connective tissue diseases. we might very well expect sars-cov- triggered development of autoantibodies. some available evidence to support this notion are discussed in the following paragraphs. injection of convalescent sera from sars patients to rhesus macaques also causes lung injury with similar histopathological features as in human disease. thus convalescent sera containing autoantibodies or other antiviral antibodies might cross-react with antigens expressed in the lung. this has to be kept in mind while recommending over enthusiastic use of convalescent sera/plasma therapy in critically ill covid cases. high titers of anticardiolipin antibodies are also seen in sars patients presenting with osteonecrosis. in vitro experiments also reveal that sera of sars patients contain autoantibodies targeting pulmonary epithelial cells and endothelial cells. another in vitro experimental study demonstrated anti-s spike antibodies in sera of sars patients that can bind to epithelial cells inducing cytotoxic injury. sera from patients with autoimmune diseases like mixed connective tissue disease, and ra were found to have higher positivity for anti-sars-cov igg and igm as compared to healthy controls. false positivity was also reported for anti-sars-cov antibodies in patients with sle. cross-reactivity between anti-sars-cov antibodies and autoantibodies targeting the same antigenic target is possible. however, autoantibodies in sars specifically bind to antigens expressed in lung tissue, and are not expected against cell nuclei (like ana), against smooth muscles (like sma) or against parietal cells (like pca). these studies show that both sars-cov and sars-cov- infections might induce expression of ana and apl antibodies. further studies are needed to delineate the spectrum and pattern of developing autoantibodies in patients with covid- . systemic vasculitis was noted in an autopsy study from patients who died from sars. simultaneous diagnosis of covid- and kd was also made in a -month-old child. outbreak of kawasaki-like diseases is reported in children, of them were positive for either sars-cov- by nasal swab or antibodies. pediatric patients who were positive for sars-cov- igg also developed cutaneous vasculitis lesions. development of cutaneous small vessel vasculits was seen in elderly female covid- patients also on the th day after onset of symptoms. sars-cov- infects endothelial cells and induces apoptosis as well as pyroptosis resulting in multi-organ dysfunction. these show hcov not only targets lungs, but can infect blood vessels, thereby causing multi-organ damage. in summary, ana and apl autoantibodies can be seen in patients with sars and covid- . theoretically, these patients may have higher chances to develop autoimmune diseases in future, like aps or a lupus spectrum disorder. rheumatologists will have to wait for the post-covid- era to witness any unfolding of events towards a rising prevalence of lupus, vasculitic process or aps. complications and fallouts of covid- disease have some similarities as well 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anticardiolipin antibodies autoantibodies against human epithelial cells and endothelial cells after severe acute respiratory syndrome (sars)-associated coronavirus infection antibody to severe acute respiratory syndrome (sars)-associated coronavirus spike protein domain cross-reacts with lung epithelial cells and causes cytotoxicity cross-reaction of sars-cov antigen with autoantibodies in autoimmune diseases analysis of false-positive associated with antibody tests for sars-cov in sle patients detection of autoimmune parameter of sars patients. zhonghua shi yan he lin chuang bing xue za zhi zhonghua shiyan he linchuang bingduxue zazhi the clinical pathology of severe acute respiratory syndrome (sars): a report from covid- and kawasaki disease: novel virus and novel case images in practice: painful cutaneous vasculitis in a sars-cov- igg-positive child cutaneous small-vessel vasculitis associated with novel coronavirus sars-cov- infection (covid- ) endothelial cell infection and endotheliitis in covid- key: cord- -tyyc uc authors: stradner, martin h.; dejaco, christian; zwerina, jochen; fritsch-stork, ruth d. title: rheumatic musculoskeletal diseases and covid- a review of the first months of the pandemic date: - - journal: front med (lausanne) doi: . /fmed. . sha: doc_id: cord_uid: tyyc uc in december , a cluster of severe pneumonia was observed in china, with the subsequent discovery of a new beta-coronavirus (sars-cov- ) as the causative agent. the elicited disease covid- is characterized by fever, dry cough, myalgia, or fatigue and has a favorable outcome in the majority of cases. however, in some patients covid- leads to severe pneumonia and sepsis with subsequent respiratory failure and gastrointestinal, hematological, neurological, and cardiovascular complications. a higher risk of infection is intrinsic to active rheumatic and musculoskeletal diseases (rmd) and the use of biological disease modifying anti-rheumatic drugs (dmards). with an increasing number of reports on covid- in rmd patients, we are beginning to appraise their risks. in this review, we summarize the published cases of covid- infections in rmd patients, including patients with inflammatory arthritis and connective tissue diseases as well as anti-phospholipid syndrome and kawasaki syndrome. overall, patients with inflammatory arthritis do not seem to be at a higher risk for infection or a severe course of covid- . risk for critical covid- in patients with systemic inflammatory diseases such as sle or vasculitis might be increased, but this needs further confirmation. furthermore, we summarize the data on dmards used to fight sars-cov- infection and hyperinflammation. since the discovery of the novel coronavirus sars-cov- causing covid- in december until june , more than , reports on the disease or the virus itself have been published. infections of patients with underlying rheumatic and musculoskeletal diseases (rmd) experiencing a complicated course of covid- have been reported. however, most of these reports are limited by the small numbers of patients. besides, several studies are still at a pre-publication stage and may therefore not yet meet the rigorous standards of scientific journals. this article summarizes current knowledge on covid- and rmd. it also reviews the general risk of viral infections in patients with rmd, the impact of disease modifying anti-rheumatic drugs (dmards) on the outcome of infections, and gives a comparison between present and previous coronavirus pandemics. the first cases of this novel pneumonia were reported in early december in wuhan, the capital city of hubei province, china ( ) . the genomic characterization of the pathogen causing this infection identified a novel enveloped positive single stranded rna beta-coronavirus, which has ultimately been named severe acute respiratory syndrome coronavirus (sars-cov- ) ( ) . coronaviruses are rna-viruses, shielded by an envelope containing membranous proteins such as spike proteins. these spike proteins give the virus a crown like appearance under an electron microscope, coining the name "coronavirus." the primary function of these proteins is host receptor binding, determining host tropism and transmission capacity ( ) . coronaviruses can cause a broad range of mostly mild infections in humans, mammals, and birds especially in the respiratory and gastrointestinal tracts, and to a lesser extent also in the nervous system. until the end of , six coronavirus strains (belonging to the alpha or beta-coronavirus genus) causing human disease had been identified ( ) . four of these viruses cause common cold symptoms, whereas the two remaining strains elicit severe illnesses, namely the severe acute respiratory syndrome coronavirus (sars-cov) and the middle east respiratory syndrome coronavirus (mers-cov) ( ) . whereas, sars-cov was the pathogen leading to the severe acute respiratory syndrome observed in / in guangdong province, china ( ), mers-cov was found to underly the severe respiratory syndrome seen since in the middle east ( ) . phylogenetically, the present sars-cov- displays an % homogeneity to two bat-derived severe acute respiratory syndrome (sars)-like coronaviruses, a % identity with sars-cov and an approximate % identity with mers-cov ( ) . an important similarity between sars-cov and sars-cov- is the mechanism of entry into cells of the lower respiratory tract, mediated by the spike protein binding to the ace protein ( ) . the novel disease, termed covid- , is marked by symptoms reminiscent of other respiratory infections: fever, dry cough, myalgia, or fatigue are commonly observed, whereas sputum production, headache, hemoptysis, and diarrhea are less prevalent ( , ) . while the majority of patients with covid- have a favorable outcome ( ) ( ) ( ) ( ) , some develop severe pneumonia eventually leading to acute respiratory distress syndrome (ards), respiratory failure, along with other organ manifestations and sepsis ( ) . covid- appears to have at least two distinct disease phases: a phase characterized by the immune response against the virus with the aim of eliminating the pathogen. some patients, even though they may have had only mild initial symptoms, subsequently develop a phase of severe "cytokine storm" (cytokine release syndrome, crs) instead of the expected phase of re-convalescence leading to fatal autoinflammation of the lung and other organs ( ) . as the disease spreads throughout the world causing the present pandemic crisis, additional symptoms have been observed, e.g., neurological changes such as anosmia or ageusia or thrombotic complications ( , ) . the development of profound thrombocytopenia ( . %) and elevated d-dimers ( . %), which are even higher in patients with severe covid- disease ( . and . %, respectively) ( ) have become a hallmark of the disease, and suggest a triggered disseminated intravascular coagulation (dic), reviewed in ( ) . there are distinctive characteristics of this new disease: it affects more men than women, causes severe disease especially in elderly people. certain comorbidities including hypertension, diabetes, prior respiratory, and cardiovascular disease as well as obesity have been associated with a worse outcome ( , ) . based on a developing cohort of , patients and a validation cohort of patients, a risk score for the development of a critical illness (defined as composite measure of admission to the intensive care unit, invasive ventilation, or death) items emerged as independent predictive factors ( ) . three of those were patient characteristics: older age, number of comorbidities, and a history of cancer. however, although immunodeficiency was one of the potential predictors in the beginning, the presence of rheumatic diseases or immunosuppressive medication was not included in the calculation, making the score less applicable from a rheumatological perspective. according to the first report of the chinese center for disease control only ∼ % of patients out of , chinese patients were critically ill, % of them suffered from a severe course, while most patients ( %) showed no or mild pneumonic symptoms ( ) . the overall case-fatality rate (cfr) was . %, which is lower than that during the earlier coronavirus outbreaks with sars-cov ( %) and mers-cov ( %) ( ) . whereas, the comorbidities and risk factors for worse outcomes of covid- are similar throughout international reports, mortality rates diverge largely from . % in singapore up to . % in france ( ) . the causes for this disparity are myriad and may include disease factors and genetic susceptibility of the populations, but also differences in health care organization (e.g., number of tests conducted, criteria for testing) and outcome reporting (particularly who is counted as deceased due to/with covid- ). immunological changes generally observed in patients with covid- include lymphocytopenia that pertains to cd +, cd +, and treg cells and increased levels of proinflammatory cytokines [e.g., interleukin (il) -β, il- ra, il- , il- , il- , il- , interferon (ifn)γ, interferon-gamma induced protein (ip) , monocyte chemoattractant protein (mcp) , macrophage inflammatory protein (mip) α, mip β, tumor necrosis factor (tnf) α, granulocyte colony stimulation factor (gcsf), platelet derived growth factor (pdgf) b, and vascular endothelial growth factor (vegf) a]. a pronounced decrease of cd + t cells and higher levels of il- , il- , and tnfα occur during the phase of hyperinflammation (crs) ( , ) . hyperinflammation is considered to initially involve activation of macrophages stimulated by damage-associated molecular patterns (damps) released from dying cells and pathogen-associated molecular patterns (pamps) such as viral rna ( ) . this leads to massive il- and il- secretion promoting the recruitment of neutrophils and cytotoxic t cells, which in turn induce pneumocyte and endothelial injury, ultimately leading to acute lung injury. pre-publication reports describe a specific monocyte population in covid- patients, not seen in healthy donors or other viral infections. these monocytes are larger, atypically shaped, vacuolated secreting high levels of both il- and il- β and expressing ace ( , ) . interestingly, interferon type i, a crucial player in the combat of viral infections, seems to be muted for unknown reasons in covid- patients ( ) , which was especially seen in critically ill patients ( ) . furthermore, alterations in the lymphocyte department, including upregulation of markers of t cell exhaustion in both lung-resident and circulating t-lymphocytes, including pd- and tim- have been described ( ) . furthermore, cd + as well as cd + t cells of covid- patients display an activated phenotype. in analogy to patients infected with sars-cov or mers-cov, cd th -cells expressing gm-csf, and il- were isolated from patients with covid- crs ( ) . in contrast, in patients in the recovery phase an effective adaptive immune response characterized by t cell clonal expansion, activation, and memory formation has also been reported in covid- ( ) . the higher occurrence of the above described th cells and the covid- -typical, highly inflammatory monocyte subset together with the inadequate ifn i answer may be determining factors leading to a worse disease course. patients with rmd are generally considered to be more prone to bacterial and certain viral infections such as the herpes zoster virus. the susceptibility to infections is multifactorial and is related to disease aspects such as disease activity, disease damage, comorbidities, as well as to treatment. in rheumatoid arthritis (ra), each . point increment of the disease activity score (das) ( ) , led to a % increase in hospital admissions and % increase of outpatient infections in a us registry of > , patients ( ) . a -fold increased susceptibility to infections was seen in systemic lupus erythematosus (sle) in an insurance database ( ) and an incidence rate of . severe infections/ , patients per year in the spanish sle-registry ( ) . current and past use of hcq seemed to have a protective effect (rr . , p = . ), whereas glucocorticoids (≥ mg/d), rituximab, mycophenolate mofetil/mycophenolic acid, and lupus activity/severity were linked to a higher risk of severe infection. according to a cochrane review, the risk of infection in patients with inflammatory arthritis [ia; including ra, psoriatic arthritis (psoa) and spondylarthritis (spa)] is higher under therapy with tnf-blockers than with conventional disease modifying antirheumatic drugs (dmards) only ( ) . a meta-analysis of other biological dmards (bdmards) used in ra indicated a . fold increased risk of serious infections in standard doses, and an almost -fold increased risk at higher doses (any dose higher than the one approved by the regularities) compared to conventional dmards ( ) . whereas, the risk of serious infections under anti-il- therapy is comparable to that of tnf-blockers; anti-il- / therapy seems to carry a lower risk (hr = . , % ci . - . ) ( ) . due to their inhibition of interferon-alpha, januskinase inhibitors (jaki) increase the risk of viral infections ( ) , especially of herpes zoster. in contrast, the overall risk for severe infections of ra patients with jaki is comparable to that of healthy controls according to a recent metaanalysis ( ) . whether there is an increased risk of coronavirus infection (or influenza) elicited by jaki used in rheumatology is currently unknown. the use of glucocorticoids (gc) in both ia ( ) and connective tissue diseases (ctd) leads ( ) to an increased rate of infections overall and viral infections in particular, especially for herpes zoster correlating with the actual gc dose, treatment duration, and the cumulative gc dose; however even doses considered relatively safe such as mg prednisone equivalent have been associated with an increased infections risk. hydroxychloroquine seems to have a protective effect for those who take gcs: in patients with gc monotherapy a hr . for severe infections was reduced to for those who took gc + hydroxychloroquine. among conventional synthetic dmards (csdmards), methotrexate has not been associated with an increased infection rate according to a study with over , ra patients ( ) . in contrast, mycophenolate mofetil, azathioprine, and cyclophosphamide were linked to a higher risk of infections particularly in patients with connective tissue diseases ( ) . we know little of rmd patients infected in the past outbreaks of coronaviruses like sars and mers ( , ). also, given the restricted geographical spread and the low number of patients affected, no special containment measures were released affecting diagnosis and the management of patients with rmd, nor were there any difficulties with drug supply for dmards. there has been a weak association of coronavirus outbreaks in the past with higher incidence of kawasaki syndrome, which is often virally triggered. kawasaki syndrome occurred in < % of patients with a coronavirus infection in some reports, however, no reports are available for sars and mers ( , ) . single cases of sars and thrombotic complications have been reported. these complications were considered to be related to the presence of anti-phospholipid antibodies ( ) . in the largest early report on patients with covid- from china there are no data on patients with rmd or any other immunological disorder ( , ). guan et al. mention two patients with immunodeficiency without a severe course of covid- infection out of a cohort of , covid- patients from china ( ) . another publication from wuhan included one patient with pre-existing connective tissue disease, who died ( ) . the cohort of , covid- patients from new york city did not list any rheumatologic disease as a comorbidity, although at least ctd should have been captured, given that the charlson comorbidity index was used in this study ( ) . likewise, no rmd was named under the comorbidities in a prospective cohort of , critically ill patients in new york city or a cohort of , covid- cases in brazil ( , ) . in a paper from lombardy, the italian region with the highest number of covid- cases ( ) , there were no patients with immunosuppressive therapy with jaki or bdmards among covid- patients with a severe course of disease. however, data from the health analytics platform opensafely, which covers % of all patients in england and holds primary care records of , , adults pseudonymously linked to , covid- -related deaths found a hazard ratio of . ( . - . ) for the combined autoimmune diseases rheumatoid arthritis, lupus, and psoriasis ( ) . additionally, arentz et al. reported patients in intensive care units in washington state. of these, one person had a "rheumatic disease" as a pre-existing condition and three had immunosuppression due to "rheumatoid disease" or transplantation. however, there is no detailed information about the precise diagnosis, medication, comorbidities, or course/outcome of these patients ( ) . in that specific cohort, it seems that there was a high rate of co-morbidities ( . %) and mortality ( %) with only a few discharges ( . %). currently available data do not imply a strikingly increased risk of infection for rmd with or without dmard therapy in general ( , ) , although differences in the individual diseases have been reported ( ) . pablos et al. investigated the prevalence of covid- in seven spanish hospitals providing medical care for a population of . million patients, and found a comparable prevalence of the infection in ra and psoa with the general population ( . %); in contrast, patients with spondylarthritis (spa) had a higher prevalence ( ) . as this was also the case for patients on bdmards or jaki, one might speculate that the higher infection rate of spa patients was due to a higher percentage of these drugs in spa patients compared to the other groups. this aspect, however, was not analyzed in the cited paper. interestingly, patients with sjögren's syndrome or systemic sclerosis showed a higher prevalence of sars-cov- infection in comparison with the general population; in contrast, prevalence in sle patients was similar to that of the reference population. this unexpected discrepancy among ctd patients might be explained at least partly by the younger age and the higher proportion of females among sle patients in this cohort. in a report from lombardy, , patients with rmd were evaluated by a survey and % were found to have either confirmed or suspected covid- , with most of them suffering from a form of inflammatory arthritis ( ) . in addition, the authors used a case-control design to investigate differences between the covid- cases with rmd and control covid- patients without rmd. no differences pertaining to comorbidities or disease course were detected. similar findings were described in a case control study from massachusetts investigating rmd patients, of which % had an ia and the remaining patients suffered from different forms of systemic autoimmune diseases. the disease course of covid- of these cases was compared with that in sex-and age-matched controls without underlying rmd ( ) . the majority of the rmd patients with covid- had active disease ( %) and a higher percentage of coronary and interstitial lung disease. whereas, the clinical presentation and laboratory values were similar in the two patient groups, rmd patients were at a higher risk for intensive care/mechanical ventilation [adjusted or for mechanical ventilation . ( % ci . - . ), p = . ]. however, this did not imply a statistically significant higher mortality ( %) or overall hospital admission rate; the higher rate of ventilation reported in rmd patients is unsettling, considering that the or was adjusted for the higher cardiorespiratory comorbidities, suggesting an intrinsic effect of the underlying autoimmune disease. nevertheless, other comorbidities, disease activity or use of immunosuppressive drugs may have contributed to the higher risk of ventilation and need to be addressed in future studies. similarly, a report from wuhan found an increased rate of respiratory failure ( %; mortality: . %) in patients with rmd with a similar distribution between ia and ctd ( ) . in comparison with this chinese study, a different cohort of rmd patients from wuhan found a lower need for ventilation ( . %) and a . % mortality rate ( ) . whereas, the disparity of the first chinese study with the cohort from the us may be attributable to a different disease population, different medication regimes, and also a different genetic background, the reason for the intra-regional difference between the two reports from wuhan remains elusive. altogether, there is a dire need for reports with larger cohorts. to address this need, the global rheumatology alliance has established a registry of rmd patients with covid- infection (https://rheum-covid.org/). this is an international initiative, supported by acr and the eular as well as by several national societies, with the possibility to include rmd patients affected by covid- from all over the world. the website is updated regularly (summarized in box , accessed on july th ) and a report of the first patients was recently published ( ) . the majority ( %) of patients were from north america or europe, female, and between and years of age. the distribution of ia vs. ctd was approximately : and % displayed moderate/high disease activity. the report described interesting findings: ( ) there was a high rate of hospitalization ( %) and mortality ( %) altogether, with sle and vasculitis patients showing a higher propensity to be hospitalized than other patients. ( ) the main factor associated with hospitalization was a prednisone dose ≥ mg/day (or . , % ci . - . ), whereas dmard therapy (csdmard monotherapy or combined with bdmards/jaki) was not associated with hospitalization. ( ) established general adverse characteristics/comorbidities as age > years, hypertension/cardiovascular disease, lung disease, diabetes as well as chronic renal insufficiency/endstage renal disease carried a higher risk of hospitalization also in rmd patients. some results, like the role and category of comorbidities, are in line with the general population experiencing covid- , others, like the protective effect of tnf-blockers were unexpected. the possible benefit of tnf-blockers was also observed in a cohort from the us ( ) and underscores the role of the overabundance of tnf in critically ill covid- patients. altogether, this report supports the recommendations issued by national and international societies not to withhold dmard treatment in rmd patients prophylactically in this pandemic. however, important questions as, e.g., a possible difference in disease course in the diverse rmds have not been answered and await clarification. the registry has also provided preliminary data on slepatients with covid- and the use of hcq. sixty-four percent of the sle-patients were taking antimalarials prior to the infection with sars-cov- and admission frequency to the hospital did not differ between hcq users and non-users [ % ( / ) vs. % ( / ), p = ns; χ -test] ( ) . this argues against a protective role of hcq (in the usually administered dose for rmd patients) in sars-cov- infection, which is also supported by pharmacological in vitro data describing a much higher level needed for effective viral inhibition ( ) . the ineffectiveness of hcq was also indicated in several rmd and sle cohorts, in which the prevalence of (confirmed or suspected) covid- was similar in patients with or without hcq treatment ( ) ( ) ( ) ( ) ( ) ( ) ( ) . in contrast, a large study using databases of general medication use and sars-cov- infection in portugal found a protective effect of chronic treatment with hcq [or . ( . - . )] for sars-cov- infection, even after adjustment for demographic characteristics and immunosuppressive treatment ( ) . due to the study design, no data on underlying diseases (including rmd) or other patient data apart from those on medication use and covid- status were retrievable precluding a definite judgement on the efficacy of hcq for preventing covid- . in particular, a selection bias with prescription of hcq preferably to patients without comorbidities could have influenced the results. other descriptions of rmd patients with sars-cov- infections are often based on case reports or case series (see tables , for summary) with different rmd and are discussed below. the disease course in a series of patients with immunemediated inflammatory diseases including cases of ia (rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis) with confirmed or highly suspected covid- was analyzed (confirmed cases: total: , of which ia). a special focus was put on the comparison of patients with an out-patient or in-patient management ( %) ( ) . seventy-two percent of patients were receiving bdmards (anti-tnf, anti-il , anti-il- , and anti-il- / agents) or jaki. among hospitalized patients, comorbidities (hypertension, diabetes, or chronic obstructive pulmonary disease) were more common than in outpatients. besides, oral gc ( vs. %), hcq ( vs. %), and methotrexate ( vs. %) were more frequently used in the former, whereas certain biologicals (mostly tnf-blockers and anti-il / agents) were more common in the latter group. whether this indicates any possible protective effect of these biologics to prevent the occurrence of a crs in rmd patients remains a matter of speculation. another possibility to learn about the course of covid- on patients with rmd is to follow prospectively a cohort of patients with a defined disease and to note symptoms and outcomes of any sars-cov- infection. in a study from northern italy with this objective, patients with ra or spa with an average age of ± years were interviewed and observed prospectively for weeks ( ). among them, there have been only incidental reports of patients with ctd besides sle: of the few cases with systemic sclerosis ( table ) , the most striking common denominator is treatment with rituximab, which seems to induce a late worsening of symptoms, leading to respiratory complications, also in patients without preexisting lung disease ( , ) . likewise, one patient from france with granulomatosis with polyangiitis, who was overweight and had hypertension under rituximab and corticosteroid therapy had a severe covid- course with ards requiring intubation. the patient was discharged after days. the disease course of french patients with sle [median age: . ( . - . )] was reported recently ( ) . sixteen of the patients had quiescent rheumatic disease, the main comorbidities were obesity ( %) and chronic kidney disease ( %); % of them had secondary antiphospholipid syndrome (aps). all were on (different doses) of hcq, % were taking glucocorticoids, most of them below mg prednisone equivalent, and % took immunosuppressants. the clinical course of these patients was more severe than expected, % of them had signs of respiratory failure needing oxygen therapy. almost half of these patients were admitted to intensive care and one third developed ards. one patient was treated with extracorporeal membrane oxygenation and two of the patients died. three patients developed acute renal injury; cardiac injury and venous thrombosis occurred in one patient each. an equally severe picture was displayed in five sle patients from michigan, were one patient died, and three needed mechanical ventilation ( ) . the reasons why these sle patients had a worse outcome than those from other reports of the general population is unclear, however, long-term glucocorticoid treatment and comorbidities might have played a role. additionally, an epigenetic dysregulation, i.e., dna hypomethylation of ace resulting in ace overexpression in t cells of sle patients, has been shown; this phenomenon could be perpetuated by the oxidative stress induced by viral infections or by sle flares and thus could contribute to the worse disease course seen in this sle cohort ( ) . in contrast, bozzalla et al. observed an overall mild course of covid- in patients of their cohort of sle patients, although the incidence of confirmed or suspected covid- was higher in this cohort than in the general population of the area; nevertheless, this might be explained by a bias of higher testing ( ) . a special aspect was observed in a -year-old chinese sle patient, who had been diagnosed with sle years earlier and had been on . mg of prednisone ever since, without other immunosuppression. she had experienced mild symptoms for weeks and was diagnosed with covid- weeks ( ) after her presumed exposure to the virus having infected two family members in the meantime. at the time of diagnosis her nasopharyngeal swabs were negative, and she showed positive sars-cov- serology, suggesting that the symptoms experienced were part of the second phase. the unusually long incubation period and the supposed prolonged viral shedding might be due to the immunosuppression with gc which also warrants caution in other rheumatic patients on long term gc treatment. apart from case series, patient surveys have been carried out in ctd patients. the largest one comprised patients in northeast italy, of which % had underlying ctd (sle, vasculitis, systemic sclerosis, myositis); . % had a high suspicion of covid- , only two patients ( . %) had a pcr confirmed infection, mortality was . the low prevalence of confirmed cases was similar to that of the general population of the veneto. overall, discontinuation of dmard therapy was rare in these patients, with higher percentages in ra ( . %) and sle ( . %) ( ) . a comparably low prevalence of covid- was seen in other surveys from milan and belgium, possibly influenced by a high percentage of patients undertaking precautionary measures early in the pandemic ( , , , ) . also in these surveys the ineffectiveness of hcq as protection against covid- was noted, as well as a negative effect of higher gc doses in terms of hospitalization and clinical features (anosmia/ageusia and diarrhea) ( ) . in conclusion, data published in the first months do not consistently describe a higher risk for infection with sars-cov- or a more severe course of covid- in patients with either inflammatory arthritis or connective tissue diseases. however, due to early case series ( , ) describing a more severe course of the infection in sle patients, more data on covid- from cohorts of sle patients with confirmed sars-cov- infection are warranted. the thrombogenic nature of covid- ( ) is shared by rheumatic diseases, which should prompt further attention on rmd-associated coagulation problems such as the aps. analogous to incidental reports of antiphospholipid antibodies (apl) in earlier coronavirus pandemics, these antibodies have occasionally been reported in covid- ( ) , albeit of an iga class, which have not yet been included in the classification criteria of aps ( ) . however, a role for iga antibodies in clinical aps not meeting the serological criteria and sle patients has been postulated ( , ) . lupus anticoagulant (lac) was present in % of a french cohort of patients with covid- without underlying rmd. in this group, % also tested positive for anti-cardiolipin (acl) or anti-beta glycoprotein (ab gp ) antibodies (mostly associated with lac) ( ) . additional data from london corroborate the higher prevalence of lac in covid- patients. in samples from covid- patients screened for coagulation abnormalities in h, were found to have a prolonged aptt, and % of the further investigated showed a positive lac, often associated with reduced factor xii levels ( ) . additionally, in a population of covid- patients on the intensive care unit (icu) % of the patients tested had a positive lac ( ) . in contrast, antiphospholipid antibodies were commonly detected to a substantially lower degree with a preponderance of antibodies, which are not part of the official classification criteria for aps [iga acl; iga ab gp ; antibodies against phosphatidylserine/prothrombin (aps/pt)] ( ) ( ) ( ) . due to their disputed role in aps in general, the significance of these antibodies for covid- is not yet clear. the occurrence of apl in viral infection is a well-known phenomenon, however, in most cases these are of igm or igg acl subtypes with an antigen recognition differing from classical aps and are less thrombogenic in nature ( ) ; in covid- the latter finding has also been observed, casting a doubt on the clinical relevance of the characterized antibodies ( ) . nevertheless, the pathogenicity of apl from covid- patients was proven elegantly in a pre-publication by zuo et al., in which igg aps/pt antibodies led to netosis in vitro, thus contributing to thrombus formation, and increased thrombus extension in vivo in a mouse model ( ) . this finding is corroborated by the observation that apl occur preferably in critically ill patients ( ) . altogether, the risk of thrombosis mediated by aps should be kept in mind and a prolonged aptt should not prevent proper anticoagulation in patients. besides, it needs to be tested whether the presence of the diverse apl is associated with an increased risk of worse outcomes in patients with covid- ( ) or vice versa, and if the emergence of lac and apl in covid- patients constitute a transient phenomenon or a persistently increased thrombogenic state. altogether, reports on patients with rmd infected with sars-cov- are still insufficient and are partly contradictory in terms of hospitalization and outcome. however, an unequivocal signal of higher risk for infection with sars-cov- or a more severe disease course of covid- has not been detected. more data, especially larger cohorts of different patient groups are needed to provide a better risk stratification of rmd patients. additionally, the question, whether patients receiving bdmards such as tnf-blockers or anti-il- / may be protected from a complicated disease course has to be clarified by future research. in conclusion, due to the generally increased susceptibility to infection, patients with rheumatic diseases should be considered at risk as long as the data remain scant. another yet unresolved question is whether covid- might trigger autoimmune diseases. although the detection of ana has been reported in a third of covid- patients in the acute stage ( ) , the persistence and clinical relevance thereof remains to be investigated. whereas, arthralgias and myalgias might be one of the symptoms of covid- , data on the incidence of ra or other ia after covid- have not been published yet. in a korean study of , newly diagnosed ra patients, published before the discovery of sars-cov- , an increased risk of developing rheumatoid arthritis after infection with other coronaviruses or influenza was reported. for an increase of % of coronavirus-infected patients, a . % increase in the incidence of ra in the following months was observed, especially in women and older patients ( ) . whether, there is direct molecular mimicry between coronavirus and host antigens or whether coronaviruses activate other, yet unclear mechanisms leading to chronic autoimmunity has to be investigated further. analogous to previous (small) peaks during sars/mers, a pediatric inflammatory multisystem syndrome displaying similarities with kawasaki syndrome has been observed in association with sars-cov- . the first child reported, a month-old infant, received a single dose of g/kg intravenous immunoglobulin (ivig) and high dose acetylsalicylic acid and was discharged shortly thereafter ( ) . the unusual accumulation of eight children suffering from a disease reminiscent of kawasaki syndrome with a combination of rash, conjunctivitis, peripheral oedema, extremity pain, and serious gastrointestinal symptoms prompted a national alert in great britain at the end of april ( ) . although only two (one of them postmortem) of the children tested positive for sars-cov- , all of them were reported to have antibodies. this suggests a sars-cov- triggered severe hyperinflammation necessitating the admission to the pediatric icu, where one -year-old child eventually succumbed to a large cerebrovascular infarct. all children experienced either cardiac complications or serositis, were treated with inotropics, and apart from the mentioned -year-old are alive at this point. since the first description, several larger case series have been published describing a clinical spectrum including fever, gastrointestinal symptoms, and rash as well as the complications of myocardial injury, shock, and development of coronary artery aneurysms ( ) ( ) ( ) ( ) . the disease associated with sars-cov- and the "classical" kawasaki syndrome differ in terms of age, inflammation markers including ferritin, which are all higher in sars-cov- , as well as more severe cardiac involvement ( , ) . an unusual and alarming treatment resistance to ivigs was seen in more than % of a french cohort of patients. this occurred especially in patients > years old with high ferritin levels and should prompt rapid escalation of immunosuppression with tocilizumab or anakinra to avoid serious cardiac sequelae ( ) . the exact relationship between the virus and the hyperinflammation has yet to be elucidated in order to optimize a treatment regimen. elevated levels of pro-inflammatory cytokines, including il- are associated with severe covid- and an increase in il- is associated with increased mortality ( , ) . an excessive increase of c-reactive protein (crp), il- , and ferritin observed in covid- cases was termed crs in analogy to similar findings in hemophagocytic lymphohistiocytosis and car-t cell therapy. blockade of the il- receptor (il- r) using tocilizumab is approved by the fda to alleviate the crs after hematological car-t cell therapy ( ) . in covid- tocilizumab may be considered in patients with high levels of inflammatory markers such as il- and worsening respiratory insufficiency, requiring non-invasive ventilation or intubation ( ) . however, evidence for these recommendations from randomized control trials ( ) . in addition, several retrospective case-control studies on the use of tocilizumab in covid- have been published ( ) ( ) ( ) ( ) ( ) ( ) , of which a majority reported a favorable outcome of tocilizumab treatment compared to controls. of these, campochiaro et al. and canziani et al. did not find a statistically reduced mortality in tocilizumab treated patients, although both studies showed a respective trend ( , ) additionally, tocilizumab treated patients required invasive ventilation statistically less frequently than controls ( ) . in line with this observation the outcomes of several studies suggest that patients may benefit most from tocilizumab if given before invasive ventilation ( , ) . in a prospective open single-arm study investigating covid- patients with signs of crs, administering tocilizumab within the first days of hospitalization was associated with a better outcome than later in the course of disease, irrespective of the intravenous or subcutaneous route of administration ( ) . preliminary results released of a large rct investigating the use of the il- receptor blocker sarilumab in covid- are ambivalent . patients with critical covid- treated with mg of sarilumab tended to less likely need mechanical ventilation and showed clinical improvement more often than the placebo group. however, in patients with non-critical, severe covid- the group receiving sarilumab trended toward unfavorable outcomes when compared to placebo. in this study, the patients had not been prestratified for high inflammatory burden. thus, in the absence of a cytokine storm where il- receptor blockade cannot be effective, adverse events may deteriorate outcomes. bacterial superinfection, septic shock, gastrointestinal perforation, and viral myocarditis have been reported in covid- after treatment with tocilizumab ( , , ) . in summary, il- blockade may be a useful option in covid- when administered early to those with signs of excessive inflammation. a clearer picture will emerge as randomized controlled studies for tocilizumab and sarilumab currently conducted in covid- will be published. the beneficial role of il- blockade in life-threatening hemophagocytic lymphohistiocytosis (hlh) syndrome has been well-established ( ) . due to the pathophysiological similarities pertaining to the macrophage activation between hlh and the hyperinflammation seen in the second phase of covid- , a small number of patients have been treated with the il- receptor antagonist anakinra. two case series report anakinra treatment in and patients, respectively ( , ) . eight out of nine and seven out of patients treated with anakinra did not require invasive ventilation. besides a slight elevation of transaminases, anakinra was well-tolerated. however, it is unclear if the patients might have improved without the blockade of il- . cavalli et al. conducted a retrospective case-control study of covid- patients treated with a high dose of intravenous anakinra ( mg/kg/day) comparing their outcome to seven patients receiving mg of anakinra subcutaneously twice daily and controls on standard treatment ( ) . all patients suffered from moderate-to-severe ards and hyperinflammation. while subcutaneous application of anakinra was terminated due to lack of efficacy, survival in those receiving high dose anakinra treatment was superior to those receiving standard care ( and %, respectively). although pathophysiologically reasonable, solid evidence from randomized control trials are needed; in this respect at least trials are underway targeting il- in covid- . corticosteroids have a broad anti-inflammatory potential and are readily available. therefore, they have been considered for use in critical covid- . high-dose corticosteroids at admission were identified as a risk factor for covid- mortality, and they are not recommended by the who for viral pneumonia ( ) . in a small observational study from china the use of corticosteroids was not associated with shorter hospitalization or more rapid virus clearance ( ) . still, corticosteroids in variable doses are currently used in critical covid- patients, especially those with ards ( ) . this practice is supported by data from the british recovery trial, a randomized, controlled, openlabel study including more than , hospitalized covid- patients ( ) . a daily dose of mg dexamethasone reduced -day mortality by one third in patients receiving invasive mechanical ventilation at randomization. patients requiring oxygen without invasive mechanical ventilation did benefit from the addition of dexamethasone to a lesser extent, while those not receiving respiratory support of any kind did not benefit at all. in addition, ards patients were reported to develop glucocorticoid resistance requiring excess corticosteroids to overcome progressive inflammation ( ) . this, suggests that dexamethasone is important in the later stage of infection, characterized by crs, and ards, whereas in the first stage, immunosuppression might not be of additional value ( ) . the antimalarial drugs chloroquine (cq) and hydroxychloroquine (hcq) have been suggested as box | overview of open and resolved research questions. -is there a role for antiphospholipid antibodies in the thrombogenicity of covid- -is there a protective role of jaki, il- , bdmards -if yes, when should they be applied -is the risk of a complicated course of ctd/ vasculitis patients heightened? issues that have been answered -there is no protective role of hq in rmd patients -rmd patients can continue most of their dmards without problems -there is no increased risk for a complicated course of covid- in patients with inflammatory arthritis. potential antiviral agents in covid- . the rationale arises from the observation that cq and hcq can prevent sars-cov- replication in vitro probably by increasing endosomal ph or increasing intracellular zinc levels ( , ) . in vivo, chloroquine did not reduce sars-cov viral titers in a mouse model ( ) . early clinical trials from china, suggested improved clinical outcomes of covid- when treated with chloroquine as compared to controls groups ( , ) . in a controversial french study, patients were treated with hcq and azithromycin and compared to a cohort receiving standard therapy. viral load decreased more rapidly in the hcq and azithromycin group. however, due to methodological shortcomings the results should be interpreted with caution ( ) . two large observational studies could not confirm these results ( , ) . in a retrospective analysis of , propensityscore-matched covid- patients, the rate of intubation or death was similar in those receiving hcq or standard care ( ) . moreover, a retrospective study comparing mortality of hcq/cq to standard therapy found higher mortality in those receiving hcq/cq ( ) . in order to find the optimal anti-viral dose of hcq, a rct compared two doses of cq ( mg bid for days or mg bid on day and once daily for days) in patients with covid- ( ) . the higher dose of hcq resulted in an increased lethality. furthermore, on electrocardiogram qtc-intervals > ms were found in and percent in the high-vs. low-dose group. other studies reported qtc-intervals > ms in - % of the patients treated with hcq and - % in those with azithromycin co-treatment ( ) ( ) ( ) ( ) . other adverse events of hcq and azithromycin co-treatment reported in . % were mild ( ) . in june an interim analysis of the hydroxychloroquine arm of the recovery trial revealed no benefit of hcq compared to standard of care ( ). a total of , patients had been randomized to hydroxychloroquine and compared with , patients receiving standard of care alone. no significant difference in the primary endpoint of day mortality could be found. in summary, evidence for clinical efficacy of cq and hcq in covid- is limited. therefore, antimalarial drugs should not be used in covid- outside clinical trials; accordingly, the fda revoked the emergency use authorization of these antimalarials in june and issued a cautionary statement in july ( ) . baricitinib was identified as a potential therapeutic agent for covid- employing a drug repurposing software. the suggested mechanism is specific for baricitinib, not constituting a class-effect of jaki, and works through the inhibition of virus uptake by the blockade of endocytosis ( ) . in line with these considerations, a retrospective study compared covid- patients receiving baricitinib to patients on standard therapy ( ) . case fatality and intensive care admission was significantly less frequent in the patients receiving baricitinib. additionally, a higher proportion of patients had negative nasopharyngeal swabs on discharge in the baricitinib arm. altogether, randomized controlled trials for the use of baricitinib in covid- seem worthwhile and are ongoing. colchicine, used in gout due to its effects on the chemotaxis of neutrophils and monocytes, has been proposed as a potential anti-inflammatory therapy in covid- since recruitment of neutrophils is a key factor in severe covid- pneumonia ( ) . evidence from a greek randomized, controlled trial on covid- patients suggests a benefit of colchicine treatment ( ) . however, the primary outcome measures were focused on inflammatory markers (crp and high sensitivity cardiac troponin) and overall clinical deterioration, which may have been biased by the open-label design. larger trials with mortality or invasive ventilation as endpoints are needed to support the findings of this initial trial. intravenous gammaglobulins (ivig) serve as an antiinflammatory rescue therapy in autoimmune diseases although the precise mode of action is elusive. in addition, commercially available ivig preparations contain antibodies cross-reactive to sars cov- or endogenous proteins, such as cytokines ( ) . therefore, ivig have been used in covid- ( ) ( ) ( ) . however, as no case-control studies or rct's have been published so far, the therapeutic efficacy of ivig in covid- is unknown. overall, data regarding covid- and rmd are sparse. an increased risk of developing covid- or a complicated course of covid- cannot be deduced from data on covid- or other infections in patients with inflammatory arthritis. further studies are needed to investigate the course of covid- in connective tissue diseases such as sle or vasculitis, particularly in patients with already existing organ damage and/or other comorbidities. a further point of consideration is the role of bdmards or jaki. however, more data are needed to disentangle a possible protective role of biological dmards or jaki against a possible crs as compared to an increased susceptibility for viral infections (summarized in box ). all authors critically evaluated the literature and wrote the paper. clinical features of patients infected with novel coronavirus in wuhan a novel coronavirus from patients with pneumonia in china bat-to-human: spike features determining 'host jump' of coronaviruses sars-cov, mers-cov, and beyond epidemiology, genetic recombination, and pathogenesis of coronaviruses coronavirus as a possible cause of severe acute respiratory syndrome mers: emergence of a novel human coronavirus genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding sars-cov- cell entry 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therapeutic option for deteriorating patients with coronavirus disease effect of regular intravenous immunoglobulin therapy on prognosis of severe pneumonia in patients with covid- recovery of severely ill covid- patients by intravenous immunoglobulin (ivig) treatment: a case series the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © stradner, dejaco, zwerina and fritsch-stork. this is an openaccess article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - ivw l authors: tufan, abdurrahman; avanoĞlu gÜler, aslıhan; matucci-cerinic, marco title: covid- , immune system response, hyperinflammation and repurposing antirheumatic drugs date: - - journal: turk j med sci doi: . /sag- - sha: doc_id: cord_uid: ivw l in the wuhan province of china, in december , the novel coronavirus (covid- ) has caused a severe involvement of the lower respiratory tract leading to an acute respiratory syndrome. subsequently, coronavirus (sars-cov- ) provoked a pandemic which is considered a life-threatening disease. the sars-cov- , a family member of betacoronaviruses, possesses single-stranded positive-sense rna with typical structural proteins, involving the envelope, membrane, nucleocapsid and spike proteins that are responsible for the viral infectivity, and nonstructural proteins. the effectual host immune response including innate and adaptive immunity against sars-cov- seems crucial to control and resolve the viral infection. however, the severity and outcome of the covid- might be associated with the excessive production of proinflammatory cytokines “cytokine storm” leading to an acute respiratory distress syndrome. regretfully, the exact pathophysiology and treatment, especially for the severe covid- , is still uncertain. the results of preliminary studies have shown that immune-modulatory or immune-suppressive treatments such as hydroxychloroquine, interleukin (il)- and il- antagonists, commonly used in rheumatology, might be considered as treatment choices for covid- , particularly in severe disease. in this review, to gain better information about appropriate anti-inflammatory treatments, mostly used in rheumatology for covid- , we have focused the attention on the structural features of sars-cov- , the host immune response against sars-cov- and its association with the cytokine storm. recover and severe cases (approximately %) that develop multi organ failure, primarily respiratory failure, requiring intensive care unit (icu) admission [ , ] . an efficient immune response against sars-cov- may be considered fundamental for the resolution of covid- . however, some studies have shown a significant relationship between the disease severity and the levels of proinflammatory cytokines and subsets of immune cells [ , ] . it has been suggested that during the response to sars-cov- , the immune dysregulation and the high level of proinflammatory cytokines could be the main cause of tissue injury. eventually, the exact pathophysiologic mechanism of covid- remains still largely unknown. on the basis of genomic and phylogenetic relationship, the subfamily orthocoronavirinae is classified into four genera: alphacoronaviruses, betacoronaviruses, gammacoronaviruses, and deltacoronaviruses [ ] . the alphacoronaviruses and betacoronaviruses tend to infect mammals and cause respiratory and gastrointestinal infection in humans like sars coronavirus (sars-cov), mers coronavirus (mers-cov), and sars-cov- , while gammacoranaviruses and deltacoronaviruses have the ability to infect birds in addition to mammals [ , ] . the betacoronaviruses comprise of sars-cov, mers-cov, human coronaviruses (hcovs), bat-sars-like (sl) coronaviruses, and lastly identified sars-cov- . sars-cov- possesses nonsegmented, single-stranded positivesense rna (+ssrna) with '-cap structure and '-poly-a tail which is a typical genomic structure of covs [ ] . the genome analyses have revealed that the genome sequence of sars-cov- is % and . % identical to the bat coronavirus termed batcov ratg , and sars-cov, respectively [ ] .therefore, the bat has been suggested as a natural host of sars-cov- and the transmission route of sars-cov- could be through unknown intermediate hosts. the genetic analyses of sars-cov- genomes from chinese patients demonstrated that this virus has been evolved into two main types; l type(~ %) and s type(~ %). l type is more aggressive and infectious than s type which is the ancestral version [ ] . the genome of cov contains six major open reading frames (ofrs) and numerous accessory genes. first ofrs (ofr a/b), which encompasses the two-third of viral rna, encode two large proteins of covs, polyprotein a (pp a) and pp ab. these polyproteins are divided into nonstructural proteins (nsps), responsible for viral rna replication and transcription, by virally encoded chymotrypsin-like protease ( clpro) or main protease (mpro) and papain-like protease (plpro) [ , ] . the remaining ofrs on the one-third of the genome encode major structural proteins, including spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins, all of which are crucial for the viral infectivity as seen in figure. covs possess a lipid bilayer envelope with s, m, and e proteins [ , ] . the n protein is composed of an amino (n)-terminal (nt) domain and acarboxy (c)-terminal cytoplasmic tail (ct) domain and located in the core of the viral particle. both domains bind to viral rna to form the helical nucleocapsid [ , ] . besides, sars-cov n protein acts as an antagonist to the interferon pathway by regulating the signaling and synthesis of type i interferon (ifn), which is one of the most important response in the innate immunity to viral infection [ ] . the m protein is the most abundant component of the viral envelope. the m protein contains a glycosylated nt ectodomain, three transmembrane (tm) domains, and a ct domain that binds to the nucleocapsid. the m protein gives the shape of the virus and promotes the membrane curvature and the virus assembly by interacting with the s protein and the ribonucleoprotein [ , , ] . the e protein is a small integral membrane protein, including an nt ectodomain, a tm domain, and a ct endodomain. the e protein facilitates the assembly, the budding, and the envelope formation as well as the m protein [ ] . moreover, the e protein has an ion-channel activity, contributing factor of the inflammasome activation. the animal study has shown that blocking the ion channel activity of sars-cov e protein by deletion of associated genes leads to the reduction of the edema and the level of inflammasomeactivated interleukin (il)- β, il- , and tumor necrosis factor (tnf) all of which have an important role in the progression of ards [ ] . the s glycoproteins on the surface of covs are the receptor binding proteins which are responsible for the attachment to host cells, viral-host cell membrane fusion, and the internalization of the virus [ ] . s genome of sars-cov- has less than % identical sequence with previously known sars-covs except for ratg which of s genome is . % identical with sars-cov- [ ] . besides, another genome analyses have elucidated that the sequence identity of s protein between sars-cov- and sars-cov is % and most variation has been seen at the n terminus [ , ] .the s glycoprotein consists of two domains: s domain which includes receptor-binding domain (rbd), interacting with angiotensin-converting enzyme (ace ) on the human host cells as sars-cov, and s domain which mediates virus-cell membrane fusion and viral entry [ , ] . the s domain comprises of three parts; a large ectodomain; a single tm domain, and a ct domain [ ] . the sequence of rbd from sars-cov and sars-cov- exhibits . % identity [ ] . the current study has indicated that the rbd of sars-cov- has lower affinity to ace than the rbd of sars-cov [ ] . however the result of the another study revealed that sars-cov -s protein binds ace with higher affinity than sars-cov [ ] . after attachment of sars-cov- with s protein to ace on the host cells, s protein is cleaved by host cell proteases to reveal the s domain for viral-host membrane fusion and viral entry which is coupled with tnf-α production [ , , ] . the effective antiviral responses of the host innate and adaptive immunity, including the production of various proinflammatory cytokines, the activation of t cells, cd and cd + t cells, are essential for controlling the viral replication, limiting the spread of virus, inflammation and cleaning the infected cells [ , ] . nevertheless, the tissue s protein attaches to angiotensin-converting enzyme (ace ) receptor on the host cell to entry. after the attachment, host endosomal proteases mediate the virus membrane-endosome fusion for the release of the viral genome. chloroquine (cq) and hydroxycloroquine (hcq) block the virus-receptor binding and virus-endosome fusion. besides cq, hcq, and intravenous immunoglobulin (ivig) inhibit the production of cytokines in macrophages and the antigen presentation in dendritic cells. in covid- , the count of neutrophils and leukosytes increase whereas the total count of lymphocytes cd + t cells, cd+ t cells, regulatory t (t reg) cells, memory t cells, natural killer cells, and b cells decrease. another beneficial effect of cq and hcq is increasing the activity of treg. the aberrant proinflammatory cytokine production is observed in covid- . several immunomodulatory therapies including interleukin (il)- antagonists, granulocyte colony-stimulating factor (gm-csf) inhibitor, il- antagonists, il- antagonists, and antitumor necrosis factor (tnf) agents might be used for this cytokine storm to resolve and limit the further inflammation and tissue damage (the yellow arrow indicates a decrease in the number of cells; the blue arrow indicates and increase in the number of cells). injury caused by the virus could induce the exaggerated production of proinflammatory cytokines, the recruitment of proinflammatory macrophages and granulocytes. this results in the cytokine storm (cs) termed as a macrophage activation syndrome (mas) or secondary hemophagocytic lymphohistiocytosis (shlh), thus leading to further tissue damage [ ] [ ] [ ] . data obtained from sars-cov- infected patients have shown that severe cases may be characterized by a cytokine storm inexorably progressing to ards [ ] [ ] [ ] . several features of covid- , such as the cytokine profile, serological markers, and clinical symptoms, resemble shlh most commonly triggered by viral infection [ , ] . furthermore, another important evidence is that the severity of covid- is related to the level of the proinflammatory cytokines and subsets of immune cells [ , ] . covid- possesses different levels of various cytokines and chemokines through the mild to severe stage of the disease. in sars-cov- infected patients, the retrospective analysis has demonstrated that initial plasma levels of il- β, il- ra, il- , il- , il- , ifn-ɣ, monocyte chemoattractant peptide (mcp)- , macrophage inflammatory protein (mip)- a, mip- b, granulocytecolonystimulating factor (g-csf), and tumor necrosis factor-alpha (tnf-α) are increased in patients with covid- . the further analysis has shown that the plasma concentrations of il- , il- , il- , il- , mcp- , mip- a, and tnf-α in icu patients are higher than non-icu patients [ ] . moreover, the plasma levels of il- , il- , il- , il- , and tnf-α, observed in severe infection, are prominently greater than those in nonsevere infection [ ] . few retrospective studies have revealed that the lung injury reported with murray score is strongly associated with the level of il- α, il- ra, il- , il- , il- , il- , ifn-ɣ, inducible interferon protein (ip)- , g-csf, and mcp- and these cytokines and chemokines excluding mcp- are positively related to sars-cov- viral load [ ] . the plasma level of il- , considered as a significant cytokine contributing to mas, increases both in mild and severe patient groups of covid- : severe patients have a significantly higher level of il- than mild or nonsevere patients [ , , , ] . furthermore, based on the assessment of pulmonary infiltration in patients with ards, the large area of lung injury (≥ %) is closely correlated with the increased level of il- and the subgroup of lymphocytes in the peripheral blood [ ] . during the infection, both innate and adaptive immune cells synergistically participate in the anti-viral response [ ] .the important increment in the number of neutrophils, leukocytes, and the neutrophil-lymphocyteratio (nlr) has been observed in severe covid- compared to mild cases. the prominent lymphopenia, indicating an impairment of immune system, develops in most covid- patients especially in severe ones [ , ] . therefore, it seems that neutrophils and leukocytes might reinforce the cs other than lymphocytes in covid- . the level of lymphocytes and subsets of t cells which play a significant role in the balancing of immune response varies according to the type of the virus due to possible viral pathologic mechanism. previous investigations have elicited that the total count of lymphocytes and the subset of t cells are reduced in patients with sars-cov infection [ , ] . data from recent studies have suggested that sars-cov- infection can lead to immune dysregulation through affecting the subsets of t cells. the significant alleviation of t cells is observed in covid- and more pronounced in severe cases. in patients with covid- , the level of helper t cells (cd +, cd +) and cytotoxic suppressor t cells (cd +, cd +), and regulatory t cells are below normal level while helper t cells and regulatory t cells in severe patients are remarkably lower than nonsevere patients. regulatory t cells are responsible for the maintenance of the immune homeostasis with suppressing the activation, proliferation, and proinflammatory function of most lymphocytes including cd+ t cells, cd+ t cells, nk cells, and b cells [ , ] . furthermore, the percentage of naïve helper t cells amplifies while the percentage of memory helper t cells and cd + cytotoxic suppressor t cells decreases in severe covid- [ , ] . the equilibrium between the naïve t cells and memory t cells is fundamental for mediating the efficient immune response [ ] . in addition to t cells, the reduction of b cells and nk cells are seen in covid- . another important result is the confirmed strong relationship between inflammatory markers, including esr, crp and il- and the subset of lymphocytes [ ] . however, previously it has been shown that there is no significant correlation between il- and subsets of lymphocytes [ ] . although these reports have indicated that cd+ /cd+ t cell ratio in sars-cov- infection is similar to the healthy group, the increase in this ratio and the decline of cd+ t cells and b cells are considered as a poor predictor for the assessment of post-treatment clinical follow-up [ , ] . taken together, these results indicate that sars-cov- is responsible for an immune dysregulation with the induction of aberrant cytokine and chemokine response, alteration in level of the subgroup of lymphocytes all of which might result in cytokine storm and further tissue damage. excessive inflammatory response with features of cytokine storm cause severe disease course and worsens the prognosis in covid- . undoubtedly, definitive and most effective treatments for covid- drugs would be the antiviral agents that directly target sars-cov- . considering the lack of proven antiviral agents and hyperinflammation caused by virus, antiinflammatory drugs used in daily rheumatology practice may constitute possible treatment options in treatment of covid- . following antiinflammatory treatments are potential candidates for covid- with their preclinical or limited clinical evidence. systemic corticosteroids have broad-spectrum actions on the immune system that may suppress the exuberant systemic inflammatory response that occurs in ards. severe multi-source systemic inflammation is associated with adverse outcomes, so one may think that corticosteroids may of benefit with their broad spectrum immunosuppressive effects. however, evidence has shown that use of corticosteroids delayed viral clearance in sars and mers infections, similarly they increased secondary infection rates, mortality and complications of steroid therapy in survivors of influenza pneumonitis [ ] . in a randomized controlled trial that included non-icu sars patients, "early" (< days of illness) hydrocortisone therapy was associated with a higher subsequent plasma viral load. therefore, corticosteroids should not be used early phases of disease unless there is a clear indication for their use [ ] . in sars infection, some patients showed severe inflammatory features despite reductions in viral load with subsequent seroconversion, suggestive of exuberant immune response independent of viral load [ ] . in two small observational study, use of corticosteroids did not show a survival benefit in covid- patients even increased mortality rates when used in high doses [ ] [ ] [ ] . moreover, corticosteroid use was prolonged sars-cov- rna shedding as observed in sars and mers infections [ ] . in the light of preliminary data, corticosteroids are more likely to function on inflammation-mediated lung injury and interstitial fibrosis at late-stage of ards [ ] . however, the dose, duration, and timing of corticosteroids must be individualized considering risk-benefit ratio, until results of ongoing well-designed prospective cohort studies obtained. at present, several studies are registered to assess the efficacy of corticosteroids in covid- . chloroquine (cq) and hydroxychloroquine (hcq) are -aminoquinoline derivatives that are approved by the u.s. food and drug administration (fda) for the treatment of malaria, systemic lupus erythematosus, rheumatoid arthritis (ra) and decades of experience in use of these disorders. they are also used in q fever and porphyria cutanea tarda. hcq has a better side effect profile than cq and is strongly recommended for the long-term treatment of lupus unless the occurrence of a severe side effect. hcq does not increase the risk of infections and has lipidlowering, antithrombotic and antineoplastic properties [ ] . in adult rheumatic diseases, the recommended dosage is to mg ( to mg base) with a cut off of . mg/kg/daily, is usually well-tolerated. the most dreaded complication is retinal toxicity which rarely occurs in long term use (five or more years). elders with kidney failure or tamoxifen users have an increased risk of retinal toxicity. cq and hcq may prolong qt interval which does not require routine ecg monitorization in recommended doses. at higher doses, these drugs have a potential risk of fatal arrhythmia or if combined with qt prolonging medications as well as those with cardiac diseases [ ] . other acute notable toxicities of -aminoquinoline derivatives are allergic reactions and neuropsychiatric events [ ] . very rarely they may cause cardiomyopathy which is thought to occur due to lysosomal accumulation with their chronic use [ ] . oral absorption of cq and hcq are very good and are excreted primarily by urine. the half-lives of cq and hcq are prolonged, ranging between and days and have a large volume of distribution, which allows for sustained sequestration in the tissues. tissue concentrations may differ with being highest in the lung tissue about -fold of plasma concentrations as shown in animal models [ ] . it has been known that cq and hcq have antiviral activity including hepatitis b, hiv, h n and zika virus [ ] . antiviral activity of hcq was first observed in hiv and the hepatitis b infections in the early s. small studies showed its favorable efficacy in combination regimens of hbv, hcv and hiv infections. cq and hcq are thought to exert antiviral activity via multiple mechanisms. first, these agents interfere with glycosylation and proteolytic maturation of proteins. by interfering with terminal glycosylation of ace , the cellular receptor for s protein, both agents block virus-receptor binding and cell entry [ , ] . second, cq and hcq both are weak bases and concentrated in acidic, low-ph organelles, such as endosomes, golgi vesicles, and lysosomes, increasing their ph [ ] . endosomal acidification is required for the activation of endosomal proteases responsible for the initiation of coronavirus/endosome fusion that releases viral particles into the infected cells [ ] . therefore, cq and hcq inhibit viral release into the host cell by blocking endosomal acidification. third, hcq inhibits protein glycosylation and proteolytic maturation of viral proteins. budding of the sars-cov occurs in the golgi apparatus and results in the incorporation of the envelope spike glycoprotein into the virion [ ] . studies have shown a resulting accumulation of noninfective viral particles of hiv, or an inability of viral particles to bud out of the host cell, reducing spread of infection. finally, antimalarial drugs act as protecting hemoglobin against invasion by malaria parasites with their effects on heme metabolism. there is abnormal heme metabolism in covid- patients. chloroquine phosphate competes with the porphyrin and binds to the viral protein, thereby inhibits the viral protein's attack on heme or binding to the porphyrin. according to a study, cq could prevent orf ab, orf a, and orf to attack the heme to form the porphyrin and inhibit the binding of orf and surface glycoproteins to porphyrins to a certain extent . in rheumatic diseases, the exact mechanism of action of hcq and effects on the immune system are largely unknown. however, beside interfering with lysosomal activity and autophagy as mentioned above, cq and hcq interact with membrane stability and alter signaling pathways and transcriptional activity, which can result in inhibition of cytokine production and modulation of certain costimulatory molecules. both drugs inhibit antigen presentation in dendritic cells, cytokine production in macrophages, and calcium, toll-like receptor (tlr) and cgas-sting signaling in b, t and other immune cells [ , ] . the major proposed immunomodulatory mechanisms of cq and hcq are the following: inhibition of cytokine production and release by t cells: il- , il- , il- , and il- , tnf-α and ifn-γ, reduced levels of chemokines, ccl and cxcl , inhibition of micro-rna expression, decreased th -related cytokines, increased in treg activity and upregulated levels of ifn-α and il- and il- , inhibition of cytotoxic t cell and self-reactive cd + lymphocyte activities, decreased dna, rna and protein synthesis in thymocytes [ ] . antimalarials have ironbinding and hydroxyl radical scavenging actions which may of benefit considering disrupted heme metabolism and oxidative stress [ ] . a strong antiviral activity of cq by using sars-cov- -infected vero cells has been documented [ ] . in a physiologically based pharmacokinetic models (pbpk) for each drug, hcq showed five-fold more potency than cq in vitro [ ] . in both studies, antiviral activity is dosedependent and can be achieved with use of routine safe dosages. although several in vitro studies report antiviral activity of cq and hcq against sars-cov- , in vivo data are promising but have considerable limitations. an expert consensus group in china suggested that cq may improve lung involvement evaluated at imaging with a shortening of the disease course [ ] . in another highly debated open-label, nonrandomized, controlled trial, a small number of patients with covid- were treated with hcq. nasal sars-cov- carriage was found to be lower on sixth day following hcq treatment as compared to non-treated patients [ ] . a recent, multicenter, openlabel, randomized controlled trial from china, did not find sars-cov- negative conversion rates between high dose hcq and standard of care in hospitalized patients, with reporting more rapid resolution of symptoms, normalization of crp and lymphopenia, however outcome data on icu need and mortality was not reported here [ ] . in another study comprising hypoxic pneumonia patients from france, hcq did not avert icu admission or mortality [ ] .the dilemma on clinical utility of cq and hcq in covid- will be solved by well-designed clinical trials in near future. in several countries, despite the weakness of clinical studies, based on strong preclinic scientific rationale and multimodal antiviral and immunomodulatory actions, cq and hcq are currently recommended for the treatment of covid- . optimal dosing is uncertain and there are several dosing regimens ( mg to as high as mg daily) as is the treatment durations ( - days). hcq was found to be more potent than cq in vitro and better tolerated. based on pbpk models, a loading dose, mg twice a day (bid), of hcq is given orally, followed by a maintenance dose of mg bid for days is the most commonly recommended strategy for sars-cov- infection, as it reached three times greater potency of cq when given mg twice daily for days in advance [ ] . all hcq recommended doses for covid- are above the routine doses used in rheumatic diseases, hence potential adverse events could be experienced also in this brief standing treatment. in vincent et al. reported that cq has strong antiviral effects on sars-cov- infection of primate cells with the use of drug either before or after exposure to the virus, suggesting both prophylactic and therapeutic use [ ] . animal models have shown that prophylactic use of cq may have an additional survival benefit in enteroviral infections [ ] . however, there is still no robust evidence for the use of cq or hcq for pre-or postexposure prophylaxis of covid- . there are several trials underway to evaluate the efficacy of cq or hcq in the prophylaxis of high-risk individuals (nct , nct ). ivig is a blood product containing polyclonal immunoglobulin g isolated and pooled from healthy donors used to treat immune thrombocytopenic purpura (itp), kawasaki disease and various inflammatory neurologic and myositis syndromes. it has immunomodulatory functions with unknown mechanism of action. one of the proposed mechanisms is the interaction of igg-fc with fc gamma receptors located on almost all immune cells, resulting in pleiotropic functional consequences including the expansion of regulatory t cell population, phagocytosis, antibodydependent cellular cytotoxicity (adcc), immune cell differentiation and maturation, apoptosis, expression of proinflammatory cytokines, and antigen-presentation [ ] . previous studies on sars and mers, found that ivig therapy was effective thus proposing high-dose ivig as an option for severe covid- patients [ ] . there are a few covid- cases which reported efficacy of high dose ivig [ ] . however, its high cost and limited supply restrict its general use. inferred from rheumatic diseases, covid- patients with pregnancy, secondary infections, marked thrombocytopenia, muscular, myocardial and neurologic manifestations would be better candidates for ivig treatment. there are several studies already registered for its use in covid- . il- receptors ubiquitously expressed in almost all immune cells, and il- acts as a master player inducing proliferation and differentiation of immune cells. in healthy individuals, the il- levels in circulation are extremely low and are in the range of - pg/ml, marked elevations reported in many inflammatory conditions including cytokine release syndrome [ ] . several therapeutic agents have been developed inhibiting the cytokine itself, the signaling via the il- receptor, or its postreceptor downstream signaling pathways (jak/stat). tocilizumab, sarilumab, siltuximab are il- antagonists with different pharmacologic properties. tocilizumab is approved for the treatment of ra, juvenile idiopathic arthritis, giant cell arteritis, cytokine release syndrome, and idiopathic multicentric castleman's disease (imcd), whereas siltuximab received approval for imcd and sarilumab for ra only [ ] . covid- patients have high plasma il- levels, especially those with more severe disease presentation [ ] . il- production can be stimulated by sars-cov- itself or by stimulation of other immune cells [ ] . indeed, it has been shown that during covid- , cd + t lymphocytes are rapidly activated to differentiate into pathogenic th cells, generating gm-csf and other proinflammatory cytokines, which further induced activation of monocytes with high expression of il- [ ] . in clinical view, there is striking correlation between serum il- levels and sars-cov- rnaaemia, which strongly indicates worse outcome . besides the cytokine storm, recent studies in experimentally infected animals suggest a crucial role for virus-induced immunopathological events in causing fatal pneumonia after coronavirus infections [ ] . hence, blocking il- would potentially reduce the detrimental immune response caused by sars-cov- . as are the other covid- treatments, there is no robust evidence to routinely suggest il- antagonists. a small clinical trial in china examined the effectiveness of tocilizumab in patients who met the criteria for severe or critical covid- , including respiratory failure, requiring mechanical ventilation, shock, or admission to the icu with multiple organ failures. tocilizumab improved hypoxemia, fever, lymphopenia, crp, and lung infiltration in most of the patients treated, without serious adverse events . recently, the favorable outcome of a patient with limited cutaneous systemic sclerosis under treatment with tocilizumab was reported [ ] . since there is an urgent need for the severe covid- treatments, based on these limited data, tocilizumab is included in the treatment algorithms of many countries. the dose and timing for infusions are not determined yet. numerous studies are ongoing to assess the efficacy of tocilizumab, sarilumab, and siltixumab in several countries. current practice is to give tocilizumab - mg/ kg (maximum mg) as single infusion. after careful evaluation of disease severity and response to initial treatment a repeat infusion can be administered at the same dose after - h. il- antagonists increase the risk of infections, therefore must be used in severe patients and at the end of the high viral load phase of covid- , along with antiviral treatments [ ] . there are other side effects including intestinal perforation and opportunistic infections. therefore, it is prudent to monitor patients for potential side effects. [ ] . jak inhibitors are currently approved for the treatment of ra and psoriatic arthritis and their use in other inflammatory disorders are continuously growing [ ] . many proinflammatory cytokines involved in cytokine storm of covid- might be inhibited by jak inhibitors. besides above mentioned common properties of jak inhibitors, baricitinib may block ap- -associated protein kinase (aak ) and cyclin g-associated kinase (gak) which are host kinases that regulate viral endocytosis, according to an artificial intelligence search of viral characteristics of sars-cov- . this effect is only restricted to baricitinib among other jak inhibitors and it may block viral entry and assembly of virus particles into pneumocytes in therapeutic doses used in ra [ ] . however, these hypothetical views merit further evidence for clinical use both for cytokine storm and covid- . currently, baricitinib (nct , nct , nct ), tofacitinib (nct ) and ruxolitinib (nct ) studies are ongoing. nod-like receptor family pyrin domain-containing (nlrp ) is a critical inflammasome in acute protection of the body against a wide variety of noxious stimuli, including rna viruses [ ] . nlrp activates caspase- , a molecule responsible for the activation and exuberant release of il- β and il- . previously sars-cov has been shown to induce nlrp by its ion channel-forming m protein and orf b [ ] . it has been shown that sars-cov- induces many cytokines including il- family [ , ] . il- family are pleiotropic cytokines, have roles in inflammation, hematopoiesis, and fibrosis. il- β and tnf-α promote vascular permeability and leakage. both il- β and il- fuel cytokine storm and mas and il- cytokines (except il- ) can be successfully inhibited by anakinra [ ] . anakinra is a recombinant antagonist of human il- and approved for the treatment of ra and certain autoinflammatory disorders with recommended doses of - mg/kg/day with a maximum daily dose of mg/kg [ ] . in terms of sepsis and mas, a previous, highly cited phase iii trial, anakinra did not improve -day survival rate in sepsis patients and terminated early [ ] . however, reanalysis of data from this trial suggested significant improvement in survival in patients with hepatobiliary dysfunction and disseminated intravascular coagulation (dic) [ ] . anakinra was administered intravenously at mg/kg/hr for h continuously in this study without safety concerns. this dose is extremely higher than those used in rheumatology routine which warrants careful monitoring. there are several anakinra studies registered for covid- , testing mg daily subcutaneous injection for days to - mg/day intravenous for - days (nct , nct ). colchicine has been approved for gout and familial mediterranean fever. in recent years, colchicine has attracted attention in the management of cardiovascular diseases by suppressing their inflammatory component [ ] . its mechanism of action is thought to be the inhibition of tubulin polymerization and microtubule generation and, possibly, effects on cellular adhesion molecules, inflammatory chemokines, and the inflammasome. colchicine may inhibit activation of nlrp inflammasome and additionally may inhibit directly the synthesis of tnf-α and il- [ ] . trials investigating the efficacy of conventional therapeutic doses of colchicine have been registered for the treatment of covid- (nct , nct , nct ). tnf-α is one of the most potent proinflammatory cytokines with broad spectrum of actions. marked elevations reported in many inflammatory conditions including cytokine release syndrome. serum tnf-α levels found elevated in covid- patients with being more pronounced in more severe patients [ ] . sars-cov viral spike protein is able to modulate tnf-α-converting enzyme (tace)-dependent shedding of the ace ectodomain, required for the viral entry which is coupled to tnf-α production [ ] . therefore, it is hypothesized that the use of tnf inhibitors might be effective in blocking viral entry and detrimental effects of exuberant tnf-α, as shown in preclinical studies on severe respiratory syncytial virus and influenza infections [ ] . anti-tnfs enhance the risk of bacterial, viral and fungal infections. therefore, their use in covid- must be supported with preclinical studies. one of the cytokines found abundant in covid- patients is il- and found associated with severe lung inflammation [ ] . il- has wide-ranging proinflammatory effects on induction of cytokines; il- β, il- , tnf-α; growth factors, g-csf; chemokines; and matrix metalloproteinases. in a mouse model, it was found that h n cause acute lung injury in an il- -dependent manner. it has been postulated that blocking this cytokine may be effective in reducing sars-cov- related organ damage [ ] . as mentioned, gm-csf is one of the key molecules involved in cytokine storm which is excessively released in covid- patients [ ] . blockage of this growth factor may halt immunopathology caused by virus. mavrilimumab is a gm-csf inhibitor developed for the refractory ra [ ] and a new trial is investigating its efficacy in covid- (nct ). mmf is widely used for the treatment of severe manifestations of connective tissue disorders and vasculitis syndromes. mycophenolate exhibited strong antiviral effects on sars-cov and mers-cov as demonstrated in vitro studies, with its interaction with viral proteases [ ] . a small clinical study reported efficacy of mmf in combination with ifn-β on mers patients [ ] . however, considering strong immunosuppressant effects of mmf, it is likely to cause more harm than benefit in covid- patients. an association between ibuprofen and worse outcome in covid- patients was speculated, with very weak evidence [ ] . another nsaid, indomethacin, reported to have direct antiviral effect on sars-cov by interfering with viral rna synthesis, independent of cyclooxygenase inhibition in an in vitro study. a registered trial, currently recruiting patients to determine efficacy of naproxen for its potential interaction with viral nucleoproteins (nct ). therefore, although evidence is limited, indomethacin or naproxen could be preferred over other nsaids when indicated [ ] . excessive inflammatory response with features of cytokine storm cause severe disease course and worsens the prognosis in covid- . undoubtedly, drugs that directly target sars-cov- would be the most effective treatments for covid- . there are hundreds of trials ongoing to find effective treatments for covid- both targeting virus and consequent hyperinflammation including newly developed agents on phase studies or drugs that are approved for other indications. until an effective treatment is found, drugs that are used in daily rheumatology practice may constitute potential treatment options in covid- patients not only by their antiinflammatory effects but also with some of their inherent antiviral properties. hence, inclusion of rheumatologists/ immunologists into covid teams would improve patient outcomes. the 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Éric; louf-durier, aurore; lachand, raphaël; murgier, martin; page, dominique; vermesch, régine; thierry, guillaume; delavenne, xavier title: towards optimization of hydroxychloroquine dosing in intensive care unit covid- patients date: - - journal: clin infect dis doi: . /cid/ciaa sha: doc_id: cord_uid: ikr eeov hydroxychloroquine (hcq) appears to be a promising treatment for covid- . however, all ongoing clinical trials with hcq use different dosing regimens, resulting on various concentrations pk studies are therefore needed to define the optimal dosing regimen. since december , an outbreak of covid- due to sars-cov- virus has spread from china. on march , , the who described the global covid- situation as a pandemic. as of march , , no treatment has demonstrated clinical efficacy against covid- . however, several treatment strategies are being considered and evaluated in numerous clinical trials. among these strategies, the use of hydroxychloroquine (hcq) appears to be a promising option, although only limited evidence is available at the present time. above all, hcq has the advantage of being widely available to a large number of patients. hcq is a well-known drug, effective in the treatment of malaria and autoimmune diseases. more recently, an in vitro antiviral effect has been demonstrated on sars-cov- . recent work by wang et al. showed that a chloroquine concentration of . mg/l decreased viral load by % in a cell model [ ] . apart from the ongoing clinical evaluation of the efficacy of hcq, little information is available concerning the modalities of administration of this drug for intensive care unit (icu) patients, especially in the context of covid- . hcq can be responsible for adverse events and probably an increased incidence of adverse events in the case of inappropriate dosing regimens. one of the most serious adverse events in this population is cardiac toxicity, characterized by prolongation of the qt interval, which can lead to arrhythmia in patients at risk. hcq also has very particular pharmacokinetic (pk) properties that require certain precautions. it exhibits strong tissue tropism, particularly for the kidney and liver, with a long half-life (several weeks). the risk of overdose is higher in the icu population with impaired renal and/or hepatic function than in other populations. to date, hcq pk parameters have been estimated from studies in patients with rheumatoid arthritis or lupus [ ] or healthy volunteers [ , ] . however, physiological changes in infused, ventilated patients with multiple organ failure may modify hcq pk parameters. for these reasons, we conducted a prospective study to evaluate the pk properties of hcq in icu covid- patients. this prospective cohort study was conducted at saint etienne university hospital (france) between / / and / / . the study was approved by the institutional review board [irbn /chuste]. all consecutive patients with laboratory-confirmed sars-cov- infection treated by hcq in the critical care unit were included. m a n u s c r i p t treatment patients received mg of oral hcq, three times daily, as suggested by a recent study [ ] . blood samples for determination of drug levels were drawn as part of routine care, with the decision to perform therapeutic drug monitoring based on medical guidance. hcq trough levels > mg/l and < mg/l were considered to be therapeutic [ , ] . the medical team received all results in real time to allow for dose adjustments, as necessary. blood samples were drawn from an arterial catheter and transferred to ethylenediamine tetraacetic acid-containing tubes. hcq blood levels were analyzed using a validated liquid chromatography-mass spectrometry method. briefly, blood samples ( µl) were spiked with µl hydroxychloroquine-d prepared ( . mg/l) in % trichloroacetic acid, and µl zinc sulfate was used for liquid-liquid extraction. the lower limit of quantification was . mg/l. to more clearly understand hcq pk and the effect of the dosing regimen, a simulation based on a pk population study in patients with rheumatoid arthritis was performed [ ] . from the variance-covariance matrix of the estimated pk parameters, monte carlo simulations were performed using mlxplore (lixsoft). a total of patients were generated receiving different dosing regimen based on ongoing clinical trials. thirteen patients were included in this prospective pk study. the median age of patients was years [ - years]. most patients were male ( %). median body weight was . kg [ - kg] and % were considered to be obese (body mass index > kg/m ). median renal function estimated by the ckd-epi formula was . ml.min - [ - ]; . % of subjects presented moderate or severe renal failure. twelve patients were mechanically ventilated. one patient was treated by ecmo and another patient was treated by renal replacement therapy. m a n u s c r i p t a total of blood levels were recorded and used for the analysis and samples were below the limit of quantification of the assay (figure ). only / patients ( %) achieved the supposed minimum therapeutic level of mg/l and / patients exceeded a concentration of mg/l. the mean time to reach the minimum therapeutic level was . days [ - . days]. four patients underwent dose de-escalation and subsequently received mg of hcq twice daily. hcq was withdrawn in two patients: due to qt interval prolongation ( to ms and to ms) on day and with hcq blood levels of . mg/l and . mg/l, respectively. the various dosing regimens used in currently recruiting clinical trials were simulated in order to determine the variability of hcq pk parameters ( figure ). treatment a achieved target levels on day . treatment b achieved target levels but resulted in blood levels exceeding mg/l on day . treatment c rapidly achieved target levels ( st dose), but blood levels exceeded mg/l after hours. treatment d achieved target levels after . days and a level of mg/l after days finally, we proposed treatment d to rapidly achieve target levels without exceeding m/l. to our knowledge, this is the first study to describe hcq pk in icu patients. in this study, we demonstrated that the mg three times daily dosing regimen is inappropriate to reach a supposed target blood level of - mg/l in this population. using this dosing regimen, the mean time to reach therapeutic levels was more than days and only % of patients reached target levels with this dosing regimen. this result is not surprising in view of the pk properties of hcq [ ] . hcq presented marked pk variability with a very long half-life ( to days), particularly due to large distribution into blood and tissues. steady-state concentrations are therefore achieved within weeks and vary from individual to individual with the same dosing regimen. however, icu patients present certain characteristics that can affect the pk of drugs. for example, the presence of ecmo may affect the already altered pk of icu patients by further increasing the volume of distribution, causing changes in clearance, and causing adsorption or absorption into the circuit [ ] . in the one patient treated by ecmo in this study, hcq blood levels increased more slowly than in the other patients. the therapeutic level of hcq in covid- patients has not yet been established. some in vitro and in silico studies have reported a virustatic effect of chloroquine and hcq and estimated the therapeutic blood level from ec , ranging from . to . mg/l [ , ] . the toxic hcq concentration has not been established, although a number of arguments suggest that a concentration of mg/l should not be exceeded to avoid ocular toxicity. however, the most dreaded adverse effect for covid- patients is cardiac toxicity. to our knowledge, the m a n u s c r i p t relationship between cardiac toxicity and hcq blood levels has not been determined, but it can be assumed that excessive hcq exposure is likely to be harmful. in this study, two patients experienced cardiac toxicity at variable hcq blood concentrations. no guidelines for administration of hcq are currently available. using physiologicallybased pharmacokinetic models, yao et al. suggested a dosage of mg twice daily for day, followed by mg twice daily for another days [ ] . this regimen could constitute an appropriate option, although the results of our study suggest that mg once daily on the first day can more rapidly reach therapeutic levels in icu patients. all of the clinical trials concerning the therapeutic use of hcq in covid- registered in clinicaltrials.gov (on march ) are using different dosing regimens. based on our simulations, we demonstrate that some of these dosing regimens will fail to reach therapeutic levels, while others will probably induce levels higher than mg/l. this work strongly suggests that the hcq dosing regimen should be optimized on the basis of pk data available in special populations. there is an urgent need for health agencies to clarify the standard dosing regimen of hcq in order to have comparable data across clinical trials, and to avoid dubious efficacy or toxicity results due to pk profiles. based on this prospective study, we demonstrate that pk studies are needed to define the optimal dosing regimen for icu covid- patients. based on our simulations, we propose a loading dose of mg once daily on day , followed by mg twice daily for days. therapeutic drug monitoring should be used to personalize the optimal dosing regimen. further pk and pd (virological) studies are also warranted. m a n u s c r i p t red dots represent hcq blood levels for a dosing regimen of mg three times daily, triangles represent hcq blood levels after discontinuation of treatment, circles represent hcq blood levels for a dosing regimen of mg twice daily. the green shaded zone represents the % simulation interval obtained with the model of carmichael et al for hcq mg three times daily [ ] . the brown line represents treatment a: mg once daily for days (nct ). the blue line represents treatment b: mg twice daily for days (nct ). the pink line represents treatment c: mg loading dose followed by mg hours later and then mg once daily for days (nct ). the red line represents treatment d: mg three times daily for days [ ] . the green line represents the recommended treatment e: mg loading dose followed by mg twice daily for days; the green shaded zone represents its % simulation interval. m a n u s c r i p t remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro population pharmacokinetics of hydroxychloroquine in patients with rheumatoid arthritis bioavailability of hydroxychloroquine tablets in healthy volunteers pharmacokinetics and bioequivalence study of hydroxychloroquine sulfate tablets in chinese healthy volunteers by lc-ms/ms. rheumatol ther in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) hydroxychloroquine blood levels in systemic lupus erythematosus: clarifying dosing controversies and improving adherence steady-state pharmacokinetics of hydroxychloroquine in patients with cutaneous lupus erythematosus pharmacokinetics and extracorporeal membrane oxygenation in adults: a literature review hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial m a n u s c r i p t key: cord- -a k lma authors: klimke, a.; hefner, g.; will, b.; voss, u. title: hydroxychloroquine as an aerosol might markedly reduce and even prevent severe clinical symptoms after sars-cov- infection date: - - journal: med hypotheses doi: . /j.mehy. . sha: doc_id: cord_uid: a k lma covid- is a new coronavirus disease first described in december . this respiratory illness is severe and potentially fatal. severe cases make up to %, lethality ranges between . and more than %. what is urgently needed is an efficient pharmacological treatment for the treatment of severe cases. during the infection of alveolar epithelial cells of the lung, the ace receptor has a central function. the antimalarial drugs chloroquine phosphate (cq) and hydroxychloroquine (hcq) impair in vitro the terminal glycosylation of ace without significant change of cell-surface ace and, therefore, might be potent inhibitors of sars-cov- infections. starting inhibition at . µm, cq completely prevented in vitro infections at µm, suggesting a prophylactic effect and preventing the virus spread hours after infection. in a first clinical trial, cq was effective in inhibiting exacerbation of pneumonia, improving lung imaging findings, promotion of virus-negative conversion, and shortening the disease. in addition, hcq, which is three times more potent than cq in sars-cov- infected cells (ec . µm), was significantly associated with viral load reduction/disappearance in covid- patients compared to controls. theoretically, cq and hcq could thus be effectively used in the treatment of sars-cov pneumonia. from a pharmacological standpoint, however, the major problems of oral treatment with these drugs are possible severe side effects and toxicity. concretely, this relates to (a) the inconsistent individual bioavailability of these drugs at the alveolar target cells, depending on intestinal resorption, hepatic first-pass metabolism and accumulation in liver, spleen and lung, and (b) the need for a relatively high concentration of - µm at the alveolar surface. therefore, we propose in a first dose estimation the use of hcq as an aerosol in a dosage of - mg per inhalation in order to reach sufficient therapeutic levels at the alveolar epithelial cells. by using a low-dose non-systemic aerosol, adverse drug reactions will markedly be reduced compared with oral application. this increase in tolerability enables a broader use for prevention and after contact with an infected person, which would be an advantage especially for the high-risk, often multi-morbid and elderly patients. empirical data on self-medication with a one-week aerosol application by two of the authors is presented. inhalation was well tolerated without relevant side effects. the -ncov pandemic is rapidly spreading with currently more than a million reported cases and . deaths worldwide ( ). while the majority of infections with covid- are asymptomatic or show a mild course ( , ), up to % of patients develop a severe and potentially fatal respiratory illness with an estimated lethality ranging between . and more than % ( ). currently, as no effective antiviral pharmacological treatment is available, lethality seems to depend on individual risk factors including age, gender and hypertonia and regarding the course of the disease in cases of severe pneumonia, on the availability of large-scale intensive care units and extracorporeal membrane oxygenation (ecmo) ( , ). regarding pharmacological treatment, several clinical trials have recently been initiated, e.g. with lopinavir/ritonavir ( ), remdesivir peptide (ek ) ( ) and interferon alpha ( ). the latter originally being used for the treatment of hepatitis b (novaferon, beijing genova biotech). however, the clinical efficacy of these new drugs for covid- has not been proven and their safety has not been extensively tested. further, cq and hcq have been discussed as promising, cost-effective and easily available agents in the treatment of covid- ( - ). these are relatively old agents, introduced primarily for prophylaxis and treatment of malaria and also for rheumatic diseases ( ). in vitro cell cultures, hcq seems to be a more potent inhibitor of infection with sars-cov- than chloroquine ( , ). both drugs, taken orally, may cause severe side effects ( ) ranging from psychiatric symptoms ( ) to ocular toxicity ( ) to myocardial dysfunction ( ) , even at only low overdosage ( ) . these side effects may be severe and, therefore, limit widespread application in vivo, at least at the present time. as drosten ( ) pointed out, a major limiting factor of oral hydrochloroquine treatment lies in the fact that the drug may not reach the target, i.e. the surface of the lungs. instead, it is metabolized internally, requiring treatment dosages which, in turn, may cause toxic and sometimes even lethal side effects. here, we propose a different approach to the investigation of the effectiveness of the hydrochloroquine drug that would imply a direct application of the active agent to the respiratory tract. we hypothesize that hcq especially as an aerosol application will prevent or at least markedly reduce the replication rate of the sars-cov- virus in the early phase of the infection and subsequently substantially lower the number of severe pneumonias and casualties. this hypothesis is new since the major assumption in ongoing clinical studies and actual recommendations is that hcq and cq should be used in oral application form in patients with severe covid- pneumonia and only when other treatment strategies have failed. however, the typical clinical course of this infection suggests that the virus load in the respiratory tract increases stepwise starting with mild symptoms and ending in up to % of patients with severe and potentially life-threatening pneumonia ( ). therefore, the treatment with a drug which inactivates the cell receptor for the virus should start after exposition with high risk, e.g., when one person was infected very recently with the virus or is in the early phase of the disease. moreover, our hypothesis differs from the standard recommendation to try hcq/cq in a late phase of the disease when other antiviral drugs failed. we believe that a respiratory virus infection should be treated very early because the severe acute respiratory syndrome is caused by ion channel activity of the viroporin a which activates the nlrp inflammasome ( ) . unfortunately, as of now, there is no evidence yet that hcq/cq has any inhibiting effect on this inflammasome activation. the idea to propose application of hcq/cq as aerosol is generated because one major objection against the clinical efficacy of these drugs is that they have to be administered in relatively high oral dosages. such high dosages may have several toxic side effects, strongly limiting their utilizability as preventive treatment. an aerosol application of drugs which are primarily intended to act on the respiratory system is well established for several drugs, e.g. in the treatment of asthma with corticosteroids, e.g. budesonide ( , ) and beta mimetica, e.g. fenoterol ( ) , and in the early treatment of influenza (during the first hours) with neuraminidase blockers like zanamivir ( ) . moreover, there are reports of undergoing clinical studies of aerosol interferon alpha (novaferon) for treatment of covid- ( ) leading us to advocate a clinical trial to evaluate also hcq/cq in this application form. it has been demonstrated that the sars-cov- virus enters ace -expressing cells including alveolar epithelial cells of the lung and in other organs ( ) ( ) ( ) , which has been shown before also for sars-cov- . therefore, during the infection of alveolar epithelial cells of the lung, the ace receptor has a central function ( ) . the antimalarial drugs cq and hcq impair the terminal glycosylation of ace without significant change of cell-surface. ace increases the local ph value, which reduces the activity of cathepsin l needed for hydrolysis of the viral s protein and might influence the generation of pro-inflammatory cytokines ( ) . therefore cq might be a potent inhibitor of sars-cov infection ( , , ) . in vitro, cq, starting with . µm inhibited and, moreover, completely prevented sars-cov infections in cell cultures at µm, suggesting a prophylactic effect and preventing the virus spread hours after infection. hcq is threefold more potent than cq in sars-cov- infected cells, resulting in the clinical recommendation to treat orally with mg at the first day and at mg on the following four days ( ) . two major points of action are postulated for cq/hcq in the treatment of covid- : ( ) they perturb the terminal glycosylation of the ace protein and inhibit cell-binding from a pharmacological standpoint, one major problem of oral treatment with chloroquine phosphate or hydroxychloroquine is that relatively high concentrations between - µm are needed to inhibit the glycosylation of the soluble ace receptor at the alveolar surface in the lung. therefore, the bioavailability of these drugs in the pulmonic target regions depends individually on intestinal resorption, hepatic first-pass metabolism and diffusion from the blood to the alveolar cells. this could in part be compensated by higher oral dosages, which are, however, limited by adverse and potentially toxic side effects. another problem can be that cq and hcq are accumulated in the liver, spleen, kidney, lungs and other organs. to times of the chloroquine plasma concentration is found in parenchyma cells and times in pigmented cells. around % of it is bound to plasma proteins (pharmaceutical product information). from a clinical standpoint, potential fatal arrhythmias and drug-induced sudden cardiac death in rare cases or in case of overdose can be a resulting problem. our hypothesis prompts to be tested in controlled randomized clinical studies. currently, an aerosol preparation of hcq is not commercially available. therefore, experimental data is needed to examine whether it is possible to achieve and maintain an antiviral concentration of - µm in the alveolar cells which has been proven effective in vitro. in a first approximation, one can assume that in the normal adult, the lungs weigh approximately . g which is . of the whole body weight of kg. although it is difficult to predict in the lung the effects of blood perfusion and on the other hand accumulation of hcq, it seems reasonable to substitute - mg since hcq/cq is an old drug with a known profile of side effects, also for much higher dosages, it might also be appropriate to perform open pilot studies with higher numbers of cases and compare (in a non-randomized study) the clinical course and final result in patients with similar symptoms but without hcq inhalation. for objective testing of the preventive potential, persons at risks like therapeutic staff following direct unprotected contact with corona patients as well as relatives of patients who have tested positive for sars-cov- and who are living in the same household should inhale hcq in comparison to placebo during the incubation period of - days without symptoms. we would predict that the number of subsequent infections of the contact persons will be significantly lower in the verum group. to test whether hcq as aerosol lowers the severity of the course, a randomized comparison should be performed including patients with beginning mild symptoms who were actually tested sars-cov- positive in order to evaluate the effect on clinical symptoms including fever, cough, beginning pneumonia and other complications. finally, hcq as aerosol could be tested preferentially as co-medication in more severely ill patients where an indication for oral hcq treatment is given. the prediction would be that those with the co-medication will have a better treatment outcome than those in oral monotherapy with hcq. since there is currently no commercial aerosol application available, two of the authors (a.k., clinical director and b.w., medical student working at an icu with acute covid- patients) decided to test tolerability and possible side effects of the inhalation of hcq, starting with a dosage of mg b.i.d (dissolved in ml of . % nacl solution and using a commercial nebulizer generating a droplet size < µm which can reach the alveolar space) which was stepwise increased up to mg per day over a period of one week. inhalation was well tolerated without relevant side effects. the only observation was after days the feeling of a transient bitter taste in the mouth which lasted - hours after the inhalations. if our hypothesis is true, hcq as an aerosol might not only reduce the side effect potential of the oral application form but can also be clinically used as an efficient antiviral agent in the early phase of covid- and eventually lower the rate of severely ill patients and fatalities. this might have great relevance for further prognosis and treatment of this often fatal disease. ( ) https://coronavirus.jhu.edu/map.html, downloaded april , ) ( are the current recommendations for chloroquine and hydroxychloroquine screening appropriate revisiting the cardiotoxic effect of chloroquine chloroquine serum concentration and side effects: evidence for dose-dependent kinetics severe acute respiratory syndrome coronavirus viroporin a activates the nlrp inflammasome adjunctive corticosteroid therapy for patients with severe novel coronavirus pneumonia (covid- ): a randomized controlled trial effect of inhaled formoterol and budesonide on exacerbations of asthma effect of aerosol and oral fenoterol on histamine and methacholine challenge in asthmatic subjects pharmacokinetics of zanamivir after intravenous, oral, inhaled or intranasal administration to healthy volunteers outcome reporting from protocols of clinical trials of coronavirus disease (covid- ): a review covid- : perspectives on the potential novel global threat ace receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia therapeutic effect of hydroxychloroquine on novel coronavirus pneumonia (covid- a multiscale and comparative model for receptor binding of novel coronavirus and the implication of its life cycle in host cells new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid- ? chloroquine as a potential treatment and prevention measure for the novel coronavirus: a review in vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine severe acute respiratory syndrome coronavirus (sars-cov- ) and corona virus disease- (covid- ): the epidemic and the challenges bioavalibility of hydroxychlorquine tablets in patients with rheumatoid arthritis the authors confirm that they have no conflict of interest. key: cord- - jqgsv authors: singh, awadhesh kumar; singh, akriti; shaikh, altamash; singh, ritu; misra, anoop title: chloroquine and hydroxychloroquine in the treatment of covid- with or without diabetes: a systematic search and a narrative review with a special reference to india and other developing countries date: - - journal: diabetes metab syndr doi: . /j.dsx. . . sha: doc_id: cord_uid: jqgsv background and aims: no drugs are currently approved for coronavirus disease- (covid- ), although some have been tried. in view of recent studies and discussion on chloroquine and hydroxychloroquine (hcq), we aimed to review existing literature and relevant websites regarding these drugs and covid- , adverse effects related to drugs, and related guidelines. aims and methods: we systematically searched the pubmed database up till march , and retrieved all the articles published on chloroquine and hcq and covid- . results: two small human studies have been conducted with both these drugs in covid- , and have shown significant improvement in some parameters in patients with covid- . conclusion: considering minimal risk upon use, a long experience of use in other diseases, cost-effectiveness and easy availability across india, we propose that both these drugs are worthy of fast track clinical trial for treatment, and may be carefully considered for clinical use as experimental drugs. since hcq has been approved for treatment of diabetes in india, it should be further researched in diabetes and covid- , a subgroup where significant mortality has been shown. novel coronavirus ( -ncov), officially known as severe acute respiratory syndrome coronavirus (sars-cov- ), the etiological agent of the (corona virus disease ) covid- , emerged in wuhan, hubei province, china. on th march , the world health organization (who) declared this disease as pandemic [ ] . chinese centre for disease control and prevention showed an increased mortality in people with diabetes ( . % vs. . %; overall vs. in patients with diabetes respectively) from a report of , cases of covid- [ ] . people with diabetes and covid- may need special attention and clinical care [ ] . in the absence of any known efficient therapy and because of the situation of a "publichealth emergency", many drugs have been tried recently in the treatment for covid- that includes a low-cost antimalarial drug chloroquine and its derivative hydroxychloroquine (hcq), along with several other antiviral drugs. because hcq has been approved in the treatment of type diabetes in india since as a third-or fourth-line drug, it would be interesting to research its impact in patients with diabetes, infected with covid- . reports gathered so far have suggested that a number of drugs could be potential candidates for the treatment of covid- , although the clinical effectiveness of these drugs have not yet been fully evaluated. the list of these drugs has been summarized in table [ e ]. in this review article, we have systematically searched the medical data base until and collated all the available evidences that have emerged so far on the efficacy of chloroquine and hydroxychloroquine, in the treatment of patients with covid- , with or without diabetes and present a perspective on both these compounds. additionally, we have also pooled the currently on-going trials with both these compounds against the covid- . we systematically searched the pubmed database up till march , using key words chloroquine and covid- , and hydroxychloroquine and covid- and retrieved a total of articles. the two articles that were written in chinese were excluded. in addition, we also accessed and retrieved the full text of the cross references of importance from these articles written in english. moreover, we also accessed the currently ongoing trials with both these compounds from clinicaltrials.gov. experimental studies have suggested that chloroquine is a proven anti-malarial drug that has the capability of inhibiting the replication of several intracellular micro-organisms including coronaviruses in vitro. it is also believed that chloroquine may have a varied mechanism of action which may differ depending upon the pathogen studied. it has been increasingly learnt that the anti-viral and anti-inflammatory activities of chloroquine may have a role in the treatment of patients with novel covid- . chloroquine increases endosomal ph and interferes with the glycosylation of cellular receptor of sars-cov and thereby it has the potential to block viral infection [ ] . in addition, chloroquine also inhibits the quinone reductase- , which is involved in sialic acid biosynthesis (an acidic monosaccharides of cell transmembrane proteins required for ligand recognition) that makes this agent a broad antiviral agent. it is important to note that both human coronavirus hcov-o and orthomyxoviruses uses sialic acid moieties as a receptor. moreover, chloroquine changes the ph of lysosomes and likely inhibits cathepsins, that leads to the formation of the autophagosome which cleaves sars-cov- spike protein. furthermore, chloroquine through the inhibition of map-kinase interferes with sars-cov- molecular crosstalk, besides altering the virion assembly, budding and interfering with the proteolytic processing of the m protein [ , ] . previous experimental studies have also demonstrated that chloroquine has potent anti-sars-cov- effects in vitro, primarily attributable to a deficit in the glycosylation receptors at the virus cell surface, so that it cannot bind to the angiotensin-converting enzyme (ace ) expressed in lung, heart, kidney and intestine. since sars-cov- utilizes the similar surface receptor ace , it is believed that chloroquine can also interfere with ace receptor glycosylation thus prevents sars-cov- attachment to the target cells [ e ]. chinese researchers who studied the effect of chloroquine in vitro (using vero e cell line infected by sars-cov- ) found chloroquine to be highly effective in reducing viral replication that can be easily achievable with standard dosing due to its favorable penetration in tissues including the lung [ , ] . since the structure and mechanism of action of chloroquine and hydroxychloroquine (hcq) are exactly same except an additional hydroxy moiety in one terminal in hcq, both act as a weak base that can change the ph of acidic intracellular organelles including endosomes/lysosomes, essential for the membrane fusion. it is believed that both the agents could be effective tools against sars-cov- and sars-cov- [ , ] . however, an important question that still remains is whether hcq has a similar effect on sars-cov- infection. some data show hcq effectively inhibited both the entry, transport and the post-entry stages of sars-cov- , similar to the chloroquine and one study found hcq to be a more potent agent than chloroquine in inhibiting sars-cov- in vitro [ , ] . in addition, hcq acts effectively on other intracellular bacterial infections such as coxiella burnetii (q fever) and tropheryma whipplei (whipple's disease) [ , ] . addition of hydroxyl molecule makes hcq less permeable to blood-retinal barrier and allows faster clearance from retinal pigment cell, thereby suggesting a lesser risk of retinal toxicity with hcq, as compared to chloroquine [ ] . furthermore, the narrow therapeutic and safety index margin with chloroquine makes hcq a safer option than chloroquine. an additional issue to be considered in severely sick patients is cytokine storm associated with disease severity of sars-cov- [ ] .the significant decrease in the production of proinflammatory markers and cytokines with hcq has made this agent a successful disease modifying anti-inflammatory agent in the treatment of various autoimmune diseases including rheumatoid arthritis, systemic lupus erythematosus and sjogren's syndrome. long-term clinical safety profile of hcq is better than that of chloroquine, that allows higher daily dose of hcq with less drugdrug interactions. the anti-viral and anti-inflammatory actions of chloroquine have led to numerous trials urgently in the face of global health emergency. a chinese study involving more than patients of covid- found chloroquine superior to the control group in reducing symptom duration, exacerbation of pneumonia including radiological improvement and promoting virus-negative seroconversion without any severe side effects [ ] . this represents the first human trial ever conducted with chloroquine against covid- . although the detailed results of this trial are not yet published and only available in a letter form, interestingly, this early result led china to include chloroquine in the prevention and treatment of covid- pneumonia. moreover, the national health commission of the people's republic of china recommended inclusion of chloroquine in the next version of the guidelines for the prevention, diagnosis, and treatment of pneumonia caused by covid- . in this study, chloroquine was given in dose of mg of chloroquine twice daily in mild to severe covid- pneumonia (see table ). the second human study which is currently available was conducted with hcq. in an open-label, non-randomized trial (n ¼ ) conducted in marseille, france, gautret et al. found that hcq alone and combination of hcq plus azithromycin was highly and significantly effective in clearing viral nasopharyngeal carriage (measured by polymerase chain reaction [pcr]) in only three-to six days in covid- subjects, compared to control. the virological clearance day- post-inclusion (primary outcome) with hcq vs. control was . % versus . %, respectively (p ¼ . ). in addition, the virological clearance at day- post-inclusion in hcq plus azithromycin, hcq and control arm was %, . % and . % respectively (p < . ). this data suggests a synergistic effect of azithromycin with hcq. indeed, azithromycin has shown an antiviral effect against zika and ebola viruses in in vitro studies; however, it is not yet known whether it has any action against covid- as well [ ] . these results of converting a potential carrier to a seronegative patient are of importance with regards to preventing community transmission of covid- . since the data from wuhan, china showed that some patients were carrier as long as up to days (usually around days), results of this study are very encouraging in the context of converting a patient to a seronegative subject within days. interestingly, this study also showed that the effect of hcq was significantly higher (p < . ) in symptomatic patients as compared to asymptomatic patients with covid- . the authors have acknowledged limitations of the study; a small sample size, dropout of six patients and limited follow-up, apart from the non-randomized and open-label nature of the trial. a close look in to this study also suggests that the cycle threshold (ct) value for nasopharyngeal swab pcr to call it as a sample negative, was lower (ct value > was deemed as negative for virus) compared to conventional ct threshold of > . ct is defined as the number of cycles to be run for the pcr test to turn positive. in other words, lower the number of ct denotes more virus is present and lesser number of cycles are required to hit the threshold. moreover, nasopharyngeal swab pcr is a less sensitive tool, compared to pcr of broncho-alveolar lavage and sputum in covid- . furthermore, exclusion of five patients ( %) receiving hcq from the overall analysis, exaggerate the final results of this study. nevertheless, based on limited available evidences to date, and given the prevailing pandemic of covid- , some of the institutions and or organizations have already recognized the utility of chloroquine and hcq [ ] . the expert consensus from the department of science and technology and health commission of guangdong province published on th february (based on in vitro evidence and still unpublished clinical experience) chloroquine phosphate tablet at a dose of mg twice per day for days for patients diagnosed as mild, moderate and severe cases of sars-cov- pneumonia in the absence of contraindication to the drug [ ] . a central clinical task force from korea who have treated cases of covid- recommend using lopinavir mg/ritonavir mg bid or chloroquine mg orally per day or hydroxychloroquine mg orally per day for e days, in moderate to severe case of covid- [ ] . similarly, the dutch center of disease control in a public document on its website, also suggested the use of chloroquine in those having severe covid- infection admitted in the intensive care unit [ ]. table summarizes the recommendation and dosing from all the various groups [ e ]. the antiviral activity of chloroquine and hcq have been identified in the in vitro studies and the growth of many different viruses have been inhibited in the cell culture line by both the agents, including the sars coronavirus. mice studies have also demonstrated activity of these agents against human coronavirus oc , enterovirus ev-a , zika virus and influenza a h n . however, no benefit of chloroquine was seen in the prevention of influenza and dengue infection in a randomized, double-blind, placebocontrolled, clinical trial [ , ] . similarly, chloroquine was active in ex vivo studies but not in the in vivo studies against ebolavirus, nipah and influenza viruses [ e ]. data of chloroquine against chikungunya virus is even more intriguing. while chloroquine had satisfactory antiviral activity against chikungunya in vitro, animal studies showed increase in virus replication, aggravation of fever and incomplete viral clearance [ ] . human trials of chloroquine showed no improvement in chikungunya acute illness and rather an increase in chronic arthralgia was observed during post-illness period, compared to the controls [ ] . the role of chloroquine against human immunodeficiency virus was inconclusive [ ] . the only viral disease where chloroquine was modestly effective so far before covid- era was chronic hepatitis c suggesting an increased virological response to pegylated interferon plus ribavirin [ ] . therefore, the results of chloroquine and hcq so far done against covid- , more promising than previous trial in other viral diseases. moreover, these drugs are of low cost, reasonably safe (see below), and widely available in countries where malaria is endemic. expectedly, some precautions will be needed while using both these drugs that include frequent monitoring of hematological parameters (rbc, wbc and platelet counts), measurement of serum electrolytes, blood glucose (because of hypoglycemic potential of hcq) and hepatic as well as renal functions. since both these drugs have the potential to prolong qtc, routine electrocardiography is essential prior to starting these drugs. coadministration of other drugs known to prolong the qtc interval (such as anti-arrhythmic, anti-depressants, anti-psychotics, antihistaminic, teneligliptin, ondansetron and moxifloxacin etc.) must be avoided [ , ] . moreover, addition of azithromycin to hcq as done in french trial by gautret et al. may increase the risk of qtc prolongation. perform ecg daily if qtc is e msec. additionally, hypoglycemia must be looked for in patients with diabetes especially with concurrent use of chloroquine/hcq and lopinavir/ritonavir. chloroquine and hcq should not be used concurrently with lopinavir/ritonavir and remdisivir for anticipated qtc prolongation. finally, pharmacovigilance on visual and mental disturbance is also closely required. although there are sporadic case reports of chloroquineinduced cardiomyopathy and reversible heart failure in the literature, many studies and large meta-analysis conducted in patients with rheumatoid arthritis pointed to a reduced cardiovascular risk with both these compounds [ ] . nevertheless, since both the drug has potential to prolog qtc, a baseline ecg should be done in patients with established cardiovascular disease. all clinicians using these drugs must know contraindication to both these compounds; hypersensitivity to these agents, retinopathy, porphyria, epilepsy, pre-existing maculopathy, g pd deficiency, recent myocardial infarction and qtc > msec. chloroquine is not contraindicated in pregnancy. table available guidelines (as of in the treatment of covid- for chloroquine and hydroxychloroquine. hydroxychloroquine mg tid for days. od-once daily, bid-twice daily, tid-thrice daily, urti-upper respiratory tract infection, pcr-polymerase chain reaction, i.v -intravenous. although evidence of chloroquine and hcq is limited (based on the experimental data and only two small human trials), considering the potentially favorable benefit-risk balance of chloroquine and hcq in absence of any other valid treatment option, we believe that such treatment could be useful in the current context of pandemic covid- outbreak. we have summarized current consideration and proposed line of management in table . the low cost of chloroquine and hcq could also be an effective strategy to counter covid- (especially in patients with diabetes and other co-morbidities in whom mortality is high) in resource constrained and covid- overburdened heath care systems in middle-and low-income counties including india. we hereby declare that we have no conflict of interest related to table based on the available evidence so far (including in vitro, ex vivo and in vivo studies e both experimental and two human studies) and considering the benefit-risk ratio as well as the low cost of chloroquine and hydroxychloroquine in indian context, we propose the following regime, until more evidence is available - proposed chemoprophylaxis no conclusive evidence so far; however, chloroquine or hcq could be researched as a prophylactic agent in endemic areas. recent guidelines from indian council of medical research recommend it as prophylactic agent (see reference for indication and dose) note: hcq can be used as an adjunct to control glycemia in adult patients with type diabetes (approved for treatment in india). however, role of such adjunctive treatment for testing its potential role as a prophylaxis of covid- in diabetes, has not been researched but could be attempted (in view of above) considering a higher mortality in patients with diabetes, as compared to non-diabetic subjects with covid- . confirmed covid- a. chloroquine phosphate: @$ . covid- urti: mg bid for days. . covid- lrti: mg bid for days. b. hydroxychloroquine: @$ loading dose: mg bid day , then maintenance dose: mg bid for e days. c. monitor and watch for side effects* @ watch for hypoglycemia in diabetes especially with concurrent use of lopinavir/ritonavir, $ should not be used concurrently with lopinavir/ritonavir and remdisivir due to increased qtc prolongation, * complete blood count, renal, hepatic profile and ecg e watch for qtc prolongation, urti-upper respiratory tract infection, lrti-lower respiratory tract infection, hcq-hydroxychloroquine, bid e twice daily. who director-general's opening remarks at the media briefing on covid- - characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention clinical considerations for patients with diabetes in times of covid- epidemic asymptomatic carrier state, acute respiratory disease, and pneumonia due to severe acute respiratory syndrome coronavirus (sars-cov- ): facts and myths learning from the past: possible urgent prevention and treatment options for severe acute respiratory infections caused by -ncov drug treatment options for the -new coronavirus ( -ncov) remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro chloroquine for the novel coronavirus glycopeptide antibiotics potently inhibit cathepsin l in the late endosome/lysosome and block the entry of ebola virus, middle east respiratory syndrome coronavirus (mers-cov), and severe acute respiratory syndrome coronavirus (sars-cov) chloroquine and hydroxychloroquine as available weapons to fight covid- design and synthesis of hydroxyferroquine derivatives with antimalarial and antiviral activities hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) bactericidal effect of doxycycline associated with lysosomotropic agents on coxiella burnetii in p d cells antibiotic susceptibility of tropheryma whipplei in mrc cells american academy of ophthalmology. recommendations on screening for chloroquine and hydroxychloroquine retinopathy clinical features of patients infected with novel coronavirus in wuhan, china breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies hydroxychloroquine and azithromycin as a treatment of covid- : results of an open label non-randomized clinical trial of chloroquine and covid- multicenter collaboration group of department of science and technology of guangdong province and health commission of guangdong province for chloroquine in the treatment of novel coronavirus pneumonia mount sinai health system treatment guidelines for sars-cov- infection (covid- interim clinical guidelines for patients suspected of/confirmed with covid- infection chloroquine for influenza prevention: a randomised, double-blind, placebo controlled trial a randomized controlled trial of chloroquine for the treatment of dengue in vietnamese adults lack of protection against ebola virus from chloroquine in mice and hamsters chloroquine is effective against influenza a virus in vitro but not in vivo chloroquine administration does not prevent nipah virus infection and disease in ferrets acceleration of viral replication and up-regulation of cytokine levels by antimalarials: implications in malaria-endemic areas paradoxical effect of chloroquine treatment in enhancing chikungunya virus infection the enigma of the clandestine association between chloroquine and hiv- infection hydroxychloroquine augments early virological response to pegylated interferon plus ribavirin in genotype- chronic hepatitis c patients chloroquine us prescribing information chloroquine us prescribing information chloroquine and hydroxychloroquine are associated with reduced cardiovascular risk: a systematic review and meta-analysis a systematic review on the efficacy and safety of chloroquine for the treatment of covid- key: cord- - p cwb e authors: guzman-prado, yuli title: recent findings on cardiovascular safety with the use of chloroquine and hydroxychloroquine for covid- . date: - - journal: am j cardiol doi: . /j.amjcard. . . sha: doc_id: cord_uid: p cwb e nan and found no significant differences in the likelihood of abnormal electrocardiogram findings (defined as arrhythmia or prolonged qt interval) between groups, however, the researchers highlight that cardiac arrest was more likely in the group treated with hcq plus az (adjusted or . ; % ci . - . ). no significant differences in in-hospital mortality were found. in order to elucidate if the finding of cardiac arrest was mediated or not by mechanical ventilation, the authors performed a stratified analysis and indicated that in the group of patients who did not receive mechanical ventilation, the risk for cardiac arrest remained increased for hcq alone compared with az alone (adjusted or . % ci . - . ). the analysis performed in this study was adjusted for likely confounders, including comorbidities, demographics and disease severity. however, due to the observational study design it is possible that some amount of unmeasured confounding remains. later on, mehra et al ( ), reported a large international observational registry including data from patients from hospitals in six continents (mainly north america and europe) who received cq (n= ); cq plus macrolide (n= ); hcq (n= ); hcq plus macrolide (n= ) versus none of these drugs (n= ) and found that the use of cq or hcq with or without macrolides was independently associated with a higher risk of in-hospital mortality and an increased frequency of de-novo ventricular arrhythmia (non-sustained or sustained ventricular tachycardia or ventricular fibrillation). the authors state that the combination of cq or hcq with a macrolide increased the hazard of de-novo ventricular arrhythmias: cq with a macrolide (hr . ; % ci . - . ); cq alone (hr . ; % ci . - . ); hcq with a macrolide (hr . ; % ci . - . ) and hcq alone (hr . ; % ci . - . ). predictors of ventricular arrhythmia were also investigated and the variables found to be independently associated with higher risk of de-novo ventricular arrhythmias were cq or hcq with or without macrolides and baseline comorbidities such as coronary artery disease, congestive heart failure, history of cardiac arrhythmia, and chronic obstructive pulmonary disease. the researchers argue that the presence of underlying cardiovascular disease could represent a partial explanation of the cardiovascular toxicity associated with the use of cq or hcq particularly with the combination of macrolides. notably, a propensity score matching analysis was conducted to mitigate potential confounders. moreover, cox proportional hazards models and a tipping-point analysis were performed to assess the robustness of the estimates. clearly, this study represents a potential landmark regarding safety and efficacy of cq and hcq, either alone or in combination with macrolides, for the treatment of covid- . however, the observational retrospective design limits the study's implications. in addition to the cardiotoxic considerations mentioned by aggarwal, et al ( ), relevant cardiovascular side effects and toxicities associated with the use of repurposing drugs for the treatment of covid- have been stated by researchers of cardiovascular disease who stress the risk of qt interval prolongation, thrombocytopenia and anaemia specifically with the use of cq and hcq ( ). in conclusion, it is not clear whether the risk of toxic effects may outweigh the benefits of potential covid- treatments. a decisive answer still awaits the results of high quality large randomized controlled trials. cardiovascular safety of potential drugs for the treatment of coronavirus disease effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus (sars-cov- ) infection: a randomized clinical trial clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in covid- patients with at least a six-day follow up: a pilot observational study the qt interval in patients with covid- treated with hydroxychloroquine and azithromycin assessment of qt intervals in a case series of patients with coronavirus disease (covid- ) infection treated with hydroxychloroquine alone or in combination with azithromycin in an intensive care unit risk of qt interval prolongation associated with use of hydroxychloroquine with or without concomitant azithromycin among hospitalized patients testing positive for coronavirus (covid- ) infection. jama cardiol clinical efficacy of hydroxychloroquine in patients with covid- pneumonia who require oxygen: observational comparative study using routine care data association of treatment with hydroxychloroquine or azithromycin with in-hospital mortality in patients with covid- in new york state hydroxychloroquine or chloroquine with or without a macrolide for treatment of covid- : a multinational registry analysis. the lancet covid- and the cardiovascular system: implications for risk assessment, diagnosis, and treatment options key: cord- -oagv q u authors: liu, peng; zhao, liwei; ferrere, gladys; alves-costa-silva, carolina; ly, pierre; wu, qi; tian, ai-ling; derosa, lisa; zitvogel, laurence; kepp, oliver; kroemer, guido title: combination treatments with hydroxychloroquine and azithromycin are compatible with the therapeutic induction of anticancer immune responses date: - - journal: oncoimmunology doi: . / x. . sha: doc_id: cord_uid: oagv q u amid controversial reports that covid- can be treated with a combination of the antimalarial drug hydroxychloroquine (hcq) and the antibiotic azithromycin (azi), a clinical trial (oncocovid, nct ) was launched at gustave roussy cancer campus to investigate the utility of this combination therapy in cancer patients. in this preclinical study, we investigated whether the combination of hcq+azi would be compatible with the therapeutic induction of anticancer immune responses. for this, we used doses of hcq and azi that affect whole-body physiology (as indicated by a partial blockade in cardiac and hepatic autophagic flux for hcq and a reduction in body weight for azi), showing that their combined administration did not interfere with tumor growth control induced by the immunogenic cell death inducer oxaliplatin. moreover, the hcq+azi combination did not affect the capacity of a curative regimen (cisplatin + crizotinib + pd- blockade) to eradicate established orthotopic lung cancers in mice. in conclusion, it appears that hcq+azi does not interfere with the therapeutic induction of therapeutic anticancer immune responses. the current epidemic of coronavirus disease- caused by severe acute respiratory syndrome coronavirus- (sars-cov- ) requires curative interventions. hydroxychloroquine (hcq) inhibits sars-cov- replication invitro, presumably through a nonspecific action on acidic organelles. this in vitro observation spurred clinical evaluation of this antimalarial drug, alone or in combination with macrolide antibiotics (in particular azithromycin, azi), which are often used for the treatment of respiratory infections. the oral administration of hcq, alone or (more so) in combination with azi, yielded encouraging results in uncontrolled observational studies. , however, hcq alone failed to reduce sars-cov- replication in vitro in susceptible animal species including ferrets and nonhuman primates (https://www.researchsquare.com/article/rs- /v ). moreover, hcq alone or in combination with azi failed to show clinical activity against covid- in randomized trials. , the polemics around the therapeutic use of hcq+azi is not closed. however, in the absence of other therapeutic options, the clinical trial "oncocovid" (accession no. nct at www.clinicaltrials.com) was launched at gustave roussy cancer campus on april , . the objective of this trial is (i) to determine the prevalence and the -months incidence of sars-cov- in cancer patients and (ii) to evaluate the covid- disease-specific mortality rate in cancer patients treated by hcq+azi. indeed, the underlying conditions (aging, obesity, smoking, . . . ), co-morbidities (metabolic syndrome, arterial hypertension, cardiopathy, immunodeficiency, . . .) and nosocomial exposure predispose oncological patients to infection by sars-cov- and the development of severe covid- , calling for therapeutic interventions against the virus that do not interfere with the clinical management of cancer patients. the success of most if not all antineoplastic treatments depends on the induction of a mostly t lymphocytemediated anticancer immune response. while this appears obvious for so-called immunotherapies, it also applies to successful chemotherapies and targeted therapies. [ ] [ ] [ ] for this reason, we investigated whether the hcq+azi combination would be compatible with the treatment of two cancers with immunogenic chemotherapy or a combination chemoimmunotherapy. c bl/ mice housed in specific pathogen-free conditions were treated with hcq (injected intraperitoneally, i.p. at a daily dose of mg/kg) or azi (given orally with the drinking water at to mg/ml for days), alone or in combination (figure (a) ). at this dose, azi caused a reduction in bodyweight that was not aggravated by hcq (figure (b) ). as to be expected [ ] [ ] [ ] hcq caused a partial blockade in autophagic flux, as indicated by an increase in the lipidated form (lc -ii) of microtubuleassociated proteins a/ b light chain b (hereafter referred to as lc ) and in the abundance of sequestosome- (sqstm , best known as p ). these alterations were observed in the liver and in the heart (figure (c-f)). thus, both hcq and azi were used at concentrations that affected whole-body physiology. in the next step, we determined whether the combination treatment with hcq+azi would affect the chemotherapeutic response of mc colon cancers to oxaliplatin (oxa) (figure (a)), the antitumor effect of which is known to entirely rely on the active contribution of t lymphocytes. [ ] [ ] [ ] [ ] systemic oxabased therapy alone (given twice i.p. on days and postimplantation of mc cells under the skin) caused a similar weight loss as did the treatment with hcq+azi, and both treatments exhibited additive toxicities (figure (b)). the oxa-based chemotherapy led to a prolonged tumor growth control that lasted beyond treatment discontinuation and that was not affected by the simultaneous treatment with hcq +azi. thus, the hcq+azi combination is compatible with the success of chemotherapy. recently, we developed a protocol for the eradication of established non-small cell lung cancers (nsclc) consisting of a combination of cis-diamminedichloridoplatinum (ii) (cddp, best known as cisplatin, a classical cytotoxicant targeting dna), high-dose crizotinib (a tyrosine kinase inhibitor) and pd- blockade. this combination treatment can be applied to mice bearing bioluminescence-detectable, luciferase-expressing tc lung cancers and induces full resolution of the disease in the majority ( - %) of cases. , we combined this therapeutic regimen with hcq+azi (figure (a)) without observing additive toxicities (figure (b) ). we then monitored the (pre-)clinical evolution of nsclc by imaging technology (figure (c,d) ). pd- blockade alone limited tumor growth, and this effect was abolished by hcq+azi. however, the combination treatment (cddp+crizotinib+pd- blockade) led to a major reduction of the growth of all tumors, and this effect was compatible with hcq+azi co-treatment ( figure ). accordingly, hcq+azi had no significant negative effect on the curative effects of the combination treatment (around % for cddp+crizotinib+pd- blockade), defined as the complete disappearance of the luciferase-dependent bioluminescence signal ( figure (a,b) ). moreover, % of the mice survived after combination therapy with cddp figure . effects of hydroxychloroquine plus azithromycin on naïve mice. tumor-free mice were treated interperitoneally (i.p.) with hydroxychloroquine (hcq), orally fed with azithromycin (azi, supplied at different concentrations in the drinking water), or their combination (combo) as illustrated in the scheme (a). the bodyweight of the mice was monitored every ~ days and the ratios to the st measurement at different time points are summarized as line chart (b), mean ± sem, n = mice/group; **p < . , and ***p < . , two-way anova test, compared to the water group). livers and hearts were excised from mice of each group at hours after the second injection of hcq and subjected to protein extraction for sds-page and immunoblot (c, d). β-actin (actb) was measured as a loading control. band intensities were measured to quantify the ratio of lc -ii to lc -i (lc -ii/lc -i) and p to actb (p /actb). data are expressed as means ± sem of three mice (e, f). statistical significance is indicated as *p < . , **p < . , and ***p < . as compared with untreated control (ctrl) (student's t-test). +crizotinib+pd- blockade irrespective of the co-treatment with hcq+azi ( figure (c,d)). thus, hcq+azi does not interfere with therapeutic efficacy. covid- is perceived as a major menace to human health, causing the lockdown of entire countries over several months. here, we evaluated a possible, but highly controversial treatment option, the combination of hcq +azi, for its possible interference with oncological treatments in preclinical models. hcq is a lysosomotropic agent, meaning that its physicochemical characteristics (lipophilic, cell membrane permeable weak base) cause it to enrich in acidic cellular compartments, such as lysosomes, endosomes, autophagolysosomes, leading to their alkalization and to a nonspecific interference with viral replication. hcq also causes the inhibition of autophagy, - a cytoprotective mechanism that is essential for cellular stress management, explaining why hcq sensitizes cells to cytotoxic agents. , however, hcq may also mediate autophagy-independent cell killing through lysosomal membrane permeabilization, causing the release of cytotoxic enzymes including cathepsins and secondary mitochondrial membrane permeabilization, unleashing the apoptotic cascade. in vivo, even high doses of hcq ( mg/kg per day) are unlikely to fully inhibit autophagy. indeed, genetically-induced autophagy inhibition (by inducible knockout or knockdown of the essential autophagy genes atg or atg ) causes a dramatic acceleration of aging that provokes the degeneration of all internal organs and kills young adult mice in - months. , no such side effects are known for hcq treatment, meaning that autophagy inhibition cannot be complete, in spite of the prolonged half-life of the molecule. autophagy induction in cancer cells is required for the induction of immunogenic cell death (icd) by oxa or the combination of cddp +crizotinib, meaning that cancer cells in which atg expression has been suppressed do not respond to these combination regimens in vivo. , , hence, the fact that hcq fails to interfere with the efficacy of icd-based therapeutic regimens might be explained by its incapacity to fully block autophagic flux. tumor immunosurveillance and protective immune responses against viruses both require functional type ifn receptor signaling. , however, hcq was associated with impaired ability of plasmacytoid dendritic cells from systemic lupus erythematosus patients to produce ifn-α and tnf-α upon stimulation with tlr- and tlr- agonists. this might explain why, to some extent, hcq reduced the efficacy of anti-pd abs as a standalone therapy against our lung cancer tumor. however, given that a sustained type ifn response causes secondary resistance to immune checkpoint blockade, hcq may actually curtail this feed -back loop, as suggested in our combinatorial regimen. azi is a macrolide antibiotic. combinations of broadspectrum antibiotics (that however did not include macrolides) capable of causing temporary sterilization of the gut interfere with the anticancer effects of oxa and pd- blockade. , there are also clinical reports suggesting that treatment with macrolides or other broad-spectrum antibiotics during the month preceding immunotherapy negatively affect therapeutic responses. [ ] [ ] [ ] however, in the specific setting that we investigated here, azi did not interfere with the efficacy of oxa or a combination therapy including pd- blockade. in sum, the preclinical data contained in this work suggest that hcq+azi do not interfere with anticancer treatments. additional work is required to determine whether hcq+azi have therapeutic utility against covid- in oncological patients (as suggested for instance in ref. or whether this treatment, which is reputed to cause an increase in the q-t interval causing potentially lethal arrhythmia should be discontinued). murine colon adenocarcinoma cell line mc was purchased from the american type culture collection (virginia, ma, usa). luciferase-expressing murine non-small lung cancer (nsclc) tc luc + cell line was kindly shared by dr t.c. wu. all cells were cultured in dulbecco's modified eagle medium (dmem) supplemented with % fetal bovine serum (fbs), u/ml penicillin, and μg/ml streptomycin, at °c in a humidified incubator supplied with % co . all cell culture media and supplements were from gibco-invitrogen (carlsbad, ca, usa) and all plastic material was purchased from corning costar (corning, ny, usa). hydroxychloroquine sulfate (phr ), oxaliplatin (y ), cisplatin (c ), and figure . influence of hydroxychloroquine plus azithromycin on the efficacy of anticancer chemo-immunotherapy. orthotopic nsclc were established by intravenous (i.v.) injection of × tc cells stably expressing luciferase (tc luc + ) and tumor incidence in the lung was detected by bioluminescence. mice (n = minimum of mice per group) were treated with crizotinib (criz) plus cisplatin (cddp) or equivalent volumes of pbs, and combined or not with monoclonal antibody to pd- (αpd- ) or isotype antibody (iso), either alone or in combination with hydroxychloroquine (hcq, i.p.) plus azithromycin (azi) in drinking water as illustrated in the scheme (a). bodyweight of mice was measured every days, and tumor size was monitored by luciferase activity every to days. bodyweight (ration to st measurement after randomization) is reported as mean ± sem (b). statistical analysis was performed by means of two-way anova test, ***p < . as compared to the pbs group (n = - mice per group). representative time-lapse images of mice from different groups are reported in (c and d). crizotinib (pz ) were purchased from sigma-aldrich (st louis, mi, usa). azithromycin was obtained from the local pharmacy ( mg tablet, pfizer france, paris, france). beetle luciferin (e ) was purchased from promega (madison, wi, usa). monoclonal antibody to lc b (# ) and p (# ) werwas purchased from cell signaling technology (danvers, ma, usa) and hrp-conjugated monoclonal antibody to β-actin (ab ) came from abcam (cambridge, uk). monoclonal antibody to mouse pd- (clone: f. a catalog#: be ) and isotype control antibody (clone: ltf- catalog#: be ) were purchased from bioxcell (lebanon, nh, usa). naïve female c bl/ mice ( -week old) were treated with hydroxychloroquine and azithromycin for two day days as described below. four hours after the nsecond treatment, mice were sacrificed and livers and hearts were snap-frozen in liquid nitrogen. for purification of tissue proteins, mg tissue was immersed in ml of ripa lysis buffer (# , invitrogen, carlsbad, ca, usa) in precellys lysing tubes (#ck _ ml, bertin technologies sas, france). the tissues were then dissociated using the precellys homogenizer (bertin technologies sas) at , rpm for seconds, and centrifuged at , g for mins to collect the supernatant that contains soluble proteins. protein concentration was quantified with the dc protein assay (bio-rad, hercules, ca, usa). the protein solution was mixed with x loading buffer, and denatured at °c for h hour before being used for western blotting. forty micrograms of protein were resolved on sds polyacrylamide gel electrophoresis (page) gels (invitrogen) and transferred to pvdf membranes (merck millipore), which were blocked in tbs containing . % tween- and % nonfat dry milk for h before the incubation with primary antibodies overnight at °c on a rocking shaker. membranes were then washed five times with tbst for min each, and incubated with hrp-conjugated secondary antibody for h at room temperature. at last, the membranes were washed five times with tbst for min each and subjected to chemiluminescence-based detection with the amersham ecl prime detection reagent kit and the imagequant las softwareassisted imager (ge healthcare, piscataway, nj, usa). band quantification was performed using the imagequant tl software (ge healthcare). all animal experiments have been approved by the ethics committee of gustave roussy cancer campus (project number: - ) and were performed in accordance with governmental and institutional guidelines and regulations. all mice were maintained in a temperaturecontrolled specific pathogen-free environment with -h light/dark cycles, with food and water ad libitum. six to eightweek-old female c bl/ mice were purchased from charles river (france) and were adapted for at least week before being used for experiments. all animal experiments were performed once with a number of animals per group sufficient for statistical analysis by means of the freely available software t u m o r g r o w t h ( h t t p s : / / g i t h u b . c o m / k r o e m e r l a b / tumgrowth). hydroxychloroquine was intraperitonially administrated at a dose of mg/kg in ul pbs daily for weeks. significance are expressed as **p < . and ***p < . as compared to the pbs group; or ## p < . as compared between indicated groups (n = - mice per group). azithromycin was dissolved in autoclaved drinking water at a concentration of mg/kg or mg/kg, and the solution was changed daily throughout the treatment period. for the toxicity test, azithromycin was supplied continuously for days; while for the treatment of tumor-bearing mice, it was used in a -day on/ -day off mode ( days with azithromycin in drinking water plus day without). oxaliplatin ( mg/kg), crizotinib ( mg/ kg), and cisplatin ( . mg/kg) wawere administrated intraperitoneally at day (when mice were randomized) and day . monoclonal antibody to pd- or an isotype control antibody was administrated intraperitoneally at day , , and after the sfirst chemotherapy, at a dose of mg/kg. for the establishment of mc colon adenocarcinoma, mice were subcutaneously (s.c.) injected with × cells/mouse in ml cold pbs in a low-flow isoflurane anesthesia vaporizer. when the tumor surface (calculated as longest dimension x perpendicular dimension x π/ ) reached around - mm , tumor-bearing mice were randomized into treatment groups as described above. tumor surface was then monitored every days and animals bearing neoplastic lesions that exceeded mm were euthanized. to establish the orthotopic nsclc model, × tc luc + cells (in µl pbs) were intravenously (i.v.) injected to -week old female c bl/ mice. tumor incidence and development were visualized by in vivo photonic imaging of tumor cells' luciferase activity. mice were i.p. injected with mg/kg beetle luciferin and mins later, photons were acquired on aa xenogen ivis bioluminescence imager (caliper life sciences inc., hopkinton, ma, usa). in vivo imaging was conducted every - days with an exposure time starting at min, and gradually reduced to min, min, min when photon saturation occurred. when tumors could be detected at min exposure, the tumor bearintumor-bearing mice were randomized into treatment groups as described above. mice showing photon saturation in less than min of exposure were euthanized. mice bearing orthotopic nsclc were treated with crizotinib (criz) plus cisplatin (cddp) or equivalent volumes of pbs, and combined or not with monoclonal antibody to pd- (αpd- ) or isotype antibody (iso), either alone or in combination with hydroxychloroquine (hcq, i.p.) plus azithromycin (azi) in drinking water. tumor incidence was monitored by luciferase activity every to days and mice that lost the diagnostic pulmonary bioluminescence signal were regarded as tumor-free. tumor-free (total influx of image photons < , ) survival rates (a, b) and overall survival rates are reported as kaplan-meier curves. statistical analysis was performed by means of the likelihood ratio test. significances are expressed as *p < . and **p < . as compared to the pbs group (n = - mice per group). coronavirus infections: epidemiological, clinical and immunological features and hypotheses in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus digesting the crisis: autophagy and coronaviruses clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in covid- patients with at least a six-day follow up: a pilot observational study covid- transmission, outcome and associated risk factors in cancer patients at the first month of the pandemic in a spanish hospital in madrid antiviral efficacies of fda-approved drugs against sars-cov- infection in ferrets hydroxychloroquine in patients with mainly mild to moderate coronavirus disease : open label, randomised controlled trial clinical efficacy of hydroxychloroquine in patients with covid- pneumonia who require oxygen: observational comparative study using routine care data caring for patients with cancer in the covid- era the anticancer immune response: indispensable for therapeutic success? immunological effects of conventional chemotherapy and targeted anticancer agents immunological off-target effects of imatinib inhibition of macroautophagy triggers apoptosis principles and current strategies for targeting autophagy for cancer treatment autophagy as a target for anticancer therapy immunogenic death of colon cancer cells treated with oxaliplatin restoration of the immunogenicity of cisplatin-induced cancer cell death by endoplasmic reticulum stress chemotherapyinduced antitumor immunity requires formyl peptide receptor a synergistic triad of chemotherapy, immune checkpoint inhibitors, and caloric restriction mimetics eradicates tumors in mice crizotinibinduced immunogenic cell death in non-small cell lung cancer crizotinib -a tyrosine kinase inhibitor that stimulates immunogenic cell death autophagy and the integrated stress response counteracting autophagy overcomes resistance to everolimus in mantle cell lymphoma repurposing drugs in oncology (redo)-chloroquine and hydroxychloroquine as anti-cancer agents mitochondrial membrane permeabilization is a critical step of lysosome-initiated apoptosis induced by hydroxychloroquine mitochondria as multifaceted regulators of cell death autophagy is required for glucose homeostasis and lung tumor maintenance temporal inhibition of autophagy reveals segmental reversal of ageing with increased cancer risk bioavailability of hydroxychloroquine tablets in healthy volunteers molecular mechanisms of atp secretion during immunogenic cell death autophagy induction by thiostrepton improves the efficacy of immunogenic chemotherapy type i interferons in anticancer immunity viral subversion of immunogenic cell death hydroxychloroquine is associated with impaired interferon-alpha and tumor necrosis factor-alpha production by plasmacytoid dendritic cells in systemic lupus erythematosus sustained type i interferon signaling as a mechanism of resistance to pd- blockade commensal bacteria control cancer response to therapy by modulating the tumor microenvironment gut microbiome influences efficacy of pd- -based immunotherapy against epithelial tumors the commensal microbiome is associated with anti-pd- efficacy in metastatic melanoma patients the microbiome in cancer immunotherapy: diagnostic tools and therapeutic strategies antibiotics are associated with decreased progression-free survival of advanced melanoma patients treated with immune checkpoint inhibitors gut bacteria composition drives primary resistance to cancer immunotherapy in renal cell carcinoma patients experience with hydroxychloroquine and azithromycin in the covid- pandemic: implications for qt interval monitoring cancer immunotherapy using irradiated tumor cells secreting heat shock protein tumgrowth: an open-access web tool for the statistical analysis of tumor growth curves gk is supported by the ligue contre le cancer (équipe labellisée); agence national de la recherche (anr) -projets blancs; anr under the frame of e-rare- , the era-net for research on rare diseases; ammica us / cnrs ums ; association pour la recherche sur le cancer (arc); association "le cancer du sein, parlons-en!"; cancéropôle ile-de- key: cord- -a wue e authors: cohen, isaac v.; makunts, tigran; moumedjian, talar; issa, masara a.; abagyan, ruben title: cardiac adverse events associated with chloroquine and hydroxychloroquine exposure in years of drug safety surveillance reports date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: a wue e chloroquine (cq) and hydroxychloroquine (hcq) are on the world health organization’s list of essential medications for treating non-resistant malaria, rheumatoid arthritis (ra) and systemic lupus erythematosus (sle). in addition, both drugs are currently used off-label in hospitals worldwide and in numerous clinical trials for the treatment of sars-cov- infection. however, cq and hcq use has been associated with cardiac side effects, which is of concern due to the higher risk of covid- complications in patients with heart related disorders, and increased mortality associated with covid- cardiac complications. in this study we analyzed over thirteen million adverse event reports form the united states food and drug administration adverse event reporting system to confirm and quantify the association of cardiac side effects of cq and hcq. additionally, we identified several confounding factors, including male sex, nsaid coadministration, advanced age, and prior diagnoses contributing to drug related cardiotoxicity. these findings may help guide therapeutic decision making and ethical trial design for covid- treatment. , and nct . despite limited efficacy data, hcq and cq are currently being administered as part of first-line treatment for sars-cov- in various hospitals across the world , . as previously mentioned, cardiac complications attributed to hcq and cq have been described in various reports. a systematic review of the literature published in june examined reports including individual cases and short series from an online database search. the report identified patients with cardiac events attributed to hcq and cq administered in the context of various inflammatory disorders with a median daily dose of mg for cq and mg for hcq . recent disproportionality analysis study observed an elevated prevalence of torsade de pointes and qt prolongation reports in cq and hcq . the cardiac aes of these therapeutics are of increased concern since a subset of patients infected with covid- present with cardiac injury, suggesting a relevant cardiovascular involvement in the pathophysiology of the disease . in a single-centered cohort study from wuhan, china, shi et al. examined the incidence of cardiac injury in hospitalized patients with covid- . among those, of the patients demonstrated cardiac injury, and higher in-hospital mortality rates were seen in patients with cardiac issues ( . %) compared to those without ( . %) . guo et al. described similar findings in a retrospective single-center case series analysis in which out of hospitalized patients suffered from myocardial injury. the mortality rate in those patients was . % compared to . % in those without cardiac injury . in support of these findings, recent studies have shown that major cardiac outcomes associated with cardiomyopathy ( %) and cardiac injury ( %) are common in critically-ill patients , . similar lines of evidence are also followed by an italian case report of a -year-old woman with lab-confirmed covid- who was admitted to the hospital for severe lv dysfunction and acute myopericarditis. this case highlights that sars-cov- can impact the cardiovascular system even in the absence of major respiratory tract involvement . other studies have observed covid- cardiac complications such as fulminant myocarditis, ventricular tachycardia [ ] [ ] [ ] . the goal of this study is to reanalyze the extensive clinical data of cq and hcq cardiac aes collected during the last years to derive the strength of the associations and, more importantly, contributing risk factors. the findings may improve the safety of these therapeutics for covid- treatment in patients that are already at higher risk of cardiac complications. fda adverse event reporting system. the study used over thirteen million ae reports available from the united states food and drug administration adverse event reporting system (faers) and its older version, adverse event reporting system (aers) data sets. at the time of the study the faers/aers set contained reports from years - , all available online at: https ://www.fda.gov/drugs /quest ions-and-answe rs-fdas-adver se-event -repor ting-syste m-faers /fda-adver seevent -repor ting-syste m-faers -lates t-quart erly-data-files data preparation. faers/aers reports are collected through voluntary reporting to the fda through medwatch and stored in quarterly data subsets with their respective parameters (age, sex, drug, ae etc.), and common case identifiers. faers data format has had changes historically, requiring each quarterly set to be individually downloaded and modified into consistent data tables [ ] [ ] [ ] . since the faers/aers set has reports from all over the world with their respective drug brand names, unique terms were recognized and translated into single generic cq and hcq names. the final data set contained , , ae reports. sle, ra, and malaria were considered for possible sources of reports, however due to low sample size of malaria treatment with hcq or cq in faers, only sle and ra were included in the analysis. cohort selection and data cleaning. , reports were obtained from the fda faers database to form three cohorts for analysis by logistic regression: cq cohort, hcq cohort and control cohort. the control cohort for both drugs was defined by reports with ra and sle patients where hcq and cq were not used (n = , ). the cq and hcq cohorts were defined by reports with ra and sle indication with cq (n = ) and hcq (n = , ) was used in addition to other therapeutics. rstudio (version . . ) and r (version . . ) were employed for data cleaning and logistic regression modeling. faers/aers data sets historically include a small fraction of duplicate reports. the set was scanned for these entries with the r package "dplyr" "distinct" function and were removed as appropriate (when records were found to have matching age, sex, weight, submission date, country of origin, drugs, indications, and outcomes). a summary of the records demographic factors is made available in table . in order to define the list of possible cardiac aes in this database a table was generated and manually checked for errors by the investigators. for a copy of this table of all aes considered cardiac related see supplementary table s . similarly, a list of non-steroidal anti-inflammatory drugs (nsaids) was generated for use in our analysis and is made available in supplementary table s . note that the number of nsaids did not include aspirin, as this was modeled separately due to expected divergent effects such as higher cardiovascular risk in the patient demographics. during the data cleaning stage, age was limited to a range of to years. for the purpose of our analysis, only sex values of "f" or "m" from fears/aers were analyzed. the r package "dplyr" function "mutate" and "str_detect" were employed for counting the number of nsaids and cardiac aes observed in each report. a subsample of the original database that included reports with non-empty values for age and sex was also prepared. for a summary of the sample size of the subgroups see supplementary table s . measured outcomes. the primary outcome of interest was the report of any cardiac ae as defined by one of terms listed in supplementary table s . this outcome was chosen over other more granular outcomes, such as qt prolongation, in order to increase the sensitivity of the analysis. the r package "glm" was employed scientific reports | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ for logistic regression modeling via the "binomial" family function. cardiac aes were the outcome of interest in logistic regression modeling and were coded as a binary (" " if occurred in the report or " " if not). covariates explored in the modeling included age (as a continuous variable expressed in years), sex (as a binary), disease state (coded as a binary of either sle or ra), nsaid usage (as a integer variable equal to the number of nsaids observed in the record), and aspirin usage (as a binary). nsaids were selected as a covariate of interest due to their high utilization among sle and ra patients. aic (akaike information criterion) and n number of records included are reported for each of the eight models presented. note that due to absence of some values in the raw data from the fda, models including age and sex have lower sample size than those without these covariates (supplementary table s ). subject weight was not used for model building due to extreme paucity of the data. models a, b, a, and b are built using the control and cq cohorts (described above and presented in tables and , and supplementary table s ). similarly, models a, b, a, and b are built using the control and hcq cohorts (described above and presented in tables and , and supplementary table s ).f coefficient estimates, standard error, adjusted odds ratio (aor), % confidence intervals ( % ci), and p values are reported (tables and ) . p values that meet the significance threshold of less than . are marked in tables with an asterisk (*). the adjusted odds ratio is defined as an odds ratio that controls multiple predictor variables table s ). these numbers of records are large, in particular for hcq, they cover a range of demographic parameters, and are sufficient to evaluate the contributors to the cardiac aes with logistic regression. the last row of table indicates an elevated number of cardiac side effectcontaining reports for both cq and hcq, . % and . % respectively, compared to . %. interestingly, the most common individual adverse effects (not grouped by the category) for each cohort were calculated and they did not include cardiac aes (see supplementary fig. s ). chloroquine. several logistic regression analyses were performed in order to evaluate whether cardiac aes were related to cq. binary logistic regression was employed to determine the confounding variables contributing to the apparent effects of the drugs on occurrence of cardiac side effects. in the simplest analysis model a ( hydroxychloroquine. hcq cardio ae analysis reveals a similar pattern to the results presented in table . table explores the same effects due to hcq in ra and sle patients. sle, nsaid use, aspirin use, male sex, and advanced age were all shown to be important factors in predicting cardiac aes (table ). in the simplest model a, hcq was shown to significantly increase cardiac aes by a modest adjusted odds ratio of . ( % ci . - . ) . interestingly, in model b this effect was lost after controlling for nsaid and aspirin use (p = . ). surprisingly, after adding age and sex into the model this effect's significance was regained (model b, aor . [ % ci . - . ]). although modest, hcq has been shown to have significant effects on reporting of cardiac adverse reactions. these trends have also been shown to be exacerbated by both clinical and demographic factors (table ). in our study we analyzed , fda adverse event reports divided into cq, hcq and control cohorts to determine their association with cardiac aes when taking into account other factors. although both drugs were significantly associated with increased cardiac ae reports, it was observed that this effect was substantially larger for cq than hcq (fig. ) . additionally, age, sex, concurrent nsaid use, and disease state were identified to contribute to cardiotoxicity reporting with both therapeutics. after controlling for these factors, it was observed that the deleterious effects of cq and hcq on cardiac aes remained significant (tables and ) . we expanded the current safety surveillance evidence by using a more comprehensive list of cardiac aes (supplementary table s ), to avoid diluting the safety signal with many individual terms. additionally, we performed multivariate analysis and identified several at-risk populations. the results of the multivariate binary logistic regression were validated by prior knowledge from literature: ( ) sex, when compared females, males are more often diagnosed with myocardial infarction, fatal coronary heart disease, among other cardiac diseases ; ( ) age, cardiovascular disease (cvd) has been shown to increase with age across multiple populations ; ( ) www.nature.com/scientificreports/ nsaids, especially preferential and selective cyclooxygenase- inhibitors, increase the risk of cvd , . two other essential factors were shown to be necessary to correct for, due to their association with cardiac side effects in the patient population used for our analysis and the current utilization of cq and hcq: ( ) sle: sle patients have been shown to have a greater risk of cardiac aes than patients with ra. this makes sense, as it is well known that cvd is one of the major complications and is one of the leading factors in mortality in patients living with sle . additionally, the host inflammatory response seen in sle may be similar to that in covid , . ( ) aspirin: it may be expected for aspirin to present a protective effect, however the opposite effect was observed. use of aspirin in itself does not increase cvd risk, in fact it is mainly used for preventing cardiac events . aspirin use in our study population is likely heavily associated prior cardiac related medical history, that is not listed in fears, and could not be otherwise accounted for in the model. aspirin increases the robustness of the model by correcting for prior treatment of cardiac disease or prevention in high risk patients. the regression model was instrumental to exclude this aspirin association from the quantification of the direct cardiac side effects of cq and hcq, which remained significant after adjustment ( generalizability of results to covid- treatment with cq and hcq. this analysis revealed a trend of increased cardiac ae reporting propensity associated with cq and hcq that likely apply to a wide range of patients. although our study was not performed on reports from covid- patients, due to absence of such reports at the time of the study, the results may still be of value because of our mode of evaluation. additionally, the cardiac complications and related mortality of sars-cov- patients are mainly attributed to the inflammatory nature of the infection , , which is also seen in sle pathophysiology , . in fact, several case reports describe the heart related mortality being associated with inflammation of the myocardium , . cq and hcq use in sle and ra cohorts for the study was a beneficial coincidence, since they are also inflammatory conditions affecting the cardiovascular system that are also treated with cq and hcq , . further clinical studies are needed to subdivide the cardiac ae's in to clinically relevant subcategories, such as arrythmia or myocardial infarction. the attempts to split the cardiac outcomes into subcategories with the current dataset met the obstacle of insufficient numbers of reports for significant conclusions. clinical studies with prospective methods may benefit from targeted monitoring of the important induvial clinical outcomes. and hcq with respect to other therapeutics used for ra and sle. the association remained significant when demographic parameters and concurrent medications were accounted for in the analysis. it may be beneficial to closely monitor patients for cardiac complications. hcq may be safer for use than cq in patients at higher risk of cardiac complications. although, when compared to cq, hcq use was associated with a lower reporting of these events, the association was still statistically significant. when available, alternative therapeutics may be safer to use for sars-cov- patients who are already at higher risk of cardiovascular complications due to age, pre-existing cardiovascular issues, concomitant medications, and the sars-cov- infection itself. due to the voluntary nature of the faers/aers reports, actual population incidences of the adverse events cannot be derived. medwatch reporting may also be biased by newsworthiness and legal variables. the safety surveillance data misses comprehensive medical records and medication history limiting the scope of the analysis. as with any association study, causality may not be derived from association, since the cases were not uniformly evaluated for causality by clinical specialists. however, the postmarketing surveillance data analysis of over , reports provides population scale evidence which can be used to identify safety signals that might go unnoticed in small scale studies. while our approach with multivariate logistic regression controls for important biases in the data, such as sex, age, and concomitant drug use, some unaccounted-for factors may remain. the outcome modeled in this study (presence of any cardiac ae) is broad; further study is necessary to investigate more granular outcomes such as arrythmias. new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid- ? chloroquine for the novel coronavirus sars-cov- targeting endosomal acidification by chloroquine analogs as a promising strategy for the treatment of emerging viral diseases hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies hhs accepts donations of medicine to strategic national stockpile as possible treatments for covid- patients remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro nct ) hydroxychloroquine treatment for severe covid- pulmonary infection (hydra trial) (hydra) nct ) hydroxychloroquine chemoprophylaxis in healthcare personnel in contact with covid- clinicaltrials.gov. 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kashour, zakariya; aldosary, oweida; riaz, muhammad; tlayjeh, haytham; garbati, musa a.; tleyjeh, rana; al-mallah, mouaz h.; sohail, m. rizwan; gerberi, dana; bin abdulhak, aref a.; giudicessi, john r.; ackerman, michael j.; kashour, tarek title: the cardiac toxicity of chloroquine or hydroxychloroquine in covid- patients: a systematic review and meta-regression analysis date: - - journal: mayo clin proc innov qual outcomes doi: . /j.mayocpiqo. . . sha: doc_id: cord_uid: rgvcimk objective to systematically review the literature and estimate the risk of chloroquine (cq) and hydroxychloroquine (hcq) cardiac toxicity in covid- patients. methods we searched multiple data sources including pubmed/medline, ovid embase, ovid ebm reviews, scopus, and web of science, and medrxiv.org from november through may , . we included studies that enrolled covid- patients treated with cq or hcq, with or without azithromycin and reported on cardiac toxicities. we performed a meta-analysis using the arcsine transformation of the different incidences. results a total of studies with a total of patients were included. the pooled incidence of tdp arrhythmia or vt or cardiac arrest was per , % ci ( - ), i = %, studies with patients. among studies of patients, the pooled incidence of discontinuation of cq or hcq due to prolonged qtc or arrhythmias was %, % ci ( - ), i = %. the pooled incidence of change in qtc from baseline of ≥ ms or qtc ≥ ms was %, % ci ( - ), i = %. mean/median age, coronary artery disease, hypertension, diabetes, concomitant qt prolonging medications, icu care, and severity of illness in the study populations explained between-studies heterogeneity. conclusions treatment of covid- patients with cq or hcq is associated with a significant risk of drug-induced qt prolongation and relatively higher incidence of tdp/vt/cardiac arrest. therefore, these agents should not be used routinely in the management of covid- disease. covid- patients who are treated with antimalarials for other indications should be adequately monitored. the severe acute respiratory syndrome coronavirus (sars-cov- ) that causes the coronavirus disease (covid- ) has spread across the globe claiming hundreds of thousands of lives and causing enormous economic losses. repurposing of approved drugs for the treatment of covid- was a logical approach before an effective vaccine is available. among the drugs that received significant early attention were the antimalarial medications chloroquine (cq) and hydroxychloroquine (hcq). cq and hcq are weak bases that increase the ph of the intracellular vesicles like endosomes. these changes could affect several stages of viral life cycles from cell entry, viral replication, and viral particle assembly to viral particle release from the host cells. additionally, these vesicle ph changes interfere with antigen processing and presentation and consequently immune cell activation and production of proinflammatory cytokines. this effect is favorable in the management of autoimmune diseases like systemic lupus erythematosus (sle) and may have favorable impact in covid- patients with cytokine storm. hcq was also reported to improve endothelial function and reverse prothrombotic state, which, is relevant to patients with covid- since they manifest significant pulmonary vascular endothelialitis and microvascular thrombosis. the successful demonstration of in vitro antiviral properties of cq and hcq against sars-cov- virus led to initiation of several clinical studies testing the therapeutic potential of cq and hcq in covid- . , the early encouraging experience of cq therapy in patients from china led to its recommendation by the national health commission of china. , another non-randomized study of covid- patients treated with hcq alone or in combination with j o u r n a l p r e -p r o o f azithromycin in france showed reduced nasopharyngeal viral carrier sate at days after the initiation of treatment. despite its serious methodological limitations, this report received exceptional attention by media and politicians and triggered a widespread off label use of cq and hcq for covid- with subsequent reports of cq-related deaths. while cq and hcq are generally considered safe, qt prolongation and torsade de pointes (tdp) ventricular tachycardia as well as other arrhythmias, myocarditis, and cardiomyopathy have been reported with chronic hcq use. [ ] [ ] [ ] [ ] recent reports confirmed the increased risk of qt prolongation among covid- patients. , the indiscriminate use of the antimalarial medications for the treatment of covid- in the absence of robust clinical evidence for their efficacy, coupled with associated potential harm, calls for rigorously conducted systematic reviews/meta-analyses of the available clinical data to present a clearer picture about their safety and efficacy and provide data-informed view regarding their utility in the treatment of covid- . in this study, we set to systematically review the literature regarding the cardiac toxicity of cq or hcq in patients with covid- . we followed preferred reporting items for systematic reviews and meta-analyses (prisma) and meta-analysis of observational studies in epidemiology (moose) guidelines for reporting systematic review and meta-analysis of observational studies guidelines. studies that reported electrocardiographic changes and/or cardiac arrhythmias in covid- patients treated with cq or hcq with and without azithromycin were included using pre-specified inclusion j o u r n a l p r e -p r o o f criteria as follows: ( ) covid- patient population, ( ) the study included more than patients receiving either one of the agents, ( ) electrocardiographic changes and/or cardiac arrhythmias were reported. in order to avoid introducing non-independence by including patients in the analysis more than once, we included results from the same study with the larger sample size if there were more than one study reporting data on overlapping patient population. the literature was searched by a medical librarian for the concepts of cq or hcq combined with covid- on several databases including pubmed/medline, ovid embase, ovid ebm reviews, scopus, and web of science. the search strategies were created using a combination of keywords and standardized index terms and were run up to may, , (supplement ). we also searched for unpublished manuscripts using the medrxiv, in addition to google scholar and the references of eligible studies and review articles. endpoints included the incidence of: ( ) change in qtc interval from baseline of ≥ ms, ( ) qtc ≥ ms, ( ) the composite of endpoint and , ( ) tdp arrhythmia, or ventricular j o u r n a l p r e -p r o o f tachycardia (vt) or cardiac arrest and ( ) discontinuation of treatment due to prolonged qt or arrhythmias. the number and percentage of subjects experiencing different endpoints were extracted from each study. due to the very low incidence of tdp and other endpoints (rare events), the arcsine transformation was used to obtain a pooled estimate of the different incidences. for metaanalyses of rare events, this transformation is more appropriate than the commonly used logit transformation, as it can accommodate studies with no observed events, without requiring a continuity correction. [ ] [ ] [ ] both fixed-effects and random-effects dersimonian & laird models with inverse variance method were considered. statistical tests for heterogeneity were performed using the cochrane's q and i-squared statistics, with the assumption of homogeneity being considered invalid for p values < . . when homogeneity could not be assumed, we reported summary estimates and forest plots from the random-effects models. meta-regression analyses were used to assess whether the incidence of different endpoints significantly varied by multiple variables specified a priori. these variables were chosen based on risk factors that are known to potentially increase the risk of cardiac arrhythmias (age, sex, cad, chf, dm, disease severity, ckd, icu care, and mortality as another surrogate of disease severity). we then performed sensitivity analyses by repeating the above analyses after excluding two studies that used high dose cq or hcq. two-tailed p values less than . were considered to be statistically significant. all statistical analyses were performed using meta and metafor packages in r statistical software, version . . . j o u r n a l p r e -p r o o f a total of studies with a total of patients, including single center and multicenter studies, were included in our systematic review. , , - figure shows the result of our search strategy (prisma flow diagram). table illustrates the general characteristics of the included studies. all included studies were of high methodological quality in terms of exposure ascertainment or outcome assessment (table ) . several important cardiovascular adverse events have been observed in our meta-analysis. however, several other events have not been reported consistently by all of the included studies and therefore, we were unable to perform a meta-analysis for all the events. the reported events that we could not meta-analyze were cardiac complications other than tdp, ventricular tachycardia and cardiac arrest. tdp tachycardia, ventricular tachycardia and cardiac arrest events were observed in patients in studies with total of patients. the pooled incidence of these events was per , % ci ( - ), i = % (figure ). however, only two episodes of tdp tachycardia were reported among patients. the pooled incidence of discontinuation of cq or hcq due to prolonged qtc or arrhythmias was %, % ci ( - ), i = % among patients from studies (figure ). among studies with patients, the pooled incidence of change in qtc from baseline of ≥ ms or qtc ≥ ms was %, % ci ( - ), i = % (figure ) . in studies of patients, the pooled incidence of change in qtc from baseline of ≥ ms was %, % ci ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , i = % ( figure ). further, the pooled incidence of qtc ≥ ms was %, % ci ( - ), i = % from studies with patients, (figure ). for tdp arrhythmia or vt or cardiac arrest, concomitant qt prolonging medications prevalence in included studies explained heterogeneity. the amount of heterogeneity accounted for concomitant qt prolonging medications was %. regarding discontinuation due to prolonged qtc or arrhythmias, age, cad, and dm explained heterogeneity. the amount of heterogeneity accounted for by age, cad and dm was: %, % and %, respectively. for the change in qtc from baseline of ≥ ms, age, cad, and htn and icu admission explained heterogeneity. the amount of heterogeneity accounted for by age, cad, htn and icu care was: %, %, % and %, respectively. on the other hand, heterogeneity for qtc ≥ ms was explained by age, cad, and htn and severity of illness. the amount of heterogeneity accounted for by mean/median age, and cad, and htn prevalence and severity of illness was: %, %, % and %, respectively. finally, age, cad, htn, concomitant qt prolonging medications, icu care and severity of illness were responsible for observed heterogeneity for the change in qtc from baseline of ≥ ms or qtc ≥ ms. the amount of heterogeneity accounted for mean/median age, and cad, htn, concomitant qt prolonging medications and icu care prevalence and severity of illness was: %, %, %, %, % and %, respectively. after excluding the studies that used high dose cq or hcq, , we did not observe an important change in pooled estimates or associated heterogeneity. in this meta-analysis, we systematically examined the risk of qtc prolongation and its associated complications in patients with covid- treated with antimalarial medications cq and hcq (cq/hcq). the majority of studies included in this analysis used hcq alone or in combination with azithromycin. our analysis revealed that treatment with cq/hcq was associated with a clinically significant increased risk of qtc prolongation and discontinuation of drug due to this risk. in addition, cq/hcq was associated with a clinically significant risk of tdp or vt or cardiac arrest of per ( % ci . - ). the incidence of critical qtc prolongation defined as qtc ≥ ms or ∆qtc ≥ ms ranged from % to %. one of the most remarkable findings is that in the study by bessière et al, % of the studied patients exhibited an increase in qtc prolongation and % had critical qtc prolongation. in our pooled analysis, critical qtc prolongation ranged between % and % with significant heterogeneity among the studies (i of up to %). several factors contributed to the observed heterogeneity were identified by the meta-regression analysis. these include age, hypertension, cad, icu admission, dm, use of other qtc prolonging agents, and covid- disease severity. these factors are concordant with the biological explanation for the observed differences, as it is well known that underlying cardiac conditions, comorbidities, and inflammatory states increase the risk of drug induced qtc prolongation. , this meta-analysis revealed low but clinically significant risk of combined endpoint of tdp, ventricular tachycardia and cardiac arrest. however, we could not perform a meta-analysis on tdp separately because there were only two reported cases of tdp among patients from studies ( . %). the low incidence of tdp is probably an underestimate. a number of factors can explain this low incidence of tdp; most importantly the precautionary discontinuation of the drugs when qtc reaches a certain threshold (qtc > ms or ∆qtc ≥ ms), short duration of therapy, and in certain instances, the therapeutic intervention for long qt using qt shortening agents as reported by saleh et al for example. indeed, in our pooled analysis, % of patients had their medication discontinued because of qtc prolongation and in one study by jain et al, % of patients had their cq/hcq discontinued because of qtc prolongation. moreover, some tdp cases could have been missed because of underreporting or misclassification. in fact, the two largest studies included in this meta-analysis did not specifically include tdp as a separate endpoint but grouped all arrhythmias under one category. the study by rosenberg et al observed arrhythmias in . % and cardiac arrest in % of patients and it is quite possible that some of these arrhythmias were tdp or some of the cardiac arrests were preceded by tdp. nonetheless, the incidence of tdp reported here are consistent with the published data on the drug induced tdp. the risk of developing tdp in association with non-antiarrhythmic drugs is relatively low; for instance, the risk for cisapride, which has been removed from the market, was . %. the risk of tdp with other non-antiarrhythmic drugs is in the range of that was reported with cisapride. the observed incidence of tdp observed in this meta-analysis ( . %) is -fold higher than that of cisapride. it is noteworthy that cisapride and terfenadine (a nonsedating antihistamine) were taken off the market because the risk of tdp even though the risk of tdp associated with their use in absolute terms was very low. this underscores the importance of taking into account the total number of potential lethal events rather than the expressed ratios when assessing the risk drug induced arrhythmias. it is also well known that the highest risk for drug-induced qt prolongation and tdp is associated with class-iii antiarrhythmic drugs, which ranges between - % over - years. the risk of tdp with sotalol therapy at a low daily dose of mg is only . %. this risk is much higher than the observed risk with cq and hcq in this study; however, the estimated risk reported for the antiarrhythmic drugs was over to years of chronic use as opposed to the risk reported here for cq and hcq over a very short-term use. in fact, this increases further the concern about the cardiac risk associated with cq and hcq treatment in covid- disease. a number of other cardiac adverse events documented in the included studies were not negligible and include, myocardial injury, acute mi, myocarditis, and others. notwithstanding, a cause and effect relationship between cq/hcq exposure and these complications cannot be j o u r n a l p r e -p r o o f inferred from these studies. however, it is noteworthy to mention that in the study by borba et al, the incidence of acute cardiac injury was higher in the high dose cq group in comparison with low dose cq ( % vs. . %) and the two patients with sustained ventricular tachycardia also occurred in the high dose cq, which could imply dose-response relationship and probable cause and effect link. . in a recent meta-analysis of hcq effects on cardiovascular system, ventricular hypertrophy was noted in % of patients while heart failure was noted in . % of patients. it is worth noting also that it has been a common practice to use hcq in combination with azithromycin for covid- during the current pandemic. azithromycin has been identified as a potential cause of significant serious cardiac arrhythmias through qt prolongation dependent and independent mechanisms and has been linked to increased risk of sudden cardiac death. , hence, the concomitant use of cq/hcq and azithromycin or other qt prolonging agents could potentially increase the risk of serious cardiac arrhythmias and death particularly in critically ill patients or those with risk factors for qt prolongation. our findings indicate that the cardiac risk imposed by cq/hcq use in covid- disease is not trivial and support the need for close monitoring of covid- patients who are treated with cq/hcq alone or in combination with azithromycin. since their efficacy in improving the outcomes of covid- patients is lacking, these agents should be used only in the context of randomized clinical trials given the potential harm that could be associated with their widespread use. this position is supported by the recent fda statement. our meta-analysis is the first comprehensive systematic review examining the risk of qt prolongation and its associated adverse events in covid- patients treated with cq/hcq. however, like any meta-analysis, it has several limitations. first, the retrospective nature of most of the included studies make them prone to incomplete or missing data. second, there were variations in the variables collected by individual studies particularly related to reporting qtc parameters and adverse events and significant differences in the patient populations enrolled by these studies. third, there was marked heterogeneity in our pooled estimates; however, we performed a meta-regression that allowed us to identify contributors to the observed heterogeneity and further determine populations at risk for cq/hcq induced qt prolongation, which further strengthens our study and its conclusions. our meta-analysis indicates that the treatment of covid- patients with cq or hcq alone or in combination with azithromycin is associated with a significant risk of drug-induced qt prolongation. cq/hcq use resulted in a relatively higher incidence of tdp as compared to drugs withdrawn from the market for this particular adverse effect. therefore, these agents should be used only in the context of randomized clinical trials in patients at low risk for drug-induced qt prolongation and with adequate safety monitoring. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. a review on possible modes of actions of chloroquine/ hydroxychloroquine: repurposing against sar-cov- (covid ) pandemic hydroxychloroquine reverses the prothrombotic state in a mouse model of antiphospholipid syndrome: role of reduced inflammation and endothelial dysfunction pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid- in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) fda approved drugs with broad anti-coronaviral activity inhibit sars-cov- in vitro breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies multicenter collaboration group of department of science and technology of 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treated with hydroxychloroquine and/or azithromycin institution-wide qt alert system identifies patients with a high risk of mortality high prevalence of corrected qt interval prolongation in acutely ill patients is associated with mortality: results of the qt in practice (qtip) study spontaneous adverse event reports of serious ventricular arrhythmias, qt prolongation, syncope, and sudden death in patients treated with cisapride drug-induced long qt syndrome an update on risk factors for drug-induced arrhythmias cardiac complications attributed to chloroquine and hydroxychloroquine: a systematic review of the literature azithromycin and the risk of cardiovascular death azithromycin causes a novel proarrhythmic syndrome fda cautions against use of hydroxychloroquine or chloroquine for covid- outside of the hospital setting or a clinical trial due to risk of heart rhythm problems the data underlying this article are available in the article and in its online supplementary material. dr. giudicessi reports equity interest-glaxosmithkline, medtronic, myokardia, and pfizer".however, these relations are not related to this work.prof. ackerman reports the following: consultant for abbott, audentes therapeutics, boston scientific, invitae, lqt therapeutics medtronic, myokardia, and uptodate. dr ackerman and mayo clinic are involved in an equity/royalty relationship with alivecor.however, these relations are not related to this work.other authors report no conflict of interest.j o u r n a l p r e -p r o o f key: cord- -mzon fd authors: ferreira, a.; oliveira-e-silva, a.; bettencourt, p. title: chronic treatment with hydroxychloroquine and sars-cov- infection. date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: mzon fd background: hydroxychloroquine sulphate (hcq) is being scrutinized for repositioning in the treatment and prevention of sars-cov- infection. this antimalarial drug is also chronically used to treat patients with autoimmune diseases. methods: by analyzing the portuguese anonymized data on private and public based medical prescriptions we have identified all cases chronically receiving hcq for the management of diseases such as systemic lupus erythematosus, rheumatoid arthritis, and other autoimmune diseases. additionally, we have detected all laboratory confirmed cases of sars-cov- infection and all laboratory confirmed negative cases in the portuguese population (mandatorily registered in a centrally managed database). cross linking the two sets of data has allowed us to compare the proportion of hcq chronic treatment (at least grams per month) in laboratory confirmed cases of sars-cov- infection with laboratory confirmed negative cases. results: out of , sars-cov- positive patients, ( . %) were chronically treated with hcq, while , ( . %) out of , negative patients were receiving it chronically (p= . ). after adjustment for age, sex, and chronic treatment with corticosteroids and/or immunosuppressants, the odds ratio of sars-cov- infection for chronic treatment with hcq has been . ( . - . ). conclusions: our data suggest that chronic treatment with hcq confer protection against sars-cov- infection. several in vitro studies have shown chloroquine phosphate and hydroxychloroquine sulphate (hcq) to be effective in both preventing and treating sars-cov- infection in isolated cells ( ) ( ) ( ) . chloroquine phosphate and hcq were also found effective in the treatment of covid- patients in small nonrandomized or unblinded clinical studies ( ) ( ) ( ) . a recent observational study has shown no association between hcq use and the composite endpoint of intubation or death in hospitalized patients ( ) . however, up until today there are no available results from well designed, randomized, double blinded, placebo controlled clinical trials showing any evidence of a therapeutic or preventive effect of these drugs in sars-cov- infection. these two drugs are also chronically used to treat systemic lupus erythematosus (sle), rheumatoid arthritis (ra) and other autoimmune diseases. sle patients are at increased risk of viral infections ( ). increased ratios of serious bacterial and viral infections have been shown or are expected in different autoimmune diseases ( - ). thus, patients with these diseases should be expected to have increased ratios of sars-cov- infection as compared to the general population. antimalarials appear to have a protective effect against infections in patients with sle ( , ) . to evaluate the effect of chronic treatment with hcq for other medical conditions in sars-cov- infection incidence. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org / . / . / patients and methods the portuguese national health service (serviço nacional de saúde -sns) has a centrally controlled electronic database where all drug prescriptions, both from public and private medical care, are systematically registered (prescrição anonymized data were extracted from these databases. by analyzing these sets of data, we were able to detect all patients with sars-cov- confirmed infections and all clinically suspected but non-confirmed patients between mars , (the date of the first portuguese case) and the moment of the analysis. all cases prescribed with hcq between january , and december , for other medical conditions were documented too. additionally, we identified all prescriptions of corticosteroids (prednisolone, methylprednisolone, dexamethasone and deflazacort) and/or immunosuppressants . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . to be considered under chronic treatment with hcq, cases had to be prescribed with at least grams of hcq per month, on average. chloroquine phosphate is not available in portugal. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . patients with, at least, months on corticosteroids and/or immunosuppressants were considered under chronic immunosuppressant treatment. for each case, the first hcq prescription was identified (index prescription). then, the total hcq dose (in grams) prescribed from the index prescription (inclusive) was quantified up until the last day of . finally, the average monthly dose of hcq was computed. the same methodology was used for corticosteroids and immunosuppressants, but the number of prescriptions (not grams) was quantified. non-residents in portugal were excluded from the study. anonymized raw data were extracted from the abovementioned databases on july , . the raw data were revised to ensure internal consistency and . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . in the set of patients with case definition, , received hcq (at least grams per month) and were prescribed with corticosteroids and/or immunosuppressants for months at least. the results of univariate analysis are summarized in table . sars-cov- positive patients were older and more frequently of the male gender than negative patients. the proportion of hcq chronic treatment was higher in negative patients is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint pcr test for hcq chronic treatment was . ( . - . ). immunosuppressive treatment was found to be positively associated with sars-cov- infection with an odds ratio of . ( . - . ). as far as we know, this is the first study retrospectively assessing the relationship between chronic treatment with hcq and sars-cov- infection in a large sample of patients at a nation-while level. in this study, we only included patients chronically prescribed with hcq during , whose prescriptions continued into . this way, we were able to exclude those who have started taking hcq aiming to treat or prevent sars-cov- infection. otherwise, this would be a confounding factor in the analysis of the data. although we have no information on comorbidities of the patients registered in the databases used in this study, it seems fair to assume that patients who chronically received hcq at a dose of at least grams per month have sle, ra or other autoimmune diseases. in fact, in portugal, hcq is approved for the treatment of sle, discoid lupus erythematosus, ra, juvenile idiopathic arthritis, and for the prophylaxis and treatment of malaria, which is not endemic (only imported cases). in the last case, the dose and duration of the treatment are much lower than the ones selected for inclusion in this study. thus, patients using hcq for prophylaxis or treatment of malaria were excluded from the study by the inclusion criteria we have used. the prevalence of sle and ra in portugal is . % and . %, respectively ( ) . these figures have allowed us to . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . patients with sle or ra have numerous cellular and humoral abnormalities that contribute to an increased susceptibility to infectious agents ( ) ( ) ( ) . sle patients have a . times higher incidence rate of severe herpes simplex virus infection than controls ( ) . sle patients treated with glucocorticoids alone also have a . -fold hazard ratio for severe infections when compared to patients treated with hcq alone ( ) . the prevalence of papillomavirus and cytomegalovirus is supposedly higher in patients with autoimmune diseases too ( , ) . besides the negative effect of the immunologic disturbance, these patients usually receive immunosuppressive drugs too, making them even more susceptible to infection ( ) ( ) ( ) . thus, the available data suggest that autoimmune patients are at increased risk of viral infections. the benefits of hcq treatment in this context, if any, depend on its ability to prevent those infections. the covid- risk scoring guide of the british society for rheumatology ( ) seems to adopt the same orientation, by scoring with zero the use of hcq while it scores with three high-dose corticosteroids and cyclophosphamide. our data give further support to this suggestion. we were able to show that patients taking hcq have had reduced odds of sars-cov- infection. this effect was most evident after adjustment for age, sex, and chronic immunosuppressive treatment, increasing the likelihood that chronic treatment with hcq actually has a protective effect against sars-cov- infection. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . a recent paper compared the proportions of hcq use in sars-cov- positive and negative patients ( ) , finding no differences between the two groups. however, as study limitation, the authors stated that the duration of treatment was not documented and only patients received hcq in the positive cases group. an indian study showed that hcq (in the dosage currently used in the prophylaxis of malaria and after four maintenance doses) was effective in preventing sars-cov- infection in health care workers ( ) , while another paper showed no statistically significant effect of hcq (in the dose of mg/day for four days, after a loading dose of mg plus mg after to hours) in postexposure prevention ( ) . the first two studies evaluated the effectiveness of hcq as pre-exposure prophylaxis, while the latter tested its effectiveness as post-exposure prophylaxis. taken together, these results suggest that a relatively long period of hcq treatment may be necessary in patients without prior contact with the virus to obtain a preventive effect. we believe our data supports this hypothesis. available data show that there was a significant increase of hcq prescriptions in march and in the first two weeks of april . additional studies are needed to understand whether this increase in hcq consumption has had any effect on the evolution of the outbreak in portugal and on the incidence of adverse events. in conclusion, our findings suggest that hcq might have a preventive effect of sars-cov- infection. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . despite having a considerable number of lab tests that were missing in the official electronic registries, we believe that we have captured all suspected confirmed and unconfirmed cases of sars-cov- infection by checking the lab data (sinave lab) against de the obligatory registry database (sinave med). furthermore, all positive and negative cases included in our sample were confirmed by pcr testing, giving consistency to our results. the fact that we do not have information on comorbidities may be a major limitation of the study, because it did not allow us to exclude a potential selection bias -for example, physicians may have decided to prescribe hcq to patients with fewer comorbidities, and because it was not possible to adjust the effect of hcq to such comorbidities. thus, the observed effect could be related to a better health status of patients and not to a preventive action of hcq. however, clinical guidelines recommend the use of antimalarials in all cases ( ) , regardless of the existence of comorbidities (namely, those that may affect resistance to infection, such as chronic renal failure). it is therefore to be expected that physicians have followed those recommendations and that no such selection bias has occurred. we do not have any data on the therapeutic compliance of patients, and this is a study limitation too. although the proportions of hcq chronic treatment are low, the difference in proportions between the two groups is unlikely to be cancelled out by the study pitfalls mentioned above. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . considering the low toxicity ( ) ( ) ( ) and the enormous clinical experience with hcq utilization, these results strongly suggest it should continue to be used in patients with autoimmune diseases, mostly in the context of the sars-cov- pandemic. whether it should be used as a pre-exposure prophylactic measure in other risk groups or in the general population is unknown. we believe that our findings suggest that at least it should be considered for this purpose. the lockdown is severely affecting the world's economy, increasing unemployment, and inducing more suffering than the disease itself. eventually, its effects on the welfare of the populations will be much worse than the potential adverse effects of hcq prophylaxis. furthermore, it cannot be maintained for an extended period. thus, while waiting for an effective and safe vaccine, a chemoprophylaxis-based strategy, using this inexpensive and safe drug, continuously monitored and modified according to the results of ongoing clinical trials, may be justified. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . negative cases - , ) due to missing data. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial efficacy of hydroxychloroquine in patients with covid- : results of a randomized clinical trial observational study of hydroxychloroquine in hospitalized patients with covid- international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity risk of severe herpes simplex virus infection in systemic lupus erythematosus: analysis of epidemiology and risk factors analysis in taiwan risk factors for cytomegalovirus disease in systemic lupus erythematosus (sle): a systematic review risk of human papillomavirus infection in women with rheumatic disease: cervical cancer screening and prevention risk of serious infection for patients with systemic lupus erythematosus starting glucocorticoids with or without antimalarials predictors of major infections in systemic lupus erythematous epidemiological study of rheumatic diseases in portugal -epireumapt immunopathogenesis and spectrum of infections in systemic lupus erythematosus. semin arthritis rheum international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted the risk of infections associated with rheumatoid arthritis, with its comorbidity and treatment infections in systemic lupus erythematosus and rheumatoid arthritis continuous hydroxychloroquine or colchicine therapy does not prevent infection with sars-cov- : insights from a large healthcare database analysis healthcare workers & sars-cov- infection in india: a case-control investigation in the time of covid- a randomized trial of hydroxychloroquine as postexposure prophylaxis for covid- update of the eular recommendations for the management of systemic lupus erythematosus antimalarials -are they effective and safe in rheumatic diseases? reumatologia it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review)the copyright holder for this preprint this version posted june , . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint key: cord- -gn hg oc authors: infante, marco; ricordi, camillo; fabbri, andrea title: antihyperglycemic properties of hydroxychloroquine in patients with diabetes: risks and benefits at the time of covid‐ pandemic date: - - journal: j diabetes doi: . / - . sha: doc_id: cord_uid: gn hg oc the antimalarial drug hydroxychloroquine (hcq) has long been used as a disease‐modifying antirheumatic drug for the treatment of several inflammatory rheumatic diseases. over the last three decades, various studies have shown that hcq plays also a role in the regulation of glucose homeostasis. although the mechanisms of action underlying the glucose‐lowering properties of hcq are still not entirely clear, evidence suggests that this drug may exert multifaceted effects on glucose regulation, including improvement of insulin sensitivity, increase of insulin secretion, reduction of hepatic insulin clearance and reduction of systemic inflammation. preliminary studies have shown the safety and efficacy of hcq (at a dose ranging from to mg/day) in patients with type diabetes over a short‐term period. in , hcq has been approved in india as an add‐on hypoglycemic agent for patients with uncontrolled type diabetes. however, large randomized controlled trials are needed to establish the safety and efficacy profile of hcq in patients with type diabetes over a long‐term period. with regard to the covid‐ pandemic, several medications (including hcq) have been used as off‐label drugs due to the lack of proven effective therapies. however, emerging evidence shows limited benefit from hcq use in covid‐ in general. the aim of this manuscript is to comprehensively summarize the current knowledge on the antihyperglycemic properties of hcq and to critically evaluate the potential risks and benefits related to hcq use in patients with diabetes, even in light of the current pandemic scenario. this article is protected by copyright. all rights reserved. since the s, the antimalarial drugs chloroquine and hydroxychloroquine (hcq) have been used as disease-modifying antirheumatic drugs (dmards) for the treatment of several inflammatory rheumatic diseases, including rheumatoid arthritis (ra) and systemic lupus erythematosus (sle). ( ) importantly, hcq has shown a good safety and tolerability profile in most patients, even during pregnancy and breastfeeding. ( , ) intriguingly, a growing body of evidence coming from studies conducted over the last three decades suggests that hcq plays also a role in the regulation of glucose homeostasis in individuals with and without diabetes. ( ) although the exact mechanisms of action underlying the glucose-lowering properties of hcq are still not entirely clear and may differ between patients with and without diabetes, pre-clinical and clinical data suggest that hcq could exert multifaceted effects on glucose homeostasis, namely: improvement of insulin sensitivity, increase of insulin secretion, reduction of hepatic insulin clearance and intracellular insulin and insulin-receptor complex degradation, increase of adiponectin levels, reduction of systemic inflammation, and/or reduction of inflammation-induced insulin resistance in adipocytes and skeletal muscle cells. ( ) ( ) ( ) in this regard, a randomized study is currently underway at washington university (metahcq, metabolic effects of hydroxychloroquine; clinicaltrials.gov identifier: nct ) to evaluate the effects of - week hcq administration (at a dose of mg/day) on insulin sensitivity (determined by this article is protected by copyright. all rights reserved. accepted article hyperinsulinemic euglycemic clamp), fasting blood glucose, lipid profile and serum biomarkers of inflammation in subjects with t d. remarkably, several studies demonstrated that hcq use is associated with a significantly reduced risk of developing diabetes in non-diabetic subjects with inflammatory rheumatic diseases, including ra, ( ) ( ) ( ) sle, ( ) psoriasis, ( ) and sjögren syndrome. ( ) in the early s, an italian -month randomized, placebo-controlled trial conducted by quatraro et al. ( ) in t d subjects first showed that the addition of hcq ( mg/day) to glibenclamide or insulin led to a significant reduction in glycated hemoglobin (hba c), accompanied by a % reduction in daily insulin dose among patients on insulin therapy. thereafter, a canadian randomized, placebo-controlled trial conducted in obese patients with t d refractory to sulfonylureas showed that the addition of hcq (up to a maximum of mg bid) reduced hba c by an absolute amount of . % more than placebo after months. ( ) no significant hcq-related side effects were reported in these studies, except for a severe hypoglycemic episode occurred in one patient treated with hcq in combination with insulin. ( ) moreover, a retrospective study conducted in diabetic patients with concomitant rheumatic diseases (ra and sle) showed that hcq, when compared to methotrexate, was associated with a significantly greater reduction in hba c from pre-treatment baseline to its lowest value within months of treatment initiation. ( ) more recently, a -week prospective randomized trial ( ) and two real-world, prospective observational studies of short duration (up to - weeks) ( , ) conducted in india have shown that the use of hcq ( mg/day) as an add-on treatment in patients with t d uncontrolled on a combination of two or more oral hypoglycemic agents (including metformin, sulfonylureas, pioglitazone, dpp- inhibitors, sglt inhibitors, and alpha-glucosidase inhibitors) was welltolerated and led to a significant improvement of glucose control (assessed by hba c, fasting-and postprandial blood glucose) from baseline (without occurrence of severe hypoglycemia). this article is protected by copyright. all rights reserved. in india, diabetes has reached epidemic proportions over the last years and newer antidiabetic drugs pose affordability challenges due to their high cost. based on the aforementioned preliminary results, hcq (at a dose of mg/day) has been approved in by the drug controller general of india (dcgi) as a third-line (add-on) hypoglycemic agent for patients with inadequately controlled t d despite lifestyle management associated with sulfonylurea and metformin combination therapy. ( , ) thereafter, a vibrant debate about hcq safety in t d has arisen within the indian scientific community. ( ) ( ) ( ) ( ) animal and human studies support the existence of different molecular mechanisms underlying the antihyperglycemic properties of hcq (and chloroquine). a study conducted by halaby et al. ( ) in cultured rat muscle cells and in a rat model of insulin resistance demonstrated that chloroquine can promote insulin-mediated glucose uptake and glycogen synthase activity by activating akt. in , powrie et al. ( ) conducted a randomized, placebo-controlled trial in patients with t d (referred to as "non-insulin-dependent diabetes mellitus") who were not receiving any antidiabetic medication and were only adopting dietary measures for diabetes management. the authors performed a hyperinsulinemic euglycemic clamp before and after a -day treatment with chloroquine ( mg administered four times daily) or placebo, using a stable isotopically labelled d-glucose to calculate hepatic glucose production (ra, rate of glucose appearance) and glucose utilization (rd, rate of glucose disappearance). notably, chloroquine led to a significant reduction in fasting plasma glucose and a significant rise in the total amount of exogenous glucose required to maintain euglycemia during the whole experiment, due to an increase in rd (without changes in ra). also, chloroquine administration resulted in a % reduction in metabolic clearance rate of insulin at low-dose insulin infusion. more importantly, fasting c-peptide levels significantly increased throughout the study after chloroquine treatment, despite the initially lower plasma glucose values. a decreased feedback inhibition of c-peptide secretion was also reported during low-and high-dose insulin infusion (by . % and . %, respectively) ( ) . overall, these data suggest that chloroquine can improve fasting glucose levels in subjects with t d by: (i) increasing peripheral glucose disposal (as evidenced by the increased overall glucose infusion rate required to maintain euglycemia, associated with an increase in rd), (ii) reducing hepatic insulin clearance, and (iii) increasing endogenous insulin secretion both in the fasting state and during hyperinsulinemia. the assumption of the authors that chloroquine has a direct insulinotropic effect on pancreatic beta cells is strengthened by the fact that c-peptide has a negligible hepatic clearance and approximately half of the produced c-peptide is metabolized by the kidneys; in particular, the majority of total cpeptide produced is degraded via peritubular uptake and approximately % is excreted unchanged in the urine. ( , ) however, these findings cannot be directly translated to hcq, even though chloroquine and hcq display similar pharmacodynamic properties. ( ) thus, further investigation is warranted in this direction. a study conducted by emami et al. ( ) in diabetic rats showed that hcq increased circulating insulin levels and reduced blood glucose levels in a concentration-dependent manner. in a subsequent study, the same authors found that hcq can inhibit cytosolic insulin-metabolizing enzyme and intracellular insulin degradation in rat liver cells. ( ) in , mercer et al. ( ) showed that hcq therapy (at a dose of . mg/kg/day) for weeks was associated with a significantly increase in insulin sensitivity index (isi) -assessed by a -min oral glucose tolerance test (ogtt) -along with trends toward reduced insulin resistance (assessed by homa-ir) in non-diabetic obese subjects without systemic inflammatory conditions. thereafter, wasko et al. ( ) confirmed similar results in a -week randomized, placebo-controlled study, showing that hcq ( mg/day) improved both beta-cell function (determined by the disposition index) and isi (assessed by intravenous glucose tolerance test) in non-diabetic overweight or obese subjects. at variance with these findings, a randomized, placebo-controlled this article is protected by copyright. all rights reserved. accepted article cross-over trial conducted by solomon et al. ( ) in non-diabetic subjects with stable ra showed that treatment with hcq for weeks (at a dose of . mg/kg/day, and not to exceed mg/day) produced no significant change in isi (assessed by -min ogtt) and insulin resistance (assessed by homa-ir) compared to placebo. these results may indicate that hcq can prevent development of diabetes in patients with inflammatory rheumatic diseases -as it has been widely demonstrated - ( ) ( ) ( ) ( ) ( ) through additional mechanisms other than improvement in insulin sensitivity. hcq is a well-known anti-inflammatory and immunomodulatory agent able to reduce the production of pro-inflammatory cytokines, and is therefore used as a dmard for the treatment of several inflammatory rheumatic diseases. ( , ) with regard to the anti-inflammatory effects of hcq in t d, amit gupta ( ) has recently shown that diabetic patients with higher baseline levels of highsensitivity c-reactive protein (hs-crp > mg/l) exhibited a more pronounced, although not significant, improvement in glucose control from baseline to weeks after the initiation of hcq therapy, as compared to patients with lower baseline levels of hs-crp (≤ mg/l). furthermore, patients with higher baseline hs-crp levels also exhibited higher (although not significant) baseline levels of hba c, fasting-and postprandial glucose levels. ( ) the previously mentioned study by wasko et al. ( ) conducted in non-diabetic overweight or obese subjects also found that hcq (at a dose of mg/day) significantly increased plasma levels of the adipokine adiponectin, which plays a key role in insulin resistance and metabolic syndrome by exerting anti-inflammatory actions and increasing insulin sensitivity. ( ) ( ) ( ) importantly, hcq use up to weeks (at a dose of up to mg/day) in patients with t d has also led to an improved lipid profile, which consisted of a significant reduction in total cholesterol, ldl-cholesterol and non-hdl cholesterol in different studies. ( , , ) a randomized, placebocontrolled cross-over trial also found a significant reduction in total cholesterol and ldlcholesterol after weeks of hcq therapy (at a dose of . mg/kg/day, and not to exceed mg/day) in non-diabetic patients with ra. ( ) nevertheless, it is still not clear whether the potential this article is protected by copyright. all rights reserved. accepted article lipid-lowering properties of hcq may depend on its anti-inflammatory actions rather than on other hcq-related mechanisms. interestingly, type diabetes (t d) trialnet international network is currently investigating the potential ability of hcq to prevent or delay the progression from normal glucose tolerance to impaired glucose tolerance or symptomatic t d in subjects with islet autoimmunity, who are at increased risk of developing t d (clinicaltrials.gov identifier: nct ). therefore, participants enrolled in this study are subjects in the stage (islet autoimmunity) of t d pathophysiology. ( ) however, the current lack of data on safety and efficacy of hcq in patients with established t d does not allow for any conclusion concerning the use of this drug in t d. altogether, these findings suggest that hcq regulates glucose homeostasis by virtue of multifaceted effects, which may allow for classifying this drug as an antidiabetic medication potentially acting as an insulin-sensitizing agent (insulin sensitizer), an anti-inflammatory agent and/or an insulinotropic agent (secretagogue) (figure ). since chronic low-grade inflammation and islet inflammation have been linked to insulin resistance and beta-cell dysfunction in t d, respectively, ( , ( ) ( ) ( ) the anti-inflammatory actions of hcq may be at the interface between its insulin-sensitizing effects and its insulinotropic properties. on the basis of pre-clinical data in animals, hcq appears to have the ability to: (i) inhibit intracellular insulin and insulin-receptor complex degradation, (ii) increase circulating insulin levels, (iii) reduce blood glucose levels, and (iv) promote glucose uptake and glycogen synthase activity in skeletal muscle cells. thus, it is tempting to speculate that hcq is able to inhibit glucagon secretion by pancreatic alpha-cells and/or glucagon action in the liver. additionally, it is still not clear whether the putative effect of hcq on endogenous insulin secretion is mediated by a direct stimulatory effect on beta-cells, and/or by indirect effects involving the reduction of islet inflammation and/or the reduction of glucotoxicityand lipotoxicity-related beta-cell dysfunction following the improvement in metabolic control. moreover, the lack of severe hypoglycemic episodes observed across the studies investigating the this article is protected by copyright. all rights reserved. accepted article use of hcq as an antidiabetic medication in t d (even when hcq was administered in combination with sulfonylureas) may underlie a putative glucose-dependent insulin secretion mechanism mediated by hcq. yet, the occurrence of severe hypoglycemic episodes cannot be excluded in these studies, since they did not employ continuous glucose monitoring. notwithstanding, we believe that all these speculations and unanswered questions may prompt the researchers to conduct future mechanistic studies in order to elucidate the exact mechanisms underlying the antihyperglycemic properties of hcq in both subjects with and without diabetes. even though hcq is considered as one of the safest dmard and has been widely used for the treatment of ra and sle, ( ) data on long-term safety and efficacy of hcq in diabetes are still lacking. therefore, the use of hcq in patients with diabetes should be carefully evaluated, particularly in subjects with established microvascular and/or macrovascular complications. the most dreaded complication deriving from hcq use is retinal toxicity. ( ) however, current evidence suggests that high-dose and long-term (> years) use represent the most important predictors of hcq-induced retinopathy. ( ) therefore, the american academy of ophthalmology recommends a maximum hcq dose of ≤ mg/kg actual body weight per day to markedly minimize the risk of retinopathy. ( ) also, hcq-mediated cardiotoxic effects (including potentially lethal heart rhythm disorders, such as prolonged qt interval, ventricular arrhythmia and torsades de pointes) have been reported. ( , ( ) ( ) ( ) thus, baseline electrocardiography to evaluate for prolonged qt interval is advisable prior to and following the initiation of hcq, particularly in high-risk subjects such as hospitalized patients and individuals taking other qt interval-prolonging drugs (e.g. macrolides such as azithromycin, quinolones, antihistamines, antiviral and antifungal drugs, antiarrhythmic medications, etc.). ( , ( ) ( ) ( ) risk factors for potentially lethal cardiac arrhythmias induced by hcq include: (i) coexisting cardiac conditions such as cardiomyopathy, left ventricular this article is protected by copyright. all rights reserved. accepted article dysfunction, ventricular hypertrophy, coronary artery disease, heart failure, or bradycardia; (ii) history of bradycardia, prolonged qt interval, ventricular arrhythmia or (unexplained) syncope; (iii) family history of premature sudden cardiac death or cardiac ion channelopathies; (iv) pacemaker and implantable cardioverter-defibrillator use; (v) electrolyte abnormalities such as hypokalemia and hypomagnesemia; (vi) genetic and autoimmune channelopathies; (vii) systemic inflammation; (viii) concomitant use of azithromycin or other qt interval-prolonging agents. ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) with regard to the renal function, chronic kidney disease can result in reduced hcq clearance, increased drug bioavailability and subsequent augmented risk of hcq-related side effects. ( ) furthermore, certain drugs (such as tamoxifen, glycosides, methotrexate and ciclosporin) can influence the pharmacokinetics of hcq. ( ) these drug interactions can increase the risk of hcqrelated side effects and therefore require careful consideration. besides the well-known contraindications to hcq use (including known hypersensitivity to aminoquinoline compounds), ( , ) other conditions or circumstances under which hcq should be contraindicated or used with caution in the context of diabetes include the following:  pre-existing retinopathy/maculopathy, history or risk for macular edema, and concomitant use of other oculo-toxic agents. ( , )  diabetes complicated by hypoglycemia unawareness, repeated episodes of severe hypoglycemia and/or malnutrition, due to the potential risk for severe hypoglycemic episodes. although hcqinduced severe hypoglycemia has mainly been documented in non-diabetic subjects, ( ) ( ) ( ) there have been a few reports of severe hypoglycemic episodes occurred with the use of hcq in patients with newly diagnosed t d ( ) and established t d treated with multiple daily injection insulin therapy. ( , )  pre-existing cardiomyopathy or heart failure, due to the possible risk of hcq-related cardiotoxicity. ( ) this article is protected by copyright. all rights reserved.  pre-existing myopathy and/or neuropathy, due to the potential risk of hcq-induced neuromyotoxicity. ( ) ( ) ( ) ( ) ( ) ( )  glucose- -phosphate dehydrogenase (g pd) deficiency, due to the potential increased risk of hcq-related hemolysis crisis. ( ) interestingly, heymann et al. ( ) found a significantly higher proportion of g pd-deficient patients among the diabetic population aged - years. however, a large retrospective study of patients with rheumatic diseases examined the relationship between hcq use and hemolytic anemia in g pd-deficient patients, showing that % of patients had g pd deficiency ( subjects, all african american) and only of them had episodes of hemolysis that occurred while not taking hcq. ( ) based on these findings, authors did not support routine measurement of g pd levels or withholding hcq therapy in african american patients with g pd deficiency. ( ) importantly, a proper risk-benefit assessment of hcq use in diabetic patients should also be considered in relation to the current coronavirus disease (covid- ) pandemic caused by the novel coronavirus sars-cov- (severe acute respiratory syndrome coronavirus ) ( , ) and declared a global pandemic by the who on march , . so far, several medications (including hcq and chloroquine) have been used in hospital settings as off-label drugs for covid- due to the current lack of proven effective therapies. ( , ) however, a recent observational study conducted on , consecutive hospitalized patients with covid- and moderate-to-severe respiratory illness showed that hcq did not have a significant impact on the risk of intubation or death over a median follow-up period of . days. ( , ) despite the observational design of this study, these findings dot not support hcq use for treatment of covid- importantly, diabetes has reached epidemic proportions over the last years, ( ) affecting approximately million people worldwide according to recent estimates. ( ) also, emerging evidence suggests that diabetes represents one of the most prevalent comorbidities in patients with covid- , ( ) ( ) ( ) as well as a major risk factor for a worse prognosis of the disease. ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) in light of these remarks, a pre-emptive and careful evaluation of all the potential risks and benefits related to hcq is critical for a proper and cautious use of this drug in subjects with diabetes, particularly in the context of covid- . over the last decades, several studies have shown that hcq plays an important role in the regulation of glucose homeostasis through multifaceted effects, including improvement of insulin sensitivity, increase of insulin secretion, reduction of hepatic insulin clearance and intracellular insulin and insulin-receptor complex degradation, and reduction of systemic inflammation, among others ( figure ). different studies have shown the safety and efficacy of hcq use (at a dose ranging from to mg/day) in patients with t d over a short-term period (up to months). ( , , , , ) in , hcq (at a dose of mg/day) has been approved in india as an addon hypoglycemic agent for patients with inadequately controlled type diabetes despite lifestyle management associated with sulfonylurea and metformin combination therapy. nevertheless, concerns on long-term safety of hcq in patients with t d still persist due to the lack of robust data. thus, large randomized controlled trials of long duration are warranted to establish the longterm safety and efficacy of this drug in the context of t d. with regard to the current pandemic scenario, emerging evidence shows that patients with diabetes have a greater risk for adverse outcomes following covid- infection. although findings from clinical studies have suggested this article is protected by copyright. all rights reserved. accepted article limited benefit from hcq in covid- in general, ( ) several randomized controlled trials are currently investigating the use of hcq for prophylaxis of covid- . ( ) moreover, guidelines from different countries have listed some investigational drugs (including hcq) as potential adjuvant treatment options, whilst cautioning to take into account the individual risk of harm. ( , ) therefore, a careful risk-benefit assessment of hcq is required for the most cautious use of this drug in subjects with diabetes. accepted article without diabetes suggests that hydroxychloroquine regulates glucose homeostasis by virtue of multifaceted effects, including increase of insulin secretion, improvement of insulin sensitivity, reduction of hepatic insulin clearance and intracellular insulin and insulin-receptor complex degradation, increase of adiponectin levels, reduction of systemic inflammation, and/or reduction of inflammation-induced insulin resistance in adipocytes and skeletal muscle cells. altogether, these actions may allow for classifying hydroxychloroquine as an antidiabetic agent by acting as an insulin-sensitizing agent, an anti-inflammatory agent and/or an insulinotropic agent (secretagogue). since chronic low-grade inflammation and islet inflammation have been linked to insulin resistance and beta-cell dysfunction in type diabetes, respectively, the 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consensus opinion: role of anti-inflammatory agents in the management of type- diabetes rssdi clinical practice recommendations for the management of type diabetes mellitus hydroxychloroquine in diabetes and dyslipidaemia: primum non nocere drug approvals in india drug approvals in india -authors' reply the marketing of unproven drugs for diabetes and dyslipidaemia in india chloroquine stimulates glucose uptake and glycogen synthase in muscle cells through activation of akt mode of action of chloroquine in patients with non-insulin-dependent diabetes mellitus the clinical utility of c-peptide measurement in the care of patients with diabetes a practical review of c-peptide testing in diabetes insulin-sparing effect of hydroxychloroquine in diabetic rats is concentration dependent inhibition of insulin metabolism by hydroxychloroquine and its enantiomers in cytosolic fraction of liver homogenates from healthy and diabetic rats hydroxychloroquine improves insulin sensitivity in obese 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the seattle region -case series comorbidity and its impact on patients with covid- in china: a nationwide analysis clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study diabetes is a risk factor for the progression and prognosis of covid- prevalence and impact of diabetes among people infected with sars-cov- diabetes mellitus is associated with increased mortality and severity of disease in covid- pneumonia -a systematic review, meta-analysis, and metaregression accepted article endocrine and metabolic link to coronavirus infection covid- pandemic, corona viruses, and diabetes mellitus coronavirus infections and type diabetes-shared pathways with therapeutic implications role of adjunctive treatment strategies in covid- and a review of international and national clinical guidelines lombardy section italian society infectious and tropical diseases -. vademecum for the treatment of people with covid- no funding received. we would like to thank dr. nathalia padilla for her contribution to the comprehensive review of the literature. none declared. this article is protected by copyright. all rights reserved.accepted article key: cord- -xg xnjf authors: dinicolantonio, james j.; barroso-aranda, jorge title: harnessing adenosine a a receptors as a strategy for suppressing the lung inflammation and thrombotic complications of covid- : potential of pentoxifylline and dipyridamole date: - - journal: med hypotheses doi: . /j.mehy. . sha: doc_id: cord_uid: xg xnjf counterproductive lung inflammation and dysregulated thrombosis contribute importantly to the lethality of advanced covid- . adenosine a a receptors (a ar), expressed by a wide range of immune cells, as well as endothelial cells and platelets, exert camp-mediated anti-inflammatory and anti-thrombotic effects that potentially could be highly protective in this regard. the venerable drug pentoxifylline (ptx) exerts both anti-inflammatory and antithrombotic effects that reflect its ability to boost the responsiveness of a ar to extracellular adenosine. the platelet-stabilizing drug dipyridamole (dip) blocks intracellular uptake of extracellularly-generated adenosine, thereby up-regulating a ar signaling in a way that should be functionally complementary to the impact of ptx in that regard. moreover, dip has recently been reported to slow the cellular replication of sars-cov- in clinically feasible concentrations. both ptx and dip are reasonably safe, well-tolerated, widely available, and inexpensive drugs. when covid- patients can be treated within several days of symptom onset, using ptx + dip in conjunction with hydroxychloroquine (hcq) and an antibiotic - azithromycin (azm) or doxycycline – might be warranted. hcq and azm can suppress sars-cov- proliferation in vitro and may slow the cell-to-cell spread of the virus; a large case series evaluating this combination in early-stage patients reported an impressively low mortality rate. however, whereas hcq and azm can promote qt interval lengthening and may be contraindicated in more advanced covid- entailing cardiac damage, doxycycline has no such effect and exerts a potentially beneficial anti-inflammatory action. in contrast to hcq, we propose that the combination of ptx + dip can be used in both early and advanced stages of covid- . concurrent use of certain nutraceuticals – yeast beta-glucan, zinc, vitamin d, spirulina, phase inducers, n-acetylcysteine, glucosamine, quercetin, and magnesium – might also improve therapeutic outcomes in covid- . generation, expression of adhesion molecules, trans-endothelial migration of neutrophils, opening of the endothelial barrier, tissue factor generation, and platelet aggregation in a ar-responsive cells. downregulation of nf-kappab activation and jak-stat signaling pathways contribute importantly to these effects of camp. importantly, neutrophils, whose activation and transit into lung interstitial tissue and alveolar space is a key mediator of the respiratory distress syndrome associated with covid- , are highly responsive to the functionally suppressive effects of a ar, as are the endothelial cells whose activation attracts and enables transendothelial passage of activated neutrophils. [ ] [ ] [ ] [ ] these considerations suggest that selective agonists of a ar may have important potential for blunting the lethality of covid- . as may be expected, such agents have shown protective effects in rodent models of inflammatory lung injury. [ ] [ ] [ ] [ ] unfortunately, these agents are not yet clinically available. however, at least two drugs are currently available -venerable, reasonably safe and well tolerated, and inexpensive -that can function to up-regulate a ar signaling: pentoxifylline (ptx) and dipyridamole (dip). although ptx is known to have broad anti-inflammatory activity, it is employed primarily in the treatment of intermittent claudication; by lessening neutrophil activation, ptx renders these cells more distensible, so that they can more readily pass through narrow capillaries in affected legs. , (upstream stenotic obstructions decrease the transcapillary pressure gradient, rendering the passage of bulky neutrophils through narrow capillaries more difficult in this syndrome.) although the clinical effects of ptx have usually been ascribed to the ability of this drug to inhibit camp phosphodiesterase -thereby boosting camp levels -this effect is only significant in vitro at millimolar concentrations that are orders of magnitude higher than the low micromolar concentrations of this drugs achieved clinically. ironically, however, it does appear that camp mediates ptx's clinical effects. within the last decade, ptx's anti-inflammatory effects have been shown to be contingent on activation of a ar. - whether ptx can act as a direct agonist for a ar is currently unclear, and some data argue against this. what is clear is that ptx can potentiate the responsiveness of this receptor to adenosine. the latter is produced extracellularly from atp released into the extracellular space, which is then converted to adenosine by the sequential activity of the cd and cd ecto-phosphatases expressed on the plasma membranes of a ar-expressing cells. , the signaling activity of extracellularly-generated adenosine is terminated by intracellular uptake of the adenosine. the platelet-stabilizing agent dip is distinguished by its ability to block this re-uptake by platelets. , hence, dip up-regulates the adenosine-mediated activation of platelet a ar, thereby boosting platelet levels of camp, which functions to suppress platelet aggregation. moreover, dip blocks adenosine uptake by a range of other a ar-expressing cell types, including endothelial cells, neutrophils, and monocytes. [ ] [ ] [ ] it is evident that ptx and dip have the potential to work in a complementary fashion to boost a ar signaling -dip can be expected to boost the extracellular levels of adenosine whose signaling activity ptx potentiates. surprisingly, only a handful of studies have evaluated this combination experimentally or clinically -with encouraging results -likely because the mechanism of action of ptx has been clarified only recently. - pre-administration of ptx is protective in rodent models of acute respiratory distress syndrome (ards) evoked by lipopolysaccharide (lps) administration or severe hemorrhage. [ ] [ ] [ ] clinically, it was found to reduce mortality, lower plasma tumor necrosis factor, and achieve clinical and radiological improvements in ards associated with cancer. a meta-analysis of clinical studies found that ptx therapy is associated with a decrease in plasma levels of both tumor necrosis factor and c-reactive protein. in chimpanzees, it was shown to blunt lps-induced activation of coagulation and fibrinolysis. in isolated lungs, ptx pre-treatment reduces the tissue injury induced by neutrophil infusion. in endothelial cells, ptx counteracts the ability of pro-inflammatory cytokines to stimulate expression of adhesion factors and chemokine production. these findings are expectable in light of the known effects of a ar signaling, and encourage the speculation that ptx could have potential for blunting the exuberant lung inflammation and pro-thrombotic effects of advanced covid- . not surprisingly, the use of ptx for treatment of ards associated with sars infection was suggested in . (presumably, this was not studied because the syndrome rapidly disappeared.) in seeming contradiction, a large multi-center study of lisofylline therapy in ards patients failed to show benefit. lisofylline is the r-isomer of a reductive metabolite of ptx, notable for its protective impact in rodent models of type diabetes. conceivably, the impact of this agent on a ar signaling -which has not been reported -is different than that of ptx. alternatively, this finding may reflect the fact that, for unclear reasons, a ar agonism is more effective for controlling ards when implemented before the syndrome becomes florid. konrad and colleagues, in interpreting this result, suggest that adenosine levels may be too low in the context of advanced sepsis. if so, the concurrent use of dip would make logical sense. most studies with dip have focused on its platelet-stabilizing effects -which presumably could provide some protection from sars-cov- 's pro-thrombotic effects -but experimental studies also show that dip can act on neutrophils to suppress superoxide production, adhesion to endothelial cells, and, in a mouse model of anti-phospholipid syndrome (a sometime feature of covid- ), netosis formation. [ ] [ ] [ ] [ ] and dip has been shown to suppress superoxide production and tissue factor expression in monocytes. of particular pertinence is this new discovery: chinese researchers have reported that, in clinically relevant concentrations as low as nm, dip slows the replication of sars-cov- in vero e cells; this effect may be mediated in part but not entirely by the binding to dip to the sars-cov- protease mpro. in a controlled pilot study, hospitalized covid- patients with respiratory difficulties were treated with either dip ( mg times daily) or placebo; of the patients who received dip, including that were severely ill, all but one recovered, and the remaining patient was in remission at time of the report. of severely ill patients in the control group, patients died and were in remission. the difference in therapeutic outcome just missed traditional statistical significance (p= . ). the response in d-dimer levels was significantly better in the treated than in the control patients. an anti-viral effect of dip is not unprecedented -cell culture studies have reported that this agent can slow the proliferation of various rna viruses, and a russian clinical report some decades ago concluded that dip administered prophylactically was effective for reducing risk for influenza and upper respiratory infections. [ ] [ ] [ ] [ ] [ ] [ ] these considerations suggest that a ptx/dip regimen might have considerable potential for control of the progression and complications of covid- . provisionally, we recommend dosage schedules for ptx and dip typically used for their approved indications: ptx mg times daily, and dip mg times daily. another venerable drug which has been suggested for use in covid- management is the anti-parasitic agent ivermectin. evidence that it can suppress proliferation of sars-cov- in cell culture is likely of little pertinence, as the ic concentration that achieves this -about um -is vastly higher than the plasma concentrations achievable by doses approved for clinical use. , nonetheless, anecdotal claims of its apparent effectiveness in late-stage covid- are encouraging clinical trials with this agent. largely overlooked is the fact that, in oral doses that are roughly analogous to the standard clinical dose in humans, ivermectin pre-administration can protect mice from a lethal dose of lps. , hence, if ivermectin proves useful in covid- , an anti-inflammatory mechanism may underlie this benefit. however, it should be acknowledged that a ar agonism also has potential for suppressing the dendritic cell activity that provides the antigen presentation necessary for developing an antiviral antibody response. the authors have been unavailable to find any studies suggesting that ptx increases infection risk -in marked contrast to the well-known literature on anti-inflammatory corticosteroids -so perhaps this is a relatively minor consideration. indeed, some studies fail to find an effect of a ar agonists on dendritic cell antigen presentation. and ptx has actually been suggested as an adjuvant to vaccination, as it boosts memory response to vaccination by increasing survival of activated t cells. nevertheless, it would seem prudent to complement ptx/dip therapy with agents such as yeast beta-glucan that specifically boost dendritic cell activity, as a compensatory measure. [ ] [ ] [ ] curiously, beta-glucan administration has been found to be protective in rodent models of sepsis-induced ards. , supplemental zinc could be another worthwhile adjuvant measure, as it has been found to decrease incidence of infection while lowering systemic markers of inflammation in elderly subjects. in early-stage ambulatory patients with covid- , it would be appropriate to consider using ptx + dip in conjunction with hydroxychloroquine (hcq). currently, this agent is the most commonly used drug for treatment of early-stage covid- . hcq decreases replication of sars-cov- in vitro in clinically relevant concentrations. , studies examining the molecular biology of sars-cov- cell-tocell spread have found that endosomal cathepsin l protease activity markedly expedites such spread, presumably because it enables sars-cov- virions taken up into cellular endosomes to fuse their membranes with that of the endosome, thereby allowing the virion to enter the cytoplasm and begin replication. it is well known that hcq functions to alkalinize endosomes, and this would be expected to inhibit cathepsin l activity. , moreover, hcq has recently been shown to inhibit activation of nadph oxidase complexes in endosomes; this would be expected to exert an anti-inflammatory effect that might complement the anti-viral activity of this agent. this model makes evident the desirability of employing hcq as early during the clinical course of covid- as feasible, as the ability of this agent to slow cell-to-cell spread may be of less utility once the lung epithelium is already widely colonized by the virus. pharmacokinetic modeling, combined with in vitro data, suggest a regimen for hcq of mg twice daily for one day, and mg twice daily for a further days; this is predicted to maintain antiviral plasma levels of hcq for at least days. hcq therapy often induces modest increases in qt interval, and concurrent administration of azithromycin can amplify this effect. such increases can increase chances for dangerous torsade de pointes arrhythmias; studies examining ecgs in hospitalized covid- patients treated with this combination reported cases of torsade de pointes in such patients. [ ] [ ] [ ] [ ] although it is very rare for these drugs to induce arrhythmias when used for their current indications, sars-cov- can directly attack the heart, and conceivably this could potentiate the pro-arrhythmic impact of hcq. , hence, monitoring of qt interval appears to be prudent when using hcq in covid- . fortunately, neither ptx nor dip have been linked to qt prolongation or torsade de pointes arrhythmias. adding an antibiotic such as azithromycin or doxycycline to early-stage treatment of covid- to prevent bacterial super-infection -as many doctors have done when employing hcq in covid- therapy -would also be a reasonable option. in addition to their antibiotic activities, azithromycin exerts anti-viral effects in vitro against various viruses, and doxycycline has anti-inflammatory properties that likely would be beneficial in sars-cov- -induced cytokine storm. , however, azithromycin might be inappropriate for late-stage therapy, as it has a greater tendency than hcq to prolong qt intervals; doxycycline does not have this effect. nutraceutical adjuvant measures that support the antigen-specific immune response -such as yeast glucan and zinc -would also appear to be indicated. nutraceuticals that might be expected to boost the interferon response evoked by sars-cov- while lessening the contribution of oxidants to lung inflammation have been proposed, including spirulina, phase inducers, n-acetylcysteine. supplemental glucosamine may likewise up-regulate the type interferon responses to viruses, while exerting anti-inflammatory effects that render it protective in rodent models of sepsis and lung inflammation induced by lps or cigarette smoke. [ ] [ ] [ ] [ ] [ ] this anti-inflammatory effect might reflect upregulated activity of the de-ubiquitinase a , which opposes traf signaling. up-regulation of type interferon induction may also play a role in the anti-viral effects of quercetin. , theoretical considerations as well as epidemiological findings suggest that good vitamin d status may also be protective. [ ] [ ] [ ] in the context of inflammation, lung epithelium and alveolar macrophages can convert circulating -hydroxyvitamin d to the active hormone calcitriol; this in turn can boost expression of the antimicrobial protein cathelicidin, which is destructive to many enveloped viruses. [ ] [ ] [ ] [ ] [ ] intracellular magnesium supports effective function of the a ar. lower serum magnesium has been associated with increased thrombotic risk and slowed fibrinolysis. 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hypercoagulation: intellectual analysis of data obtained from a sample of patients aged - years from medical and preventive facilities in russia magnesium as a predictor of acute stent thrombosis in patients with st-segment elevation myocardial infarction who underwent primary angioplasty antithrombotic effects of magnesium sulfate in in vivo experiments low intracellular magnesium levels promote plateletdependent thrombosis in patients with coronary artery disease does hypomagnesemia impact on the outcome of patients admitted to the intensive care unit? a systematic review and meta-analysis hypomagnesemia in the magnesium deficiency promotes secretion of high-mobility group box protein from lipopolysaccharide-activated macrophages in vitro magnesium deficiency and increased inflammation: current perspectives key: cord- -qdzhncs authors: choi, min joo; kang, minsun; shin, so youn; noh, ji yun; cheong, hee jin; kim, woo joo; jung, jaehun; song, joon young title: comparison of antiviral effect for mild-to-moderate covid- cases between lopinavir/ritonavir versus hydroxychloroquine: a nationwide propensity score-matched cohort study date: - - journal: int j infect dis doi: . /j.ijid. . . sha: doc_id: cord_uid: qdzhncs objectives: we aimed to compare the antiviral effect of hydroxychloroquine (hcq) and lopinavir/ritonavir (lpv/r) in patients with covid- . methods: nationwide retrospective case-control study was conducted to compare the effect of hcq and lpv/r on viral shedding duration among patients with mild-to-moderate covid- using the reimbursement data of national health insurance service. after propensity score matching (psm), multivariate analysis was conducted to determine statistically significant risk factors associated with prolonged viral shedding. results: overall, , patients with mild-to-moderate covid- were included. patients were categorized into three groups: lpv/r (n = , ), hcq (n = ), and standard care without hcq or lpv/r (controls, n = ). the median viral shedding duration was (iqr – ), (iqr – ), and (iqr – ) days in the lpv/r, hcq, and control groups, respectively. even after psm, the viral shedding duration was not significantly different between lpv/r and hcq groups: (iqr, – ) days versus (iqr, – ) days. on multivariate analysis, old age, malignancy, steroid use, and concomitant pneumonia were statistically significant risk factors for prolonged viral shedding. conclusion: the viral shedding duration was similar between hcq and lpv/r treatment groups. there was no benefit in improving viral clearance compared to the control group. transmission by reducing the viral shedding duration, as shown from the effect of oseltamivir for influenza (beigel et al., , meschi et al., . owing to the highly transmissible property of sars-cov- , even asymptomatic and presymptomatic patients have transmitted the virus to their family members and colleagues , gandhi et al., . moreover, as the pandemic has progressed, the number of deaths in high-risk groups has increased dramatically. therefore, the treatment strategy for covid- needs to be approached in two ways: reduction of mortality through combined antiviral therapy for severe patients and blockage of transmission through early antiviral treatment for patients with mildto-moderate cases. in the early pandemic periods, the most hopeful antiviral candidates were hydroxychloroquine (hcq) and lopinavir/ritonavir (lpv/r), which had already been on the market for decades with other indications , sanders et al., . both candidates were expected to interfere viral replication theoretically , sanders et al., , and showed good in vitro activity against sars-cov- , ul qamar et al., . under urgent needs, many clinical trials using either candidate have been conducted, but there are still insufficient data to recommend hcq or lpv/r use . furthermore, most studies mainly focused on patients with moderate-to-severe covid- , and antiviral agents were administered at more than days later from symptom onset (borba et al., , hung et al., . it is necessary to comparatively evaluate the effect of viral suppression when the antiviral agent is administered in the early stage of symptom development. if effective for viral suppression, it has a very important meaning in terms of infection control and treatment (gautret et al., , li et al., . this study aimed to compare the effect of hcq and lpv/r on the viral shedding duration among patients with mild-to-moderate covid- cases using south korea's national health j o u r n a l p r e -p r o o f insurance service (nhis) database. this study used reimbursement data from the national health insurance service (nhis) of south korea for the period from january , to may , . the nhis covers - % of the population ( million people). data included age, sex, dates of admission and discharge, diagnoses coded according to the international classification of disease and related health problems, th edition (icd- ), and prescription of medications covered by nhis. currently, the nhis aggregates datasets for real-time reverse transcriptase polymerase chain reaction (rrt-pcr)-confirmed covid- cases from information provided by the epidemiological investigation of korea centers for disease control and prevention (kcdc). all subjects with kcd- codes for covid- were classified into the categories according to the centers for disease control and prevention (cdc) interim guidance: critical (extracorporeal membrane oxygenation, death), severe (mechanical ventilator), moderate grade (high flow oxygen therapy), moderate grade (oxygen therapy), and mild (remaining laboratory confirmed subjects) (supplementary table ) . this nationwide retrospective study included patients with laboratory-confirmed covid- diagnoses who were discharged during the study period from january , to may , . among these, only mild-to-moderate grade patients were included in the analysis, and the effect of lpv/r or hcq use on viral shedding duration was evaluated ( figure ). we strictly included patients with mild or moderate grade covid- , excluding severe patients for the j o u r n a l p r e -p r o o f following reasons: in severe cases, anti-viral agents might have been administered following late aggravation after initial supportive standard care and hospital stay may have been extended due to complications, although sars-cov- rrt-pcr test converted to be negative. furthermore, the following two inclusion criteria should be met: i) adults aged ≥ years and ii) hospitalization within week after laboratory diagnosis for covid- . the criteria of ≤ week from diagnosis to hospitalization is needed to assess the effect of early antiviral treatment. exclusion criteria were as follows: i) concomitant lpv/r and hcq treatment; ii) patients on lpv/r or hcq medication prior to diagnosis; or iii) those who received other antiviral agents thought to be effective against camostat, nafamostat, remdesivir, ribavirin, or interferon) . for patients with multiple episodes of hospitalization, the first admission was only included for the analysis. all included patients were categorized according to lpv/r or hcq exposure: lpv/r-group, hcq-group, and control group (supportive standard care only). lpv/r or hcq use was defined as at least one prescription being recorded in the claim data. data on the prescription of lpv/r, hcq, or other drugs were extracted using drug codes based on the anatomical therapeutic chemical classification in the claim data of the study periods. comorbidities were identified using icd- codes entered within year prior to covid- diagnosis (supplementary table ). charlson comorbidity index (cci) was also calculated to assess the general health status of study subjects (supplementary table ) . a subgroup analysis was conducted for mild cases only, moderate grade cases only, and patients with pneumonia (defined as icd- codes). we defined patient's length of hospitalization as viral shedding duration, which was assessed using rrt-pcr. this is reasonable, because all covid- patients in south korea required undetectable rna from two consecutive nasopharyngeal swab specimens ( hours apart) to be discharged, according to the regulation of kcdc (choi w. s. et al., ) . the data are presented using descriptive statistics for continuous and categorical variables. differences between groups were analyzed with an analysis of variance (anova) for continuous variables and chi-square tests for categorical variables. considering the significant differences in baseline characteristics among study groups, propensity score matching (psm) was taken between two groups to be compared. to compare the viral shedding duration, we created propensity scores for the lpv/r-group, hcq-group, and control group. all sets of propensity scores were estimated via multinomial logistic regression using baseline covariates including age, sex, comorbidities, disease severity, and concomitant pneumonia. to compare the viral shedding duration, three data sets were made (lpv/r-group vs. hcq-group; lpv/r-group vs. control group; hcq-group vs. control group), and each of the sets were propensity score matched in : proportion. age and sex were perfectly matched, and greedy nearest neighbor matching was used for other covariates on the logit of the propensity score. after psm, multiple linear regression was used to determine statistically significant factors associated with viral shedding duration. variables included in the models were age, sex, comorbidities, disease severity, concomitant pneumonia, concomitant use of steroid, azithromycin or oseltamivir, and elapsed days from laboratory diagnosis to hospitalization. all tests were two tailed, and results were considered statistically significant at p-value < . . sas version . (sas institute inc, cary, nc) was used for the analyses. during study periods, a total of , covid- patients were discharged (or death occurred) in south korea. most cases (> %) were hospitalized within a day from laboratory diagnosis, and more than % were hospitalized within days. regarding disease severity, children and adolescents were milder in severity: . % ( / ) were mild, . % ( / ) were moderate grade , and none were moderate grade or severe (supplementary table ). similarly, mild cases accounted for the large portion in adults aged - year ( . %, / ). however, in the elderly, less than two-thirds of cases ( . %, / ) were mild, while the rest required oxygen therapy; one-third of cases on oxygen therapy required high flow oxygen supply or mechanical ventilation. since the first emergence of covid- in south korea, the prescription trend of lpv/r and hcq is shown in supplementary figure . overall, lpv/r and hcq prescription tended to decrease, and preference appeared to change significantly depending on the literature published at that time. sequentially, lpv/r was replaced by hcq and supportive standard care. a total of , patients with mild-to-moderate grade covid- were included in this study. patients were categorized into three different groups: those treated with lpv/r (lpv/rgroup, n= , ), those treated with hcq (hcq-group, n= ), and those with supportive standard care without hcq or lpv/r (control group, n= , ) (supplementary table ). there were some significant differences among the three groups in the baseline characteristics. compared to lpv/r or hcq-groups, the control group was significantly younger, had fewer j o u r n a l p r e -p r o o f comorbidities, and included more males. the oseltamivir combination rate was less than . % in all groups. the median time of viral rna shedding was (iqr - ) days in the lpv/r-group, (iqr - ) days in the hcq-group, and (iqr - ) days in the control group. there was no significant difference between the lpv/r-group and the hcq-group, but the viral shedding duration was estimated to be significantly longer in both treatment groups compared to the control group. as the baseline characteristics showed significant difference across the three groups, we computed propensity scores for lpv/r use and hcq use based on age and sex. after psm, most of the baseline characteristics were similar, including comorbidities. however, the disease severity and proportion of accompanying pneumonia were still significantly higher in the lpv/r and hcq-group, especially in the lpv/r-group (table ) . total dosage of lpv/r was , / mg on average, which was considered to have been administered for about days when calculated based on / mg/day as recommended by the guidelines . hcq was used on average of , mg, which was equivalent to dosage for - days, calculated based on mg/day recommended by the guideline. the median time of viral rna shedding was not significantly different between the lpv/r and hcq-group: (iqr, - ) days versus (iqr, - ) days (table ) . neither agent shortened the viral shedding duration compared to the control group. on multivariate analysis using propensity score-matched data sets comparing each antiviral group versus control group, lpv/r or hcq still showed a significantly longer viral shedding duration compared to the control group. however, the significance due to the use of antiviral agents disappeared in the subgroup analysis which includes only moderate cases or pneumonia cases (supplementary table , ). on multivariate analysis using dataset comparing lpv/r and hcq groups, neither of the agents showed a significant difference in terms of the viral shedding duration. the factors that significantly influence the viral shedding duration were age, malignancy, steroid use, and concomitant pneumonia (table ) . as the elapsed time from diagnosis to hospitalization is longer, in-hospital shedding duration was much shorter. in the subgroup analysis for patients with moderate grade severity or concomitant pneumonia, cardiac disease was identified as a factor that significantly increased the viral shedding duration. currently, no specific antiviral agent is available for the prevention or treatment of covid- , so drug repurposing has been considered as a promising approach to rapidly identify an effective therapy. hcq and lpv/r are the candidates at the forefront of drug repurposing. this nationwide retrospective study was conducted to evaluate the antiviral effect of hcq and lpv/r in the treatment of patients with mild covid- using the nhis reimbursement dataset. in this study, the viral shedding duration of sars-cov- was similar between hcq and lpv/r treatment groups. when analyzing the effect of antiviral agents, the timing of antiviral therapy is an important issue to be considered. early (within days from symptom onset) initiation of antiviral therapy may be critical in reducing sars-cov- viral load, as previously noted (fu keyaerts et al., . due to the limitation of our database, it was difficult to know the initiation timing of antiviral treatment in each individual patient; however, the government of south korea had launched a series of aggressive measures to perform tight contact tracing and mass screening tests, which enabled early diagnosis within - days from symptom onset (ryu et al., ) . based on the expert's guideline, most patients were treated within days after symptom development . as shown in this study, > % were hospitalized within a day from laboratory diagnosis. it took more than week to be hospitalized in only a small number of patients, and those were excluded in this study considering the timing issue. hcq or lpv/r monotherapy showed no benefit for improving viral clearance compared to the control group. the viral shedding duration seemed to be rather prolonged in the treatment groups (median viral shedding duration, - days in the antiviral treatment groups vs. days in the control group). however, it would be possible that the viral shedding duration in the control group was estimated to be shorter than it really is because of the following reasons. first, community treatment centers (ctc), which were introduced in korea as a measure to efficiently distribute limited medical resources during the declared epidemic starting in early march , could make a bias in the claim database. some patients with mild symptoms were transferred from the hospital to ctcs if they were medically stable but needed to maintain isolation (choi w. s. et al., ) . although supportive care is maintained in ctcs, some ctcs might not claim reimbursement due to neglectable medical costs and complex process, potentially contributing to the shortened length of hospitalization in the control group. second, during the earlier period of the covid- pandemic in south korea, many mild covid- patients diagnosed at the airport quarantine received supportive care without antiviral treatment at the ctc. actually, a majority of them had symptoms for more than a week before traveling, j o u r n a l p r e -p r o o f so the viral shedding duration might have been estimated shorter. in the previous studies including mild covid- patients in ctcs, the mean viral shedding duration from symptom onset was - . days, which is longer than the results of our control group , noh et al., . when we compared the viral shedding duration between the hcq or lpv/rgroups and the control group in subgroup analyses including only moderate cases or those with concomitant pneumonia, there was no significant difference, which reflected the selection bias of mild cases who were mainly included in the control groups. one of the most effective treatment strategies would be to stop the viral replication at the beginning, thereby minimizing the peak viral load and shedding duration (chu et al., . it is unclear why hcq or lpv/r did not show favorable antiviral effect in this study. one possible reason is that a higher dose is required to successfully suppress sars-cov- in patients as shown in vitro cytotoxicity test , keyaerts et al., . in particular, the in vitro antiviral activity of hcq at concentrations commonly used in humans was reported minimal (kang et al., ) . insufficient data are available regarding the optimal dose to ensure the safety and efficacy of both drugs for covid- . another possible reason is the inadequate target tissue concentration of those antiviral agents. lpv/r is an anti-hiv drug, innovated to get high plasma and lymphatic tissue concentration, not lung tissue (freeling et al., ) . there are no pharmacokinetic data on respiratory tract concentration of lpv/r. although pharmacokinetic data indicate hcq exhibits extensive tissue distribution, the tissue concentration of the respiratory tract might be variable depending on intestinal resorption and hepatic first-pass metabolism (klimke et al., , maharaj et al., . in comparison, there are several encouraging reports of hcq on reducing mortality (arshad et al., , catteau et al., , di castelnuovo et al., , mikami et al., . the inverse association of hcq with mortality was more evident in elderly, in patients j o u r n a l p r e -p r o o f who experienced more severe manifestation or especially having elevated c-reactive protein. furthermore, the beneficial impact was observed even in the late treatment groups, suggesting that the anti-inflammatory and anti-thrombotic potential of hcq may have had more important role rather than its antiviral properties. on multivariate analysis, old age, malignancy, steroid use, and concomitant pneumonia were identified as risk factors for prolonged viral shedding in this study, consistent with previous studies (fu et al., , zhou et al., . old age, comorbidities, and steroid use might blunt the host immune response, thereby promoting viral replication. in the subgroup analyses, chronic neurologic diseases were also associated with increased risk of prolonged viral shedding in the cases with mild covid- , while cardiovascular disease was identified as a risk factor in the moderate cases or cases with concomitant pneumonia. since ace , the sars-cov- binding receptors, is widely expressed in the various organ including the lungs, heart, and vessels, it is possible that greater number of ace receptors-along with blunted host response encountered in many comorbid conditions-might promote viral replication, resulting in prolonged viral shedding. recent studies suggested that the negative outcomes in patients with underlying cerebrovascular disease might be due to elevated expression of ace (choi j. y. et al., ) . besides prolonged viral shedding, pre-existing cardiovascular diseases were associated with worse outcomes of covid- (fu et al., ) . although unclear, covid- might trigger acute coronary syndrome, arrhythmia, or acute exacerbation of heart failure, similar to influenza viral infection (madjid et al., ) . sars-cov- itself might induce new cardiac pathology or exacerbation of underlying cardiovascular diseases under the systemic and/or localized inflammatory host response, resulting in cytokine storm in some severe cases (madjid et al., cardiotoxicity might be considered (di girolamo et al., , nord et al., . in this study, cardiovascular disease was a significant risk factor for longer hospitalization only in the analyses including the hcq-group. although generally safe when used for approved indications, including autoimmune inflammatory rheumatic diseases or malaria, the safety and benefit of hcq treatment are poorly evaluated in covid- , and potential safety hazards have been announced recently, especially among severe patients and/or high-dose users (borba et al., , mehra et al., . this study has some limitations. first, this was a retrospective cohort study. although large number of cases were included, with propensity score matched for relevant variables, we could not rule out residual confounders. second, we did not compare the viability of sars-cov- with the duration of infectivity. this study focused on the comparison of rrt-pcr-based viral clearance between lpv/r and hcq. third, this study did not include cases with severe covid- , having a limitation in evaluating the potential anti-inflammatory impact of hcq or lpv/r in the prevention of complications and fatality. fourth, environmental factors were not considered in this study. environmental factors such as temperature, humidity and food might influence transmission, severity and mortality of covid- (eslami and jalili, , roviello and roviello, ) . as the severity of covid- and the effect of antiviral treatment may vary by region with different environments, further studies from various countries or regions would be required. fifth, there was significant differences in baseline characteristics between hcq and lpv/r groups. to overcome the differences, this study used psm and subgroup/multivariate analyses. finally, because of the limitation of study design using claim database, data on drug concentration and related metabolic factors were not available. in conclusion, we compared the antiviral effect of lpv/r and hcq in patients with mild-tomoderate covid- using a large sample size health insurance database. the viral shedding duration was similar between hcq and lpv/r groups. neither hcq nor lpv/r monotherapy showed benefits in improving viral clearance compared to the control group. given such a limited effectiveness of hcq or lpv/r monotherapy, a combination strategy needs to be considered. in fact, studies have shown beneficial effects when combining ribavirin and interferon rather than lpv/r alone ( ), and several combination therapies have been tried. this study protocol was exempted for review by the institutional review board of the korea university guro hospital according to the exemption criteria (irb no. gr ). we declare no conflict of interest. the same superscript letters indicate non-significant differences between groups based on post-hoc analysis. j o u r n a l p r e -p r o o f interim clinical guidance for management of patients with confirmed coronavirus disease (covid- ) infection control guidlines for healthcare professional about covid- national institutes of health (nih) treatment with hydroxychloroquine, azithromycin, and combination in patients hospitalized with covid- presumed asymptomatic carrier transmission of covid- effect of oral oseltamivir on virological outcomes in low-risk adults with influenza: a randomized clinical trial effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus (sars-cov- ) infection: a randomized clinical trial a trial of lopinavir-ritonavir in adults hospitalized with severe covid- low-dose hydroxychloroquine therapy and mortality in hospitalised patients with covid- : a nationwide observational study of participants altered covid- receptor ace expression in a 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medicinal plants remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro covid- ) weekly epidemiological update factors associated with prolonged viral shedding and impact of lopinavir/ritonavir treatment in hospitalised noncritically ill patients with sars-cov- infection factors associated with prolonged viral shedding and impact of lopinavir/ritonavir treatment in hospitalised non-critically ill patients with sars-cov- infection prolonged sars-cov- viral shedding in patients with covid- was associated with delayed initiation of arbidol treatment: a retrospective cohort study lpv/r = lopinavir/ritonavir key: cord- -tg kfiot authors: hulme, oliver j; wagenmakers, eric-jan; damkier, per; madelung, christopher fugl; siebner, hartwig roman; helweg-larsen, jannik; gronau, quentin; benfield, thomas lars; madsen, kristoffer h title: a bayesian reanalysis of the effects of hydroxychloroquine and azithromycin on viral carriage in patients with covid- date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: tg kfiot gautret and colleagues reported results of a non-randomised open-label case series which examined the effects of hydroxychloroquine and azithromycin on viral load in the upper respiratory tract of severe acute respiratory syndrome coronavirus (sars-cov- ) patients. the authors report that hydroxychloroquine (hcq) had significant virus reducing effects, and that dual treatment of both hcq and azithromycin further enhanced virus reduction. this data has triggered speculation whether these drugs should be considered as candidates for the treatment of severe covid- . however, questions have been raised regarding the study's data integrity, statistical analyses, and experimental design. we therefore reanalysed the original data to interrogate the main claims of the paper. here we apply bayesian statistics to assess the robustness of the original papers claims by testing four variants of the data: ) the original data; ) data including patients who deteriorated; ) data including patients who deteriorated with exclusion of untested patients in the comparison group; ) data that includes patients who deteriorated with the assumption that untested patients were negative. to ask if hcq monotherapy is effective, we performed an a/b test for a model which assumes a positive effect, compared to a model of no effect. we find that the statistical evidence is highly sensitive to these data variants. statistical evidence for the positive effect model ranged from strong for the original data (bf ~ ), to moderate when including patients who deteriorated (bf ~ . ), to anecdotal when excluding untested patients (bf ~ ), and to anecdotal negative evidence if untested patients were assumed positive (bf ~ . ). to assess whether hcq is more effective when combined with az, we performed the same tests, and found only anecdotal evidence for the positive effect model for the original data (bf ~ . ), and moderate evidence for all other variants of the data (bf ~ . ). our analyses only explore the effects of different assumptions about excluded and untested patients. these assumptions are not adequately reported, nor are they justified in the original paper, and we find that varying them causes substantive changes to the evidential support for the main claims of the original paper. this statistical uncertainty is exacerbated by the fact that the treatments were not randomised, and subject to several confounding variables including the patients' consent to treatment, different care centres, and clinical decision-making. furthermore, while the viral load measurements were noisy, showing multiple reversals between test outcomes, there is greater certainty around other clinical outcomes such as the patients who seriously deteriorated. the fact that all of these belonged to the hcq monotherapy group should be assigned greater weight when evaluating the potential clinical efficacy of hcq. randomised controlled trials are currently underway, and will be critical in resolving this uncertainty as to whether hcq and az are effective as a treatment for covid- . experimental methods. the experimental details are reported in the original published paper (gautret et al. ) which we henceforth refer to as the original paper. statistical analysis. bayesian statistical analyses of the data were performed in jasp (version . , jasp-stats.org ) . we note that caution should be taken with reanalyses of this data set because there are some discrepancies between different pre-prints and published versions. the analysis file, including the raw data we transcribed from the original paper, and all materials, are available on the open science framework at https://osf.io/ dgmx/ . all references to strength of evidence refer to standard conventions for the evidentiary support of bayes factors (bf) such that - is classed as anecdotal, - as moderate, - as strong, and - as very strong (jeffreys ) . for bayes factors below , the reciprocal can be taken to obtain the strength of evidence in the opposite direction. our initial analyses attempted to reproduce the findings of the original paper using the same data. we then performed the same analyses again, but with modified assumptions for how to treat excluded patients and untested patients. this can be considered a form of sensitivity analysis, of the sort recommended by a statistical review of the original paper (dahly et al. ) . unless otherwise stated, we focus on the primary outcome of viral carriage on day relative to inclusion point into the study. treatment groups . for brevity we deviate from the nomenclature of the original paper. hcq mono refers to treatment with only hcq. hcq +az refers to treatment with both az and hcq. hcq group refers to all patients treated with either hcq mono or hcq +az . comparison group refer to those patients not receiving either treatment. note that these are erroneously referred to as controls in the original paper. note that the statistical analysis file available for this reanalysis paper refers to the comparison group as controls. data. the experimental details are reported in the original paper. raw data was not available at the time of writing but was transcribed from the supplementary table of the original paper. we assess the robustness of the original paper's claims by testing four variants of the data, which vary assumptions pertaining to deteriorated and untested patients: ( ) data orig is the data as originally reported. this is the original data, as reported in the original paper. ( ) data det includes deteriorated patients . it is questionable to exclude several patients who could not complete the treatment because their condition deteriorated. this could introduce a selection bias that inflates the effect of the treatment. we therefore modified the original data as follows: of the hcq mono group, six were originally described as being excluded. one patient died, three deteriorated into intensive care, one patient stopped because of nausea and one left the hospital. these can be considered counterfactual cases that are necessary to entertain for a conservative estimate of the effects of hcq mono . in the following, we add the patients who died or entered intensive care to the positive test cases for day . this is tabulated in fig. a . we exclude both the patient that stopped treatment due to nausea, and the one patient who left the hospital, due to the ambiguity of their cases. this means that four cases are added to the hcq mono that tested positive for sars-cov- . ( ) data xcon includes deteriorated patients and excludes the untested patients. on day there were patients who were untested, even though the day test outcome was the primary outcome. the untested patients were assumed to be positive in the original paper, and for this data variant we simply exclude them. this can be motivated by the fact that the tests have some level of stochasticity, as can be seen from the fact that there are a total of transitions from negative tests on a given day, followed by positive tests the next day. for of the patients, they tested positive on day , and for of the patients the tests were not performed either on day or day . hence it is not known with any certainty what the test outcomes would have been in the five untested patients had they been tested on day . this is especially problematic since all of these untested patients belonged to the comparison group. for this reason it is important to analyse data that excludes these untested patients. ( ) data negcon includes the deteriorated patients and assumes untested patients test negative. given the problem with untested patients, we perform an analysis to evaluate what would happen to the results had these patients been tested and they were . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april , . . https://doi.org/ . / negative, rather than positive as assumed in the original data. this is included as a data variant not because it is the most likely case, but because it is the most conservative possible outcome, given the uncertainty of the reported data. main effect of hcq mono on viral carriage reduction. the original paper compared hcq group ,which is a composite of two groups (hcq mono and hcq +az ) with different drug treatments, to the comparison group. a more appropriate test for this question would be hcq mono versus comparison group. here we perform the test hcq mono versus comparison group, and assess its sensitivity to the variants of the data under different assumptions regarding deteriorated and untested patients. fig. a shows the number and proportion of patients testing positive for sars-cov- grouped by hcq mono or comparison group. here we perform analyses to quantify the degree to which hcq mono reduces viral carriage of sars-cov- (viral carriage hence). we conducted a bayesian a/b test (gronau & wagenmakers, ; kass & vaidyanathan ) that considered three rival models. the first model is a null model h which states that the viral carriage in hcq mono is equal to that of the comparison group. this entails that the log odds ratio ψ, for viral carriage reduction is equal to . the second model is a positive effect model h + which predicts that the effect of hcq mono exceeds that of the comparison group, and is thus indicative of a beneficial effect of hcq mono on viral carriage. under this model, ψ is assigned a positive-only truncated normal prior distribution n + (μ,σ). the third model is a negative effect model hthat predicts that the effect of hcq mono is smaller than that of the comparison group, which would indicate a harmful effect of hcq mono on viral carriage. under this model ψ is assigned a negative-only truncated normal prior distribution n -(μ,σ). for all models in this paper, we perform a default analysis in which the parameters of the normal distribution are set such that μ= and σ= . the results are tabulated in fig. (b,d,f, and h) for each of the four data variants. the bayes factors that we report indicate how likely the data is under each model. thus for each data variant one can use these factors to find which model finds most support from the data. for all data variants, the prior probabilities of each model were the same, namely that h is assigned a probability of . , h + and hare each assigned a probability of . . given these priors, for each of the three models, the corresponding posterior model probabilities are computed p(model | data) as can be seen in the tables of fig. (b,d,f, and h) . for all data variants (except data negcon ) it is the positive effect model h + that receives most support from the data. for data original the evidence is strong (bf + = . ) meaning that the data is approximately times more likely under h + than under h . for data det the evidence is moderate when including the deteriorated patients (bf + ~ . ), and for data xcon the evidence is anecdotal when excluding untested patients (bf + ~ ). for data negcon there was anecdotal evidence against the positive effect model if untested patients were assumed positive (bf + ~ . ). as is evident from this, the strength of the evidence for the positive effect of hcq mono over the comparison group is highly sensitive to the assumptions regarding what to do with the deteriorated or untested patients. the more conservative the assumptions that were made, the lower the strength of the evidence for the viral reduction effect of the hcq treatment. in other words, different choices in the pre-processing of the data can sway the evidence from strong evidence for a positive effect of hcq mono to anecdotal evidence against such an effect. this analysis provides interval estimates that were missing from the original report, which allow us to assess the size of the odds ratios, and their plausible ranges under different assumptions about the data. this sensitivity to assumptions is expressed in the credibility intervals for the odds ratios of the treatments. for data original the % credibility interval for the odds ratio has a lower bound of ~ . and an upper bound of ~ . for the data negcon however the same intervals run from a lower bound of ~ . to an upper bound of ~ . . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / main effect of combined treatment of az and hcq on viral carriage reduction. fig. a shows the number and proportion of patients testing positive for sars-cov- for each of the hcq treatment subgroups, hcq mono and hcq +az . here we quantify the degree to which combining az with hcq reduces viral carriage, over and above the effects of hcq on its own. we again conducted an a/b test that considered three rival models. the first model is a null model h which states that the viral carriage in hcq mono is equal to that of the hcq +az , thus offering no clinical benefit or harm, in terms of viral carriage. this entails that the log odds ratio ψ, for viral carriage reduction is equal to . the second model is a positive effect model h + which predicts that the effect of hcq +az exceeds that of the hcq mono , and is thus indicative of a beneficial effect of adding az to hcq to reduce viral carriage. under this model ψ is assigned a positive-only normal prior distribution n + (μ,σ). the third model is a negative effect model hthat predicts that the effect of hcq +az is smaller than that of hcq mono , which would indicate a harmful effect of adding az to hcq in terms of viral carriage. under this model ψ is assigned a negative-only normal prior distribution n -(μ,σ). the results are tabulated in fig. for each of the two data variants, data original and data det . as with the previous model, for both data variants, the prior probabilities of each model were the same, namely that h is assigned a probability of . , h + and hare each assigned a probability of . . given these priors, for each of the three models the corresponding posterior model probabilities are computed p(model | data), as can be seen in the tables of fig. b&d . for both data variants it is the positive effect model h + that recieves most support from the data. note that only two data variants are computed since the comparison group is not part of this test. for data original the evidence is anecdotal (bf + = . ) meaning that the data is approximately times more likely under h + than under h . this level of evidence is sometimes referred to as "barely worth mentioning" (jeffreys, ) . this would appear to temper the conclusions of the original paper, which inferred that this was a clinically important result, and one that was central to the medical recommendations of the paper. given the priors described above, the corresponding posterior model probabilities would be h ( . ), h + ( . ) and h -( . ). as can be seen in fig. c the % credibility interval for the odds ratio has a lower bound of ~ . and an upper bound of ~ . this indicates that there is also large uncertainty in the size of the positive clinical effect. the lower bound represents a % reduced chance of viral clearance having been improved by adding az to hcq. the upper bound of this estimate represents a % improved chance. the large uncertainty in this estimate of the odds ratio is due to the small sample size obtained in the original findings, in which the hcq +az group had only members. for data det the evidence is moderate when including the deteriorated patients (bf + ~ . ), meaning that the data is approximately times more likely under h + than under h . this demonstrates that the more conservative exclusion criteria actually increases the strength of evidence for the superiority of hcq +az over hcq mono . this is because including the deteriorated patients negatively impacts on the proportion of negative tests for the hcq mono group but not the hcq +az group. if the null model h were assigned prior probability . and the h + and hwere each assigned a probability of . , the corresponding posterior model probabilities would be h ( . ), h + ( . ) and h -( . ). as shown in fig. e the % credibility interval for the odds ratio has a lower bound of ~ . and an upper bound of ~ , indicating a large uncertainty in the positive clinical effect. the lower bound represents a % reduced chance of viral clearance having been improved by adding az to hcq. the upper bound of this estimate represents a % improved chance. again, the large uncertainty in this estimate is due to the small sample size obtained in the original findings, in which the hcq +az group had only members. as is evident from this analysis, the strength of the evidence for the positive effect of hcq +az over hcq mono is sensitive to the assumptions regarding what to do with the deteriorated patients. different choices change the evidence, from anecdotal based on the original data, to moderate under more conservative exclusion criteria. this analysis provides interval estimates that were missing from the original report, which importantly allow assessment of the odds ratios, and their plausible ranges under different assumptions about the data. for both data variants there is large uncertainty in the odds ratios, ranging from moderate reductions in the chance of improvement, up to very large chances of improvement. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint the strength of evidence for all statistical comparisons for all data variants is shown in fig. . note that we have focused on the comparisons of hcq mono versus comparison group, and hcq +az vs. hcq mono , because these answer the questions set out in the original paper. we computed two other comparisons for completeness, hcq group versus the comparison group, and hcq +az versus the comparison group. focus on these last two tests were downgraded, as because the first test aggregates two different treatments, and the second test confounds the effect of az. the full analysis details are available in the supplementary materials. as can be seen for both of these additional tests, the evidence is sensitive to the assumptions pertaining to the inclusion of deteriorated patients as well as to the status of untested patients. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / summary . using a complementary (bayesian) statistical framework, we evaluated the strength of the statistical evidence for the main claims of gautret et al., and we asked how robust this evidence is to different assumptions about how to treat deteriorated and untested patients. though we were able to qualitatively reproduce a positive effect of hcq on viral load reduction, and a further improvement by adding az, the strength of the evidence was highly sensitive to variations in these assumptions. we discussed these in detail, and provided a broader context for evaluating the quality of evidence offered by the original paper. in the original paper, the main test for the effect of hcq was performed by comparing a group of two different treatments (monotherapy hcq mono and the combination therapy hcq +az ) against the comparison group. this test does not directly answer the question of what is the clinical effect of hcq on upper respiratory tract sars-cov- viral load reduction, and to answer this one needs to compute the effect of hcq mono against the comparison group. it is regrettable that this test was not reported because it is the test that is necessary to evaluate the effect of hcq on viral reduction. performing a bayesian a/b test, we found that for the original data, there was strong statistical evidence for the positive effect of hcq mono improving the chances of viral reduction when compared to the comparison group. however, we found that the level of evidence drops down to moderate evidence when including the deteriorated patients, and it drops further to anecdotal evidence when excluding the patients that were not tested on the day of the primary outcome (day ). for context, anecdotal evidence is generally considered 'barely worth mentioning' (jeffreys, ) . we were able to qualitatively reproduce the finding of an improvement of hcq +az over hcq momo . however, although this finding was statistically significant in the original finding, our reanalysis revealed only anecdotal evidence for the positive effect of hcq +az over hcq momo . however, when we included the deteriorated patients into the analysis, this evidence increased to moderate. we also performed another test, which is to compare hcq +az against the comparison group. it should be noted that this test is not the most relevant test because it varies two drugs at the same time. nevertheless, the statistical evidence for the positive effect of the combined treatment over no treatment is very strong for the original data, and drops down to moderate when excluding the untested patients (fig. ) . common to both of these sets of analyses is the fact that they are highly sensitive to the assumptions that are made about the exclusion of patients, and of the test status of untested patients. that these assumptions are not well justified, nor adequately reported gives reason to be cautious in the interpretation of the evidence obtained from the original paper. furthermore, this uncertainty is generally reflected in the credibility interval estimates for the odds ratios, which can range from moderate decreases or small increases in the chances of improvement, through to very large chances of improvement. these uncertainties stem from the small sample sizes for each subgroup. indeed, the original paper has been criticised for being underpowered due to its relatively small sample size ( comparison group, treated). however, we argue that, from the perspective of statistical evidence, this criticism is less relevant once the data are observed. firstly such criticisms, though commonly espoused, should be made with reference to the effect size they are underpowered for. small sample studies can be well powered for detecting large effect sizes. more importantly, although estimating power is useful in planning experiments, it can be misleading when making inferences from observed data (wagenmakers et al. ) . in this reanalysis we rely on bayes factors, which are an extension of likelihood ratios beyond point hypotheses. these methods of inference do not average over hypothetical replications of an experiment, but instead condition on the data that were actually observed. for instance, the fact that a small sample can reveal strong evidence for an effect indicates that the effect size could be relatively large. in this way, bayes factors rationally quantify the evidence that a particular dataset provides for or against the null, or any other hypothesis. recourse to claims about the power of an experiment can be displaced by considering the strength of the evidence for one model over other models. this is clinically important because the strength of this evidence is not apparent from the statistical reporting of the original paper (which only reported p-values). put simply, the findings of the original paper cannot be dismissed solely on the basis of being "underpowered". experimental design and pre-registered protocol. the most fundamental problem with the original paper is that there was no randomisation of the treatment, which means it is vulnerable to differences in baseline risk between the subgroups. in the original . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / paper, the treatment groups are confounded by several variables including whether or not they met the exclusion criteria, which centre implemented treatment, and differences in consent (the comparison group were composed of those that met the exclusion criteria, or did not consent to treatment). for a full statistical review of these considerations see dahler and colleagues . most importantly, the comparison between hcq +az and hcq mono is confounded by the unreported clinical reasons for which the physicians decided to add the az treatment to some patients but not to others. if these reasons were important enough to warrant different treatment, then they are important enough to impact on the comparability between the two groups. whilst we refrain from making formal inferences, it is relevant to note that the hcq group patients were older than the comparison group patients (median age . and . years respectively, table ). it is also worth mentioning, that the comparison group included five cases aged or younger, which should again warrant caution when comparing outcomes between groups. we briefly comment on the existence of putative deviations from the pre-registered protocol, available at https://www.clinicaltrialsregister.eu/ctr-search/trial/ - - /fr . outcomes specified in advance included evaluation of upper respiratory tract viral carriage at , , and days, and yet the primary outcome reported in the paper was on day . this has been interpreted by some as outcome switching, however we would, in the absence of further information, suggest the possibility that this is an issue of how the days are numbered, whether one starts counting from zero or one. the day outcome was presumably not included such that the report could be published days earlier, which is defensible given the urgency of the pandemic at the time of writing. the secondary outcomes registered in the protocol are not adequately reported or analysed. finally, the raw data tables changed between different versions of the preprint and the published paper, and thus questions can be asked about data integrity. clearly, accommodation must be made for the speed at which the original report was published, and the conditions under which the data were presumably collected. the integrity of the reanalysis presented here is explicitly predicated on the assumption that all these possible deviations and data integrity issues can be adequately resolved. good clinical practice inspection for the sake of patient safety and data transparency would help to resolve such issues. it is important to note that the pcr based test uses a threshold of cycles (ct) to distinguish between pcr positive and pcr negative, some pcr positive patients in particular in the hcq treatment group show ct numbers that are quite close to this threshold indicating that the status might be somewhat ambiguous during the test. furthermore, a number of patients are later tested positive after being tested negative (occurring a total times in patients) which may further question the use of a hard threshold on the number of cycles. for these reasons using duplicate sample analysis and confirmatory tests and eventually developing quantitative pcr tests for assessment of treatment effects would be recommended for future studies. also note, that the current recommendation for a fda-emergency approved test is that negative pcr results do not preclude presence of sars-cov- infection and recommend that such results be accompanied by clinical observations, patient history, and epidemiological information. finally, it is important to determine whether sars-cov- virus nucleic acid detected by pcr is replication competent or not. at the time of writing, detailed clinical outcome data was not available, precluding any analysis relevant to a clinical outcome other than change from a positive to a negative pcr-based test. clinical safety. while the viral load measurements were noisy, showing multiple reversals between test outcomes, there is greater certainty around other clinical outcomes such as the patients whose condition seriously deteriorated. it is important to stress that all of these belonged to the hcq mono group, a fact that did not adequately temper the central claims of the original paper regarding the clinical potential of hcq. another way to state this would be that, though there is varying degrees of evidence for an effect of hcq on viral load, it is known with greater certainty that all of the deteriorations occurred in the hcq treatment group. greater weight should be placed on this fact, when stating the possible clinical benefits of hcq in the treatment of covid- . we find that computing the appropriate statistical tests for the effect of hcq on viral load reduction, yields results that are highly sensitive to the assumptions about which patients are included and how. while this evidence is strong for the assumptions made by the original paper, for more conservative assumptions, the evidence is substantially weaker than originally reported. performing the same analysis approach to the question of whether az improves hcq treatment, we find moderate https://diagnostics.roche.com/global/en/products/params/cobas-sars-cov- -test.html . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april , . . https://doi.org/ . / statistical evidence for a positive effect. whether this is a meaningful comparison however is questionable, based on the fact that it is confounded by undisclosed clinical decision making, that lead to some being treated with az and others not. to be clear, our analysis does not resolve the uncertainties that follow from the original experimental design, nor does it address concerns that have been raised about the study's data integrity. the only way to resolve these will be via the randomised controlled trials (rct) that are already underway. statistical review of hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial informed bayesian inference for the a/b test a power fallacy acknowledgements. due to the rapid pace of this analysis we solicited the assistance of a large number of informal reviewers.many of these we cannot identify simply because they were anonymised by the collaborative text editing software. they know who they are, and we thank them. those whose identities we know are elisabeth bik, andrew a love, gaetan burgio, and birgitte madsen, and we thank them too. it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / key: cord- -aiuiotds authors: afsin, abdulmecit; ecemis, kenan; asoglu, ramazan title: effects of short-term hydroxychloroquine plus moxifloxacin therapy on corrected qt interval and tp-e interval in patients with covid- date: - - journal: j clin med res doi: . /jocmr sha: doc_id: cord_uid: aiuiotds background: limited data are available regarding hydroxychloroquine (hcq) and moxifloxacin (mox) in patients with possible coronavirus disease , (covid- ). both drugs may increase risk of malignant ventricular arrhythmias associated with prolongation of qt interval. methods: a total of subjects with chest tomography findings compatible with covid- pneumonia were enrolled in the study. standard -lead electrocardiogram (ecg) was repeated on days and in patients receiving a combination of hcq + mox. heart rate, qt interval, tp-e interval, and tp-e/qt ratio were measured. results: the mean age of the patients was . ± . years and % had hypertension. compared to day , ecg on day showed significant increases in qt interval ( . ± . vs. . ± . , respectively, p = . ), corrected qt (qtc) interval ( ( - ) vs. ( - ), respectively, p < . ), tp-e interval ( ( - ) vs. ( - ), respectively, p < . ), ctp-e interval ( . ± . vs. . ± . , respectively, p < . ). moreover, a slight decrease in tp-e/qt ratio was observed ( . ± . vs. . ± . , p = . ). qtc was > ms in % of the patients, and % of patients had an increase in qtc interval > ms. tp-e/qt ratio was > . in % of patients. five patients died due to pulmonary failure without evidence of ventricular arrhythmia. no ventricular arrhythmia events, including torsades de pointes (tdp), were observed. conclusions: hcq + mox combination therapy led to increases in qtc interval, tp-e interval, and ctp-e interval. however, this therapy did not cause ventricular arrhythmia in the short-term observation. a pneumonia epidemic, which is considered to have developed due to a new coronavirus, was detected in wuhan, hubei province, the people's republic of china, in , which could not be brought under control and spread around the world within a short time causing a pandemic [ ] . the etiological agent has been reported to be novel coronavirus- ( -ncov) belonging to the coronaviridae family, which the world health organization (who) subsequently named serious acute respiratory syndrome coronavirus (sars-cov- ), and the associated disease was named coronavirus disease (cov-id- ) [ , ] . various agents considered to be effective in the treatment of covid- have been used since the beginning of the pandemic. chloroquine and hydroxychloroquine (hcq) are quinine-derived drugs that have long been used in the treatment and prophylaxis of malaria and chronic rheumatic diseases. chloroquine and hcq have been reported to be effective in many in vitro experiments against viruses, including sars-cov- [ ] . in addition, clinical studies conducted in china demonstrated that chloroquine had marked efficacy against covid- -associated pneumonia and acceptable safety [ ] . in a small-scale study conducted in france, hcq was reported to be effective in the treatment of covid- , and its efficacy was also suggested to be increased when administered along with azithromycin (az) [ ] . however, hcq can extend the qt interval and increase the risk of torsades de pointes (tdp). moxifloxacin (mox), from the fluoroquinolone group, is a broad-spectrum antibiotic widely used in atypical pneumonia. mox has also been reported to extend the qt interval and cause tdp [ ] . the qt interval measured on -channel electrocardiogram (ecg) shows regional heterogeneity of ventricular repolarization. the time from the peak of the t wave to the end point (tp-e) measured on ecg indicates the global dispersion of ventricular repolarization [ ] . studies have shown that increases in both regional and global ventricular repolarization heterogeneity predispose the patient to cardiac arrhythmia [ ] . although hcq and mox are well-tolerated drugs commonly used in clinical practice, both can cause prolongation of corrected qt (qtc). there are insufficient data in the literature on whether prolongation of qt and tp-e intervals can occur when hcq and mox are used together in the treatment of possible sars-cov- pneumonia. drug-induced qt prolongation is an important measure of drug safety as it is associated with increased mortality. this study was performed to evaluate qt and tp-e intervals, which are ventricular repolarization parameters measured on -lead superficial ecg to predict ventricular arrhythmia, in cases of possible covid- with negative results on the polymerase chain reaction (pcr) test receiving a combination of hcq and mox. we analyzed the data of patients who presented to our hospital and were diagnosed with suspected covid- between march and april . a total of consecutive subjects with chest computed tomography (ct) findings compatible with covid- pneumonia were enrolled in the study. diagnosis of covid- was made according to the who interim guidelines [ ] . hcq treatment ( mg twice daily for day, followed by mg twice daily for days) was determined as days according to the guideline published by the ministry of health, republic of turkey [ ] and mox ( mg infused once daily for > days) were started as empirical treatment in patients with a possible diagnosis of covid- . mox treatment was discontinued in covid- cases confirmed by the pcr test. therefore, confirmed cases of covid- were excluded from the study. patients who were using medications that could affect qrs, qt, and tp-e intervals, including tricyclic antidepressants, antihistamines, and antipsychotics, those with implantable cardioverter-defibrillators, those with a previously known branch or atrioventricular (av) nodal block, and those with a negative and/or biphasic t wave on their ecg were also excluded. those with a heart rate < beats/min or > beats/min, and those with congenital long qt syndrome were also excluded. finally, noncardiac exclusion criteria were pregnancy and breast-feeding. all patients presenting to the emergency department or outpatient clinic underwent complete medical assessment, physical examination, laboratory investigation, and ct of the chest. epidemiological, clinical, laboratory, and radiological findings were obtained from the electronic medical records and noted in data collection forms. blood samples were obtained from the cephalic vein by traumatic venipuncture and mixed with edta on hospital admission. complete white blood cell counts, including neutrophils and lymphocytes, were measured using an automated hematology analyzer (cell-dyn ruby; abbott diagnostics, abbott park, il) and expressed as × cells/mm . hemoglobin and platelet counts were also calculated. glucose, creatinine, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, bilirubin, sodium, potassium, calcium, ferritin, and c-reactive protein (crp) levels were analyzed using an architect c chemistry system (abbott diagnostics) with commercial kits (abbott diagnostics). troponin i and d-dimer assay levels were analyzed using an immunoassay analyzer (aqt flex; radiometer, copenhagen, denmark). the chest ct findings were as follows [ ] : multiple, patchy, sub-segmental or segmental/lobar ground-glass opacity in unilateral/bilateral lungs; multiple, patchy, or large patches of consolidation in unilateral/bilateral lungs, with slight grid-like or honeycomb-shaped interlobular septal thickening, especially in the middle and lower lobes. in addition, nasopharyngeal swab samples were collected by professional healthcare personnel in a special sampling room. the ecg recordings of the patients were acquired from telemetric monitors or a standard -lead ecg device. rhythm recordings were obtained from telemetric monitors (intellivue mp ; philips, hamburg, germany) using mm/s as the flow rate of the telemetric device and standard derivation (dii derivation) [ ] . the -lead ecg recordings ( mm/s, mm/mv) were obtained in the supine position using a cardiofax s device (nihon kohden, tokyo, japan). resting heart rate was measured using the ecg data. qt and tp-e intervals were calculated manually by two cardiologists using the ecg data. calipers and magnifying glasses were used to reduce measurement errors. the qt interval was calculated as the time from the start of the qrs complex to the end of the t wave. the tp-e interval was measured as the time from the t wave peak to the t wave end point. the measurements were performed on lead ii and lead v , and the longest qt and tp-e intervals were used for the analyses [ ] . the measured values were corrected according to the heart rate using bazett's formula. qtc interval and corrected tp-e interval (ctp-e) were obtained, and the ratios of tp-e/qt and corrected tp-e/qt (tp-e/qtc) were calculated. the interobserver and intraobserver variation coefficients for the tp-e/qt ratio were . % and . %, respectively, and those for the tp-e/qtc ratio were . % and . %, respectively. in addition, the tisdale score was calculated to evaluate the risk of qtc prolongation. the -lead ecg was repeated on days and in patients receiving a combination of hcq + mox. this study was conducted in compliance with the ethical standards of the responsible institution on human subjects as well as with the helsinki declaration. research ethics approval was obtained from the ethics committee of adiyaman university medical faculty (approval number: / - ). all analyses were performed using spss for windows . (spss inc., chicago, il). categorical variables are presented as numbers and percentages. continuous variables were tested for normality of the distribution normality with the kolmogorov-hcq plus mox on qtc and tp-e in covid- j clin med res. ; ( ): - smirnov test. continuous variables are presented as the mean ± standard deviation or median ( th - th interquartile range) and were compared using the paired t-test if the data were normally distributed and wilcoxon's rank sum test if the data were not normally distributed. pearson's and spearman's correlation methods were applied for relationship analysis. in all analyses, p < . was taken to indicate statistical significance. a total of patients were eligible for the study. patients with ecgs that could not be analyzed (n = ), patients with branch block in ecg (n = ), covid- patients with a positive pcr test (n = ), and those without follow-up ecg (n = ) were excluded. in addition, patients with a hospitalization period < days (n = ) were also excluded. the remaining patients were included in the study. the demographic findings and comorbidities of the study group are shown in table . the mean age of the patients was . ± . , and ( %) were women. the most common comorbidity in patients was hypertension ( %), followed by diabetes mellitus ( %), and chronic lung disease ( %). five patients died during their hospital stay due to pulmonary failure without evidence of ventricular arrhythmia. the laboratory parameters of the patients at the time of presentation are shown in table . table presents the ecg parameters of the study group. there was no statistically significant change in heart rate during follow-up. ecg showed statistically significant increases in qt interval ( . ± . vs. . ± . , respectively, p = . ), qtc interval ( ( - ) vs. ( - ), respectively, p < . ), tp-e interval ( ( - ) vs. ( - ), respectively, p < . ), and ctp-e interval ( . ± . vs. . ± . , respectively, p < . ) on day compared to day . furthermore, the ratio of tp-e/qt ( . ± . vs. . ± . , respectively, p = . ) was decreased significantly from day to day . in clinical follow-up, four ( %) patients had qtc > ms, while ( %) had an increase in qtc interval > ms. three ( %) patients had tp-e/qt ratio > . . no atrial arrhythmia and ventricular arrhythmia events, including tdp, were observed in any patient (table ). in addition, five patients had nausea without vomiting due to hcq side effects. three patients had dizziness due to mox therapy. age and troponin i showed significant positive relations with qtc interval (r = . , p = . ; r = . , p = . , respectively). d-dimer showed also significant positive correlations with ctp-e interval (r = . , p = . ) and tp-e/qtc ratio (r = . , p = . ). the correlations between ecg and clinical parameters are presented in table . we evaluated the ecg parameters showing heterogeneity our results showed that qtc increased from a mean baseline of ms to a maximum of ms after days, with approximately % of the patients developing qtc > ms. there was also increase in the ctp-e interval from ms to ms. no ventricular arrhythmia and atrial arrhythmia events were observed. long qt syndromes are cardiac repolarization disorders characterized by prolonged qt interval on ecg. these repolarization disorders can lead to rapid polymorphic ventricular tachycardia, known as tdp, syncope, or sudden cardiac death. mox, a member of the fluoroquinolone group, is now widely used in treatment of atypical pneumonia and can result in acquired long qt syndrome. mox blocks the rapid activating delayed rectifier potassium current (ikr) encoded by the human ether-a-go-go-related gene (herg) in a dose-dependent tp-e interval ≥ ms manner, thereby causing the prolongation of the qt interval and tdp [ ] . it has been known for many years that hcq has antiviral activity. there have been a number of studies on the effectiveness of both hcq alone and hcq + az combination in covid- . discussion continues in the medical community regarding the effectiveness of hcq in the treatment of cov-id- . in vitro and preliminary clinical studies have shown that the use of hcq alone or in combination with az is useful in the treatment of sars-cov- . however, these clinical trials usually consisted of a small number of patients [ , ] . in a study including , patients, million et al [ ] reported that the combination of hcq and az was safe to administer in patients before the development of covid- complications and was associated with a very low mortality rate. in contrast, other groups have stated that hcq is not effective in the treatment of covid- . for example, in an observational study in , patients with covid- , geleris et al reported that hcq administration was not associated with either lowered or increased risk of the composite end point of intubation or death [ ] . due to its immunomodulatory characteristics, hcq has been used for a long time in treatment of autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus, and its cardiovascular safety is good [ ] . moreover, information about cardiac side effects of hcq is limited to a few case reports. adverse events, such as prolonged qrs, prolonged qt interval, tdp, and ventricular arrhythmia, have been observed in individuals receiving hcq due to autoimmune diseases [ , ] . chloroquine causes prolonged qt by increasing the ventricular myocardial action potential, by affecting na + and ca + channels involved in depolarization, ikr channels involved in repolarization and, especially, inward rectifier k + current (ik current), which stabilizes the resting membrane potential [ , ] . the value of the qt interval for predicting tdp risk is low, but risk is known to be greater when qt > ms [ ] . however, case reports and small patient series with drug-induced tdp have shown that qtc exceeding the ms threshold or > ms prolongation in qtc compared to baseline ecg are associated with increased cardiac risk [ ] . therefore, when using two or more drugs that prolong qtc in combination, in the treatment of covid- , there are concerns about cardiac arrhythmia and sudden cardiac death related to prolonged qt. the preliminary safety results of a randomized, doubleblind, phase iib clinical trial (clorocovid- study) comparing the use of chloroquine at a high dose (a total dose of , mg over days) and a low dose (a total dose of , mg over days) in the treatment of sars-cov- have been published. the outcome of the first patients in the high-dose chloroquine arm showed a higher rate of qtc > ms ( %) and a trend toward higher lethality ( %) compared to those given the lower dose [ ] . therefore, the researchers stopped enrolling patients in the high-dose chloroquine arm. in addition, hcq is less toxic than chloroquine and, according to in vitro studies, the former is three times more potent than the latter [ ] . there have been a number of clinical studies to address the safety of the hcq + az combination in covid- pa-tients, and no serious cardiac toxicity was observed in these studies [ ] [ ] [ ] [ ] . in a clinical study of covid- patients treated with a combination of hcq + az, the results indicated that % of patients had qtc > ms and new development of acute renal failure was a strong predictor of extreme qt prolongation, while the baseline qtc was not a predictor of severe qtc prolongation [ ] . in a study of patients, saleh et al [ ] reported that the maximum qtc was ms in patients who received combined hcq + az treatment and ms in those undergoing monotherapy. the authors did not observe any ventricular arrhythmia. in a population of patients who tested positive for covid- by pcr, mercuro et al [ ] treated patients with hcq and patients with the combination of hcq + az. seven of the patients receiving hcq alone and of the patients receiving combination therapy had prolonged qtc ≥ ms. furthermore, one patient who received the combination therapy developed tdp. in a study performed in patients with a positive pcr test, bessiere et al [ ] reported that the number of patients with qtc ≥ ms was higher in the hcq + az combination group, but they did not detect any ventricular arrhythmia. in this study, while only four cases had qtc interval > ms, patients had prolongation of qtc > ms in comparison with the previous ecg. in addition, no ventricular arrhythmia, including tdp, was observed during the follow-up of these patients. we observed a similar risk of arrhythmia in patients with a positive pcr test who received hcq + az combination therapy compared to several previous studies. the absence of malignant ventricular arrhythmias may have played a major role in the short duration of treatment. our study showed that this combination therapy can be administered safely in covid- patients with close ecg monitoring. randomized controlled trials are needed to investigate the effects of hcq + mox combination on qt interval. numerous experimental and clinical studies have reported the poor performance of qt interval for demonstrating proarrhythmic sensitivity [ , ] . as the t wave shows ventricular repolarization on ecg, studies have also investigated the relationship between drug-induced arrhythmia and changes in t wave morphology. it has been reported that ecg parameters, such as the tp-e interval and tp-e/qt ratio, reflect the transmural dispersion of repolarization and show better repolarization heterogeneity than the qt interval. it has been reported that increases in tp-e interval and tp-e/qt ratio indicate increased dispersion of repolarization, which is predictive of ventricular arrhythmia and cardiovascular death [ , ] . in addition, tp-e and qt interval have been reported to be affected by body mass index and heart rate, while the tp-e/qt ratio is unaffected by these factors, and it is more sensitive for predicting ventricular arrhythmia [ ] . while the normal range of tp-e interval is - ms, that of the tp-e/qt ratio has been reported to be . - . [ , ] . in a recent meta-analysis, tse et al [ ] reported that the tp-e intervals were longer and the tp-e/qt ratios were higher in patients showing adverse events, such as tdp, than in those without such events among those with acquired qt prolongation. in subgroup analysis of the study, they found that the tp-e interval was ± ms and the tp-e/qt ratio was [ ] reported that tp-e interval ≥ ms was associated with ventricular arrhythmia. yamaguchi et al [ ] determined that in patients with acquired long qt syndrome, tp-e/qt ratio > . increased the risk of tdp. the effects of hcq on tp-e interval and tp-e/qt ratio remain unclear. taubel et al [ ] reported that mox caused prolongation of tp-e interval. in the present study, we found that patients had increased tp-e and ctp-e intervals and tp-e/ qt ratio during follow-up. on day of follow-up, none of the patients had tp-e or ctp-e interval ≥ ms, and the increases in these intervals were within the respective normal range. three patients had tp-e/qt ratio > . , and the tp-e/ qt ratio of these patients increased only slightly. there were no patients with ctp-e/qt ratio > . . our results showed that short-term doses ( days) of hcq + mox combination therapy were accompanied by slight increases in tp-e interval and ctp-e interval, which were not related to ventricular arrhythmia. in addition, the evaluation of these parameters with qtc may be more valuable in predicting drug-induced ventricular arrhythmia. randomized controlled studies are required to validate the results and determine the cut-off values for tp-e interval and tp-e/qt ratio in predicting drug-induced ventricular arrhythmia. there have been a number of reports regarding the monitoring and management of qt prolongation caused by hcq in sars-cov- . some groups have suggested that a baseline ecg should be performed in all patients and repeat ecgs should be performed during the period of hospitalization, while others recommend ecg monitoring only in high-risk patients [ ] . our study showed that ecg monitoring once or twice is sufficient. this study has several limitations. this was a retrospective and observational study conducted in a single center with a small number of patients. use of a holter monitor for follow-up of qtc and ctp-e intervals might have been useful in determining cardiac arrhythmia in these patients. however, the risk of covid- complicates this procedure. duration of the combination therapy was limited to days in this study. however, longer combination therapy is more useful to determine its adverse effects. tisdale score was calculated based on the ecg results from day and not the basal ecg. the results of this study showed that in possible covid- pneumonia patients, hcq + mox combination therapy led to increases in qtc and ctp-e intervals; however, short-term hcq + mox combination therapy did not cause ventricular arrhythmia. nevertheless, the possibility that dysrhythmic activity may increase synergistically with the use of this drug combination should always be taken into consideration. to evaluate the risk of ventricular arrhythmia in the treatment of covid- patients, it may be safer and more appropriate to evaluate the qtc and ctp-e intervals together with the tp-e/ qtc ratio. due to the risk of infection, it may be sufficient to perform ecg monitoring on days and rather than daily during the follow-up of covid- patients. virology, epidemiology, pathogenesis, and control of covid- severe acute respiratory syndrome coronavirus (sars-cov- ) and coronavirus disease- (covid- ): the epidemic and the challenges the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro breakthrough: chloroquine phos-hcq plus mox on qtc and tp-e in covid- phate has shown apparent efficacy in treatment of cov-id- associated pneumonia in clinical studies hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial severe qt interval prolongation associated with moxifloxacin: a case report the meaning of the tp-te interval and its diagnostic value diastolic electromechanical coupling: association of the ecg t-peak to t-end interval with echocardiographic markers of diastolic dysfunction clinical management of severe acute respiratory infection when novel coronavirus (ncov) infection is suspected the management of coronavirus disease (covid- ) ventricular arrhythmia risk due to hydroxychloroquineazithromycin treatment for covid- -american college of cardiology guidance on minimizing risk of drug-induced ventricular arrhythmia during treatment of covid- : a statement from the canadian heart rhythm society drug-induced inhibition and trafficking disruption of ion channels: pathogenesis of qt abnormalities and drug-induced fatal arrhythmias antiviral treatment of cov-id- early treatment of cov-id- patients with hydroxychloroquine and azithromycin: a retrospective analysis of cases in marseille, france observational study of hydroxychloroquine in hospitalized patients with covid- clinical efficacy and side effects of antimalarials in systemic lupus erythematosus: a systematic review chronic hydroxychloroquine use associated with qt prolongation and refractory ventricular arrhythmia life threatening severe qtc prolongation in patient with systemic lupus erythematosus due to hydroxychloroquine assessment of proarrhythmic activity of chloroquine in in vivo and ex vivo rabbit models drug-induced life-threatening arrhythmias and sudden cardiac death: a clinical perspective of long qt, short qt and brugada syndromes prevention of torsade de pointes in hospital settings: a scientific statement from the american heart association and the american college of cardiology foundation effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus (sars-cov- ) infection: a randomized clinical trial in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) the qt interval in patients with covid- treated with hydroxychloroquine and azithromycin effect of chloroquine, hydroxychloroquine, and azithromycin on the corrected qt interval in patients with sars-cov- infection risk of qt interval prolongation associated with use of hydroxychloroquine with or without concomitant azithromycin among hospitalized patients testing positive for coronavirus disease (covid- ) assessment of qt intervals in a case series of patients with coronavirus disease (covid- ) infection treated with hydroxychloroquine alone or in combination with azithromycin in an intensive care unit nonclinical proarrhythmia models: predicting torsades de pointes instability and triangulation of the action potential predict serious proarrhythmia, but action potential duration prolongation is antiarrhythmic the terminal part of the qt interval (t peak to t end): a predictor of mortality after acute myocardial infarction qt ratio as an index of arrhythmogenesis predictive value of t peak -t end indices for adverse outcomes in acquired qt prolongation: a meta-analysis the morphology of the qt interval predicts torsade de pointes during acquired bradyarrhythmias t wave peak-to-end interval and qt dispersion in acquired long qt syndrome: a new index for arrhythmogenicity effects of the fluoroquinolones moxifloxacin and levofloxacin on the qt subintervals: sex differences in ventricular repolarization qt prolongation, torsades de pointes, and sudden death with short courses of chloroquine or hydroxychloroquine as used in covid- : a systematic review none to declare. none to declare. the authors declare no potential conflict of interest. not applicable. aa contributed to conceptualization, analysis, writing, and editing; ke contributed to literature search and critical editing; ra contributed to data curation, analysis, supervision and resources. the data supporting the findings of this study are available from the corresponding author upon reasonable request. key: cord- - f k ce authors: prodromos, chadwick; rumschlag, tobias title: hydroxychloroquine is effective, and consistently so used early, for covid- : a systematic review date: - - journal: new microbes new infect doi: . /j.nmni. . sha: doc_id: cord_uid: f k ce introduction hydroxychloroquine (hcq) has shown efficacy against covid- in some but not all studies. we hypothesized that systematic review would show hcq to be: effective against covid- , more effective when used earlier, not associated with worsening, and safe. methods we searched pubmed, cochrane, embase, google scholar, and google for all reports on hydroxychloroquine as a treatment for covid- patients. this included pre-prints and preliminary reports on larger covid- studies. we examined the studies for efficacy, time of administration and safety. results hcq was found consistently effective against covid- when used early, in the outpatient setting. it was found overall effective also including inpatient studies. no unbiased study found worse outcomes with hcq use. no mortality or serious safety adverse event was found conclusions hcq is consistently effective against covid- when used early in the outpatient setting, it is overall effective against covid- , it has not produced worsening, it is safe. there is a need for effective treatment for covid- infection. hydroxychloroquine (hcq), with or without azithromycin, has been found to have efficacy as a treatment for covid- in some studies [ , ] , while other studies have not shown efficacy [ , ] . while we do not prescribe hcq to typical patients, we do treat various forms of inflammatory arthritis in patients taking hcq prescribed from outside providers. some physicians have stated that hcq has greater efficacy if given earlier in the course of the disease [ , ] . several studies showing negative efficacy have been withdrawn due to methodological improprieties [ ] . we hypothesized that hcq clinical studies would show significant efficacy more often than not for covid- ; and that efficacy would be greater if hcq were used earlier in the course of the disease. we also hypothesized that some studies that failed to show efficacy would be biased against positive efficacy and that no unbiased studies would show worsening. we also hypothesized that hcq would be found to be safe. we searched pubmed, cochrane, embase, google scholar, and google for all reports on hydroxychloroquine as a treatment for covid- patients. this included pre-prints and preliminary reports on larger covid- studies. we included papers with hcq alone as well as in combination with azithromycin and/or zinc. we excluded papers that studied chloroquine. while chloroquine has shown efficacy it has a higher side effects profile than hcq. for this reason, and because hcq is inexpensive and widely available we believe that future treatment will and should focus on hcq. it was thus our priority to examine hcq as fully as possible. we excluded papers that only examined hydroxychloroquine as a means to decrease transmission of coronavirus since our focus was on demonstrated clinical efficacy. reports were analyzed for efficacy, type of study, time of intervention with hcq during the covid- disease course, and for adverse events. our final search was performed this study has four important findings. the first is that hcq appears to be consistently effective for the treatment of covid- when used early in the course of disease in the outpatient setting, and is generally more effective the earlier it is used. the second is that overall hcq has had efficacy against covid- in a majority of studies. the third is that there are no unbiased studies showing a negative effect of hcq treatment of covid- . the fourth is that hcq appears to be safe for the treatment of covid- when used responsibly. timing of hcq use: it was striking that % of the studies which used hcq early in the disease on an outpatient basis showed positive results. in two of the studies [ , ] the benefit was only a trend. however the effects were clinically important: in mitja's study resolution of symptoms was decreased from to days; in skipper's study the rate of hospitalization was decreased by %. it is likely that with higher powering statistical significance would have been reached. in the other studies hcq was given on an inpatient basis with more advanced disease. the studies were divided into early, late and icu administration times. the early use, within hours of admission showed of or % of the studies to have positive efficacy. the two later groups, after hours admission and in the icu showed of or % to have positive efficacy. thus, from % for early outpatient, to % for early hospital, to % for later hospital use, there appears to be a relationship with time of initiation of treatment, and better results the earlier hcq is used. strength is the critical methodological study analysis heretofore not attempted to our knowledge for these studies. one weakness is the heterogeneity of study designs which rendered comparison of study results challenging. another perceived weakness of the study could be that these include reports made outside of peer-reviewed literature. multiple papers reporting both improvement and no efficacy using hydroxychloroquine that have been included in the study are either pre-prints or preliminary results of larger trials. because of the unprecedented and time sensitive nature of the sars-cov pandemic the scientific community has shared data and studies on a level unseen prior to this emergency. we believe that these reports hold valuable information and decided to include them regardless of the way in which they were published. in addition we found that both the peer-reviewed and non-peer reviewed papers showed a similar breakdown between studies showing efficacy vs not so that bias was not introduced. significance: we believe our findings have substantial societal global importance since there have been numerous edicts either preventing hcq use for covid- or limiting it to the inpatient setting, which we believe have unintentionally resulted in many unnecessary deaths. our findings showing efficacy and safety of hcq against covid- indicate that hcq should be freely available to patients and physicians who choose to use it. and it should especially be freely available to be used on an outpatient basis before hospitalization where it appears to be more effective and where early fears of fatal heart arrhythmias have been shown to be unfounded [ ] . this is particularly important because of the other drugs to show efficacy, remdesivir, has shown no significant benefit in a recent study [ ] . it is also expensive and not widely available. and dexamethasone has only been shown effective in critically ill j o u r n a l p r e -p r o o f hospitalized patients [ ] . convalescent plasma has shown benefit [ ] but even this is not well validated and plasma is not available in large numbers of doses. thus hcq with proven efficacy and safety, a cost of cents per pill and thus a total treatment cost of under dollars[ ], versus , dollars for remdesivir[ ], as well as wide supply chain availability, would appear to be the best covid- treatment option available and needs to be widely promoted as such. unfortunately the controversies surrounding hcq have resulted in physicians being afraid to prescribe it for reasons which have nothing to do with medicine, and in patients being afraid to take it due to spurious reports of danger, or fears that it is not effective. it is hoped that this study will disabuse the medical community of these misapprehensions about efficacy and validate that it is both efficacious and safe, and needs to be freely prescribable. we also do not believe that randomized controlled studies are necessary before hcq is authorized for general use because the efficacy seen in studies already done indicates that control patients in such studies might die unnecessarily; and because the time delay to do any such study would cause yet more deaths by preventing hcq use when it is most needed -which is immediately. our study has shown that good evidence of efficacy exists; and there is no safety, cost, or supply reason to not treat now. unnecessary death from delayed treatment is too high a price to pay for greater certainty of knowledge. many may have already died unnecessarily due to inaccurate hcq information, and it is imperative that we do not further add to the toll. hydroxychloroquine has been shown to have consistent clinical efficacy for covid- when it is used early in the outpatient setting, and in general would appear to work better the earlier it is used. overall effectiveness of hydroxychloroquine in covid- disease: a done and dusted situation? azithromycin and hydroxychloroquine accelerate recovery of outpatients with mild/moderate covid- hydroxychloroquine is associated with slower viral clearance in clinical covid- patients with mild to moderate disease: a retrospective study, medrxiv hydroxychloroquine alone or in combination with azithromycin to prevent major clinical events in hospitalised patients with coronavirus infection (covid- ): rationale and design of a randomised early treatment of covid- patients with hydroxychloroquine and azithromycin: a retrospective analysis of cases in marseille, france two coronavirus studies retracted after questions emerge about data empirical treatment with hydroxychloroquine and azithromycin for suspected cases of covid- followed-up by telemedicine hydroxychloroquine for early treatment of adults with mild covid- : a randomized-controlled trial hydroxychloroquine in nonhospitalized adults with early covid- : a randomized trial efficacy of hydroxychloroquine in patients with covid- : results of a randomized clinical trial treatment with hydroxychloroquine, azithromycin, and combination in patients hospitalized with covid- barbot, nimpact of medical care including anti-infective agents use on the prognosis of covid- hospitalized patients over time hydroxychloroquine treatment in covid- : a descriptive observational analysis of cases from a single center in wuhan compassionate use of hydroxychloroquine in clinical practice for patients with mild to severe covid- in a french university hospital no evidence of clinical efficacy of hydroxychloroquine in patients hospitalized for covid- infection with oxygen requirement: results of a study using routinely collected data to emulate a target trial observational study of hydroxychloroquine in hospitalized patients with covid- association of treatment with hydroxychloroquine or azithromycin with in-hospital mortality in patients with covid- in new york state beneficial effects exerted by hydroxychloroquine in treating covid- patients via protecting multiple organs low dose of hydroxychloroquine reduces fatality of critically ill patients with covid- treatment response to hydroxychloroquine and antibiotics for mild to moderate covid- : a retrospective cohort study from south korea, medrxiv effect of hydroxychloroquine in hospitalized patients with covid- : preliminary results from a multi-centre, randomized, controlled trial, medrxiv effects of hydroxychloroquine on covid- in intensive care unit patients: preliminary results, hydroxychloroquine and tocilizumab therapy in covid- patients-an observational outcomes of hydroxychloroquine treatment among hospitalized covid- patients in the united states-real-world evidence from a federated electronic outcomes of hydroxychloroquine usage in united states veterans hospitalized with covid- utilization of covid- treatments and clinical outcomes among patients with cancer: a covid- and cancer consortium (ccc ) cohort study doxycycline and hydroxychloroquine as treatment for high-risk covid- patients: experience from case series of patients in long covid- in iran, a comprehensive investigation from exposure to treatment outcomes, medrxiv observational study of the efficiency of treatments in patients hospitalized with covid- in madrid, medrxiv hydroxychloroquine and azithromycin plus zinc vs hydroxychloroquine and azithromycin alone: outcomes in hospitalized covid- patients, medrxiv early hydroxychloroquine is associated with an increase of survival in covid- patients: an observational study hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in covid- patients with at least a six-day follow up: a pilot observational study lopinavir-ritonavir versus hydroxychloroquine for viral clearance and clinical improvement in patients with mild to moderate coronavirus disease treatment response to hydroxychloroquine, lopinavir/ritonavir, and antibiotics for moderate covid : a first report on the pharmacological outcomes from south korea, medrxiv outcomes of , covid- patients treated with hydroxychloroquine/azithromycin and other regimens in marseille, france: a retrospective analysis hydroxychloroquine with or without azithromycin and in-hospital mortality or discharge in patients hospitalized for covid- infection: a cohort study of covid- outpatients-early risk-stratified treatment with zinc plus low dose hydroxychloroquine and azithromycin: a retrospective case series study early administration of lopinavir/ritonavir plus hydroxychloroquine does not alter the clinical course of sars-cov- infection: a retrospective cohort study, medrxiv hydroxychloroquine use in hospitalized patients with covid- : an observational matched cohort study no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe covid- infection nih halts clinical trial of hydroxychloroquine, national institutes of health, nih.gov hydroxychloroquine in patients with mainly mild to moderate coronavirus disease : open label, randomised controlled trial hydroxychloroquine is protective to the heart, not harmful: a systematic review effect of remdesivir vs standard care on clinical status at days in patients with moderate covid- : a randomized clinical trial dexamethasone in hospitalized patients with covid- -preliminary report effectiveness of convalescent plasma therapy in severe covid- patients hydroxychloroquine prices, coupons, and patient assitance programs remdesivir priced at more than $ , for a course of treatment treated group. as described above both studies had clinically important trends toward positive results, although were underpowered and did not reach statistical significance. the other five rcts were in hospitalized patients later in disease where efficacy seems to be less. there was positive [ ], no- effect [ , , ] , and negative effect [ ] studies. the negative effect study, however, was biased, as described below ("negative effect studies"), such that any negative or no-effect result would not be the third study showing worse results with hcq was a highly powered non-peer reviewed study whose primary outcome of day mortality actually showed no difference between the hcq treated group and the usual treatment group. two of the secondary results did just barely reach significance negatively. [ ] . however the reporting of results was flawed as follows. % of the treatment group patients did not receive hcq at all; and the median number of days of treatment for all treated patients was only out of a prescribed . these facts mean that less than half of patients received the full treatment regimen or even two thirds of the full treatment regimen, with in receiving no treatment at all. these untreated and undertreated patient outcomes were however grouped with the fully treated patient outcomes. if hcq has any positive effect which we believe is well established, this undertreatment would invalidate their borderline negative secondary results. in addition treatment was initiated more than hours after admission when our aggregate data has shown a high incidence of no-effect results. the study was not blinded introducing a potential undertreatment bias toward patients who were known by the staff to be treated with hcq. this study most reasonably is actually a no effects study, which is common in already hospitalized patients such as these treated more than hours after admission. adverse events: there have been fears among some that the increased qtc seen in some patients treated with hcq or azithromycin would predispose to torsades de pointes (tdp) and then death from ventricular fibrillation. we found no such deaths, or death from any cause related to hcq treatment, and indeed only case of tdp at all -which resolved spontaneously without treatment and without sequelae. this is consistent with our prior study showing an absence of tdp mortality with hcq use [ ]. all of the adverse events which seemed attributable to hcq treatment in the studies were side effects known to occur with hcq. these included nausea, vomiting, diarrhea, stomach pain, headache, key: cord- -ewo vvlr authors: jha, sujeet; batra, nitish; siddiqui, samreen; yadav, ashutosh; misra, archa; loomba, menka; sethi, sumeet; waghdhare, swati title: hcq prophylaxis in covid- did not show any qtc prolongation in healthcare workers date: - - journal: indian heart j doi: . /j.ihj. . . sha: doc_id: cord_uid: ewo vvlr background: hcq is a commonly recommended drug for the prophylaxis of covid- . one of its rare side-effect includes qtc prolongation. methods: this was a prospective, cross sectional and observational study conducted on hydroxychloroquine (hcq) among healthcare workers (hcws) at max super speciality hospital, saket, new delhi, india. a -lead ecg (only limb leads, it does not require chest leads) was performed. the qtc cut offs were pre decided, qtc < ms for males and < ms for females was considered within the normal limits and anything above this was regarded as qtc prolongation. results: there were hcws enrolled into the study, including males and females. majority of the hcws were young and had a mean age of . ± . years. out of these, were taking hcq as per the indian council of medical research (icmr) guidelines. the median cumulative dose being taken was mg and the median qtc of these participants was ms in males and . ms in females. subsequently, participants were followed-up and found to have a median qtc of ms and a cumulative dose of hcq as mg. conclusion: in conclusion, ours is a first study in the middle of the pandemic which showed that hcq prophylaxis in young hcws without comorbidities did not show any qtc prolongation. in the current era of rapidly rising cases of coronavirus illness (covid- ) infection, multiple treatment and prophylaxis options have been tried with various degrees of success rates as well as accompanying side effects. hcq is one of the commonly recommended medications for the population at high-risk of exposure, as it has both anti-inflammatory and antiviral effects. ( , , , , , , ) the indian council for medical research (icmr), national task force for covid issued recommendations for hcq to be used as a prophylaxis for the asymptomatic exposed healthcare workers (hcw) in late march . ( ) since these and similar recommendations have come up, several questions have been raised in regards to its cardiac toxicity particularly qtc prolongation. hcq is recommended as a prophylaxis as it is a long acting drug with a terminal halflife of more than days. ( ) there is a high-risk of side-effects on cumulative dosing. the most common side effects of hcq include gastrointestinal symptoms, pruritus, hypoglycaemia, retinopathy, neuropsychiatric effects and dermatological changes that can occur in up to % of the patients. ( , ) the most severe side effects have low incidence, which includes neuromyopathy of proximal muscles, cardiotoxicity, and irreversible retinopathy. a rarer side effect of hcq is prolonged qtc interval leading to arrhythmias and on rare occasions, life threatening arrhythmia torsades-de-pointes (tdp). even though occurrence of tdp is very uncommon, the risk is increased in patients receiving medications that have an additive effect of prolonged qtc, such as anti-infective drugs (macrolides, azole anti fungals, bedaquiline, lopinavir, and ritonavir, etc.), psychotropic drugs (chlorpromazine, haloperidol, etc.), ondansetron, formetrol, etc. ( ) however majority of these side effects have been studied in treatment doses but none in prophylactic doses. essentially all the drugs that create lqts (long qt syndrome) act by blocking the rapid delayed rectifier channel (ikr) current interceded by the potassium channel encoded by the kcnh quality. ( ) we decided to proceed quickly to understand qtc prolongation due to hcq as large work force in our institution was recommended hcq for prophylaxis as per the icmr guidelines. this was a prospective, cross sectional and observational study conducted on hcq among hcws at max super speciality hospital, saket, new delhi, india. the institutional ethics committee approved the study. we enrolled healthcare workers (hcw) coming to the hospital. the inclusion criteria were hcws, > years of age and consenting to participate. the qtc cut offs were pre decided, qtc < ms for males and < ms for females was considered within the normal limits and anything above this was regarded as qtc prolongation. ( ) a process was set up to meet the cardiologist if the qtc was prolonged. we decided to follow up a subset of the study population to see the effects of cumulative doses of hcq on those hcws who continued to take it. the follow-up was done on participants and a repeat ecg was recorded after an interval of one-month of their baseline ecg. we enrolled hcws out of which were taking hcq prophylaxis as per the guidelines. the mean age of participants was . ± . years. the demographics of the participants is shown in the table . none of the study participant was found to have covid- during the study period. however during this period the hospital prevalence was also low ( . %). ( ) the hcws were at different doses of prophylaxis at this cross-section of time. majority of participants were on hcq and they had taken it as per icmr guidelines i.e. a dose of mg twice a day for day only, followed by mg once a week for weeks. of the who were on hcq, ( . %) were males and ( . %) were females. mean age of these participants was . ± . years. maximum number of participants had taken it upto weeks. the median dose taken by participants was mg. the median qtc was ms in males and . ms in females. figure shows the distribution of participants in different qtc ranges. we did not have any participants having any structural/congenital heart disease and none of them the present study was aimed to find any effect of hcq prophylaxis on qtc prolongation amongst the hcws. we found that majority of the hcws were young ( . ± . years) and had no co-morbidities. at the time of this study, the cumulative dose taken by most of the participants was mg over weeks' time. we did not observe any qtc prolongation in any of the participants. as the pandemic and its prophylaxis continued, we followed-up participants with ecg and found the cumulative dose of mg as the median. interestingly, there was no qtc prolongation at this dose also. ever since the icmr recommendations for hcq use as prophylaxis for asymptomatic hcws has come, many concerns regarding its association with qtc prolongation have been raised. however, we did not come across any such findings. some studies done by icmr ( ) and closon et al ( ) concluded that in this era of sars-cov- , hcq provided significant advantage as a part of treatment/prophylaxis for the same, even though there would be more studies required with larger number of subjects to come to a definite conclusion. ( ) j o u r n a l p r e -p r o o f this is the first study on prophylaxis and qtc prolongation, in contrast to a study in nature medicine showing qtc prolongation in patients who were on treatment (hcq+ azithromycin). ( ) with this study, our purpose was neither to recommend nor discourage the use of hcq as a prophylaxis. ours is a first study in the middle of the pandemic which showed that hcq prophylaxis in young hcws without comorbidities did not show any qtc prolongation. however large population based studies would further validate our results. randomised control studies are being conducted worldwide to determine the safety and efficacy of hcq as prophylaxis in covid- . the sample size was small, however it was well representative of the hcws population. the follow up of the patients need to be done further upto at least weeks. there are no conflicts of interest involved in this study j o u r n a l p r e -p r o o f new insights into the antiviral effects of chloroquine. the lancet infectious diseases in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid- ? hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial of chloroquine and covid- could chloroquine /hydroxychloroquine be harmful in coronavirus disease (covid- ) treatment? clinical infectious diseases observational study of hydroxychloroquine in hospitalized patients with covid- advisory on the use of hydroxy-chloroquine as prophylaxis for sars-cov- infection. mohfw; bioavailability of hydroxychloroquine tablets in healthy volunteers pharmacologic treatments for coronavirus disease (covid- ) hydroxychloroquine and covid- prevalence of flu-like symptoms and covid- in healthcare workers from india advisory on the use of hydroxy-chloroquine as prophylaxis for sars-cov- infection chloroquine and hydroxychloroquine as available weapons to fight covid- a systematic review of the prophylactic role of chloroquine and hydroxychloroquine in coronavirus disease- (covid- ) the qt interval in patients with covid- treated with hydroxychloroquine and azithromycin j o u r n a l p r e -p r o o f key: cord- - ld wv authors: mitjà, oriol; corbacho-monné, marc; ubals, maria; tebe, cristian; peñafiel, judith; tobias, aurelio; ballana, ester; alemany, andrea; riera-martí, núria; pérez, carla a; suñer, clara; laporte, pep; admella, pol; mitjà, jordi; clua, mireia; bertran, laia; sarquella, maria; gavilán, sergi; ara, jordi; argimon, josep m; casabona, jordi; cuatrecasas, gabriel; cañadas, paz; elizalde-torrent, aleix; fabregat, robert; farré, magí; forcada, anna; flores-mateo, gemma; muntada, esteve; nadal, núria; narejos, silvia; gil-ortega, aroa n; prat, nuria; puig, jordi; quiñones, carles; reyes-ureña, juliana; ramírez-viaplana, ferran; ruiz, lidia; riveira-muñoz, eva; sierra, alba; velasco, césar; vivanco-hidalgo, rosa maria; sentís, alexis; g-beiras, camila; clotet, bonaventura; vall-mayans, martí title: hydroxychloroquine for early treatment of adults with mild covid- : a randomized-controlled trial date: - - journal: clin infect dis doi: . /cid/ciaa sha: doc_id: cord_uid: ld wv background: no therapeutics have yet been proven effective for the treatment of mild-illness caused by sars-cov- . we aimed to determine whether early treatment with hydroxychloroquine (hcq) would be more efficacious than no-treatment for outpatients with mild covid- . methods: we conducted a multicenter, open label, randomized controlled trial in catalonia (spain) between march , and may , . eligible covid- cases were non-hospitalized adult patients with recently confirmed sars-cov- infection and less than five days of symptoms. patients were assigned to receive hcq ( mg on day , followed by mg once daily for days) or no antiviral treatment (not-placebo controlled). study outcomes were the reduction of viral rna load in nasopharyngeal swabs up to days after treatment start, patient disease progression using the who scale up to days, and time to complete resolution of symptoms. adverse events were assessed up to days. results: a total of patients were eligible for intention-to-treat analysis: in the control arm and in the intervention arm. the mean age was . years (sd . ), mean viral load at baseline was . (sd . ) log( ) copies/ml, and median time from symptom onset to randomization was days. no significant differences were found in the mean reduction of viral load at day (- . vs. - . log( ) copies/ml in the control and intervention arm, respectively; difference . [ % ci - . ; . ]) or at day (- . vs. - . ; d – . [- . ; . ]). this treatment regimen did not reduce risk of hospitalization ( . %, control vs. . %, intervention; rr . [ . ; . ]) nor shortened the time to complete resolution of symptoms ( days, control vs. days, intervention; p = . ). no relevant treatment-related aes were reported. conclusions: in patients with mild covid- , no benefit was observed with hcq beyond the usual care. since the emergence of the novel sars-cov- coronavirus in december , various drugs have been proposed as antiviral agents for treating the coronavirus disease- (covid- ) , including the aminoquinolines chloroquine and hydroxychloroquine (hcq) [ ] . at the time this work started, the us fda and eu ema had given emergency approval for the use of chloroquine and hcq in covid- patients [ , ] . chloroquine and hcq have been extensively used for treating malaria and various autoimmune diseases, although other therapeutic effects, including antiviral effects, have been increasingly recognized [ , ] . in-vitro studies showed that both drugs can block the viral replication of sars-cov- in cell cultures [ , ] , but a high-level assessment suggested that calculated extracellular lung concentrations are well below the in vitro efficacy values and therefore the drug has low potential for in vivo activity at standard dosing regimens [ ] . as of june , , publicly available clinical data on the effectiveness of chloroquine and hcq for treating covid- were limited to two small randomized clinical trials [ , ] and six observational studies [ ] [ ] [ ] [ ] [ ] [ ] . several studies were seriously flawed in important methodological respects and lacked internal validity [ , [ ] [ ] [ ] ] . a randomized trial with patients found that hcq administration did not result in a significantly higher pcr negative conversion ( % vs. %) by days [ ] . however, the trial design raised concerns about the long delay between the onset of symptoms and the initiation of treatment (median . days) because antiviral therapy needs to be initiated early to have an impact on viral shedding. two large observational studies of hospitalized patients with covid- treated with hcq at physician's discretion found no significant reduction in the risk of death/intubation compared with no specific treatment [ , ] . because the metrics for each trial were chosen rapidly due to the emerging threat, the measured outcomes were different from one study to the next. the clinical characterization and management working group established by who recently agreed on a minimal outcome set to facilitate study design and data sharing, including viral burden (i.e., quantitative viral rna or cycle threshold from nasopharyngeal swabs), clinical outcome (i.e., progression scale: ambulatory, hospitalized, death) and survival (i.e., all-cause mortality) [ ] . we assessed the efficacy and safety of hcq initiated early for treating outpatients with mild covid- using the who core outcome set. [ ] . trained physicians identified from that registry and selected for study participation non-m a n u s c r i p t hospitalized patients of any kind (health worker, household contact, etc.) recently diagnosed. reasons for non-enrollment were recorded. adult patients aged years or more were eligible if they had mild symptoms of covid- (i.e., fever, acute cough, shortness of breath, sudden olfactory or gustatory loss, or influenza-like-illness) for less than five days before enrollment, were non-hospitalized, and had a positive pcr test for sars-cov- in the baseline nasopharyngeal swab. patients were excluded if they had moderate-to-severe covid- disease (e.g., required hospitalization), any condition that might preclude following the study procedures safely (e.g., mental disability), known allergy or hypersensitivity to study drugs, known retinal and severe liver or renal diseases, history of cardiac arrhythmia, known qt prolongation or other diseases that could be exacerbated by study drugs (e.g., psoriasis), active treatment with medications that are contraindicated with study drugs, or known hiv infection. females who were pregnant (verbally declared or positive pregnancy test) or breastfeeding were also excluded. the study protocol and subsequent amendments were approved by the institutional review board of hospital germans trias pujol, and the spanish agency of medicines and medical devices. written informed consent was obtained from all patients. this trial was a secondary study of the barcelona postexposure prophylaxis study against sars-cov- (bcn pep cov- study) registered in clinicaltrials.gov, nct . participants were randomized ( : ) using a computer-generated random-number list to either the control arm (no treatment aside from usual care) or the intervention arm (hcq -dolquine ® , mg on day , followed by mg once daily for six days). initially, the protocol included the use of hcq and cobicistat-boosted darunavir (drvc) combined treatment, but it was adapted to hcq alone after the recommendation of the pharmaceutical company not to use drvc for the treatment of covid- due to lack of activity in-vitro [ , ] and the negative results in human clinical trials of closely related hiv protease inhibitors [ ] . the study medications were dispensed by the hospital pharmacy and provided free of charge to the patients at the first home visit by dedicated outbreak field teams of trained nurses aided by trained paramedical staff. random allocation was done remotely by a member of the study team not involved in participants' enrollment. masking was not possible because a placebo could not be prepared due to the emergency nature of the trial. laboratory technicians were unaware of participants' treatment allocation, treatment response, and previous pcr results at all time points. participants were assessed on day (baseline, hcq was started), and days , , , and . on day , patients were home visited for baseline assessment and patient enrollment. outbreak field teams verified the selection criteria for eligibility, obtained patients' signed informed consent, assessed specific symptoms associated with covid- , and collected relevant epidemiological information from a structured interview. disease progression, safety, and self-reported treatment compliance were monitored by the clinical trials unit (ctu) of hospital germans trias pujol at days and (home visits), and (phone visits). compliance was assessed using self-reports in a telephone interview (e.g., number of doses taken between interviews). adverse events (ae) were defined as any new symptom or worsening of pre-existing symptoms and were followed until complete resolution of symptoms or up to day after enrollment. serious adverse events (sae) were defined m a n u s c r i p t as any medical event that required hospitalization or caused patient death; saes were graded for causality and expectedness and reported immediately to the contract research organization of the study sponsor and the trial pharmacovigilance consultancy (asphalion, barcelona) for independent adjudication of relatedness. study data were recorded electronically by the ctu during phone interviews, and on paper case record forms by the outbreak field teams during home visits and then entered into an electronic database by the data entry team of the sponsor. data validation and cleaning were done by trial researchers with the support of a trial data management consultancy (trial form support, barcelona). for each patient, serial oral and nasopharyngeal swab samples were planned to be obtained on days and . however, preliminary analyses revealed a possible delay for a significant viral load reduction beyond day ; therefore, we amended the study protocol to extend the collection of nasopharyngeal swabs to an additional sample on day (a -day window was allowed for patients who could not be assessed on day ). the presence of sars-cov- was investigated from nasopharyngeal swabs, and viral load was quantified in all rt-pcr positive cases (all time points collected). details on laboratory methods for sars-cov- identification and quantification are provided in supplementary material. the primary outcome was the reduction of viral rna load in nasopharyngeal swabs at days , and after treatment start. the secondary outcomes were clinical progression measured by a simplified version of the who progression scale [ ] ( , not hospitalized with or without resumption of normal activities; , hospitalized, requiring supplemental oxygen; , hospitalized, requiring invasive mechanical ventilation; and , death), and time from randomization to complete resolution of symptoms within the -days follow-up period. resolution of symptoms was assessed sequentially using a symptoms questionnaire designed to gather information on the type of symptom and last day experienced; complete resolution was considered when no covid- -related symptoms were reported at all. safety outcomes included ae that occurred during treatment, sae, ae of special interest (i.e., cardiac), and premature discontinuation of therapy. ae were classified according to the national cancer institute common terminology criteria for adverse events, version . . all unexpected sae were notified through eudravigilance. we estimated that a sample size of patients would provide the trial with % power to detect a difference of . log in the mean reduction of sars-cov- viral load at a two-sided significance level of α = . , assuming an expected standard deviation of . [ ] . a . log copies/ml difference in reduction was chosen to represent the minimal threshold for a biologically relevant change for our analyses [ ] . considering the open-label design and the possibility of side effects caused by the study medication, the primary efficacy analysis was performed on the intention-totreat (itt) population. sensitivity analyses were performed with the per-protocol (pp) population. safety was assessed in the safety population, which included all participants who received any therapy, including usual care. efficacy was determined by comparing the mean reduction of the viral load from baseline to days and , with the use of a mixed effects regression model taking into account the randomization group and repeated measures within each individual. the viral load was provided in logarithmic scale; a c c e p t e d m a n u s c r i p t specimens with undetectable viral load at a given follow-up assessment were assigned a value of log copies per ml (i.e., lower limit of detection) for the purpose of statistical analysis. the secondary clinical outcome regarding between-group differences in disease progression were assessed using risk ratio (rr) for the predefined events. the time to clinical improvement was analyzed using kaplan-meier survival functions and hazard ratios (hrs), calculated using a cox proportional hazards regression model based on the assumptions of proportional risks. kaplan-meier estimates were compared using the log-rank test. the significance threshold was set at a twosided α level of . unless otherwise indicated, and all analyses were conducted in r version . . [ ] . between march and april , , we assessed for eligibility confirmed covid- patients. figure summarizes the recruitment and follow-up flowchart of study participants. four-hundred ( . %) of did not meet the selection criteria and were therefore not enrolled. additionally, ( . %) participants were finally excluded from itt analysis because of negative rt-pcr at baseline, missing rt-pcr at all follow-up visits, or consent withdrawal, yielding an itt population of covid- patients. during follow-up, participants had a protocol deviation ( were screening failure due to history of more than five days since start of symptoms, was severely ill at baseline, were taking contraindicated medication, were lost-to-follow-up, and had treatment compliance under %) and were excluded from pp population. the two study arms had similar characteristics at baseline (itt population), including age, gender, comorbidities, frequency of symptoms, and nasopharyngeal viral load ( table ). the mean age of patients was . years (sd . ), and ( . %) of them were women. the median time from symptom onset to enrollment was days (iqr - ). a total of . % of the patients ( of ) reported chronic health conditions. fever, cough, and sudden olfactory loss were the most common presenting symptoms. the mean viral load in the nasopharyngeal swab at baseline was . (sd . ) log copies/ml. most patients were healthcare workers ( [ . %] of ). for the primary outcome of reduction of the viral load in nasopharyngeal swabs, there were no significant differences between the control arm and the intervention arm at day or . the mean differences in viral load from baseline to day were - . and - . log copies/ml in the control and intervention arm, respectively (difference [d] . [ % ci - . ; . ]) (table and figure ). the comparative analysis of the reduction of the viral load followed a similar trend at day : - . and - . in the control and intervention arm, respectively (d - . [- . ; . ]). the sensitivity analysis in the pp population also showed no difference between groups (table s , supplementary material). sub-group analysis comparing the viral loads of patients treated with hcq plus drvc did not reveal differences compared with hcq alone (table s ) . m a n u s c r i p t the clinical outcome of risk of hospitalization was similar in the control arm ( . %, / ) and the intervention arm ( . %, / ; rr . [ % ci . ; . ]) ( table ) . no patients required mechanical ventilation or died during the study period. median time from randomization to the resolution of covid- symptoms was not significantly different in the control arm ( . days, iqr - ) and the intervention arm ( . , iqr - ; log-rank-test for survival analysis p = . ; figure ). in the safety population / ( . %) patients in the control group and / ( . %) in the intervention group experienced at least one ae during the days of follow-up ( table ). the most frequent treatment-related aes among participants given hcq were gastrointestinal (e.g., diarrhea, nausea, and abdominal pain) and nervous system disorders (e.g., drowsiness, headache, and metallic taste). twenty sae were reported, in the control arm and in the intervention arm, none of them related to hcq (table s ). the results of this randomized controlled trial convincingly rule out any meaningful virological or clinical benefit of hcq in outpatients with mild covid- . we found that hcq initiated within five days from symptom onset (median days) was not effective in reducing viral shedding compared with no antiviral therapy. the quantification of the viral load in the upper respiratory tract provides strong evidence on the capacity of the treatment to affect the pathogen burden. furthermore, this treatment regimen did not reduce the risk of hospitalization-though the trial was underpowered for this outcome-nor shortened the time to complete resolution of symptoms. the much higher proportion of participants with aes in the hcq arm suggests poor tolerability of the treatment; however, no major aes related to the study drug were observed. of participants who were treated with hcq, % self-reported mild-to-moderate side-effects that were mainly gastrointestinal. only eight patients presented a sae within days of hcq treatment initiation, all related to disease progression. no cardiovascular events nor syncope/palpitation/dizziness suggestive of arrhythmia were reported. this finding is particularly important because it does not corroborate the concern for harm associated with hcq therapy, particularly cardiac disease [ ] . our study has several limitations. first, clinical assessments on day were not originally scheduled and therefore the number of patients analyzed for viral positivity at this time point was lower compared to day . while who has recommended a measure of viral burden in covid- clinical trials, they have neither set up the optimal time for measurement nor the minimal threshold for significant reduction between arms. we recommend the time for viral load reduction assessment to be long enough to capture a relevant decrease-ideally, days or longer-and the significant reduction threshold to be set at . log decrease or greater. second, we had originally chosen to combine hcq with the hiv protease inhibitor drvc because in silico molecular docking studies had predicted that drvc might have therapeutic effect on sars-cov- [ ] and the better safety profile compared to other hiv protease inhibitors. however, in-vitro results showing no activity that became available after the start of our study, prompted the decision to drop drvc [ , ] . the m a n u s c r i p t concomitant administration of drv in some participants may have slightly increased plasma levels of hcq, thereby leading to increased hcq effect because drvc is a weak inhibitor of the metabolic enzyme of hcq, cyp d . therefore, we do not expect the use of drvc might have reduced the effect of hcq. third, owing to the urgency, the trial could not be masked with a placebo, which may affect the rate of ae declared (aes are less often reported in a control, non-placebo group). nevertheless, it did not affect the attrition numbers in the control arm. moreover, to minimize the detection bias of the primary outcome (i.e., the viral load), the laboratory staff remained unaware of participants' allocation. finally, the regional nature of the trial and overrepresentation of healthcare workers (> %) may limit the generalization of our findings. therefore, cautiousness should be taken when extrapolating our data to other countries or settings. hcq and chloroquine have garnered unprecedented attention as potential therapeutic agents following inconclusive clinical trials in combination or not with azithromycin [ , ] , uncontrolled case series [ ] , and public figure endorsements [ ] . while there is a growing body of scientific data against using hcq for treating covid- that includes a concern for harm, particularly cardiac disease, the potential for the treatment of mild covid- with hcq has been explored in this trial to provide definite evidence. our results indicate no impact on viral burden up to days nor symptoms resolution or hospitalization rate up to days following diagnosis. the added value of our study is the randomized-controlled design and the use of the agreed minimal outcome set for covid- clinical trials, including rt-pcr to conclusively determine the viral burden. our findings provide the scientific community and policymakers with essential insights on the inefficacy of hcq as a therapeutic candidate for sars-cov- , at least in similar settings and conditions to ours. specimens with negative pcr (undetectable viral load) were assigned a value of log copies per ml for the purpose of statistical analysis. **estimated using a mixed effects regression model. none of the estimated mean differences and risk ratios were statistically significant. d: mean difference. rr: risk ratio. sd: standard deviation. se: standard error. m a n u s c r i p t ( . %) ( . %) injury, poisoning and procedural complications ( . %) ( . %) metabolism and nutrition disorders ( . %) ( . %) musculoskeletal and connective tissue disorders ( . %) ( . %) nervous system disorders ( . %) ( . %) psychiatric disorders ( . %) ( . %) renal and urinary disorders ( . %) ( . %) reproductive system and breast disorders ( . %) ( . %) respiratory, thoracic and mediastinal disorders ( . %) ( . %) skin and subcutaneous tissue disorders ( . %) ( . %) vascular disorders ( . %) ( . %) *none of the serious adverse events (sae) were adjudicated as related to hcq by the pharmacovigilance consultants. spanish council for scientific research (csic) germans trias i pujol research institute (igtp) centre of epidemiological studies of hiv/aids and sti of catalonia (ceeiscat) xarxa sanitària i social santa tecla, tarragona, spain . gerència territorial de barcelona discovering drugs to treat coronavirus disease (covid- ) covid- : chloroquine and hydroxychloroquine only to be used in clinical trials or emergency use programmes covid- : us gives emergency approval to hydroxychloroquine despite lack of evidence new insights into the antiviral effects of chloroquine hydroxychloroquine: from malaria to autoimmunity remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) connecting hydroxychloroquine in vitro antiviral activity to in vivo concentration for prediction of antiviral effect: a critical step in treating covid- patients efficacy of hydroxychloroquine in patients with covid- : results of a randomized clinical trial hydroxychloroquine in patients with mainly mild to moderate coronavirus disease : open label, randomised controlled trial no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe covid- infection hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in covid- patients with at least a six-day follow up: a pilot observational study observational study of hydroxychloroquine in hospitalized patients with covid- breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies association of treatment with hydroxychloroquine or azithromycin with in-hospital mortality in patients with covid- in new york state a minimal common outcome measure set for covid- clinical research catalan ministry of health. catalan epidemiological surveillance system lack of antiviral activity of darunavir against sars-cov- lack of evidence to support use of darunavir-based treatments for sars-cov- a trial of lopinavir-ritonavir in adults hospitalized with severe covid- centers for disease control and prevention (cdc). cdc -novel coronavirus ( -ncov) real-time rt-pcr diagnostic panel. . available at temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov- : an observational cohort study estimating the benefit of an hiv- vaccine that reduces viral load set point r: a language and environment for statistical com-puting risk of qt interval prolongation associated with use of hydroxychloroquine with or without concomitant azithromycin among hospitalized patients testing positive for coronavirus disease (covid- ) molecular modeling evaluation of the binding abilities of ritonavir and lopinavir to wuhan pneumonia coronavirus proteases therapeutic management of covid- patients: a systematic review we thank gerard carot-sans, phd, for providing medical writing support during the revisions of the subsequent drafts of the manuscript, the personnel from the fights aids and infectious diseases foundation for their support in administration, hr and supply-chain management, eric ubals (pierce ab) and Òscar palao (opentic) for website and database management, Óscar camps and openarms non-governmental organization for nursing home operations, anna valentí and the human resources department of the hospital germans trias i pujol for telephone monitoring. we are very grateful to marc clotet and natalia sánchez who coordinated the joemcorono crowdfunding campaign. we thank the institutional review board of hospital germans trias pujol and the spanish agency of medicines and medical devices (aemps) for their prompt action for consideration and approvals to the protocol. this work was mainly supported by the crowdfunding campaign joemcorono (https://www.yomecorono.com/) with the contribution of over , citizens and corporations. the study also received financial support from laboratorios rubió, laboratorios gebro pharma, zurich seguros, synlab barcelona, and generalitat de catalunya. laboratorios rubió also contributed to the study with the required doses of hydroxychloroquine (dolquine®). a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t m a n u s c r i p t a c c e p t e d m a n u s c r i p t figure key: cord- -ncyhvkwl authors: rowland yeo, karen; zhang, mian; pan, xian; ban ke, alice; jones, hannah m; wesche, david; almond, lisa m title: impact of disease on plasma and lung exposure of chloroquine, hydroxy‐chloroquine and azithromycin: application of pbpk modelling date: - - journal: clin pharmacol ther doi: . /cpt. sha: doc_id: cord_uid: ncyhvkwl we use a mechanistic lung model to demonstrate that accumulation of chloroquine (cq), hydroxychloroquine (hcq) and azithromycin (az) in the lungs is sensitive to changes in lung ph, a parameter that can be affected in patients with covid‐ . a reduction in ph from . to in the lung, as observed in respiratory disease, led to ‐, . ‐ and . ‐fold increases in lung exposure of cq, hcq and az, respectively. simulations indicated that the relatively high concentrations of cq and hcq in lung tissue were sustained long after administration of the drugs had stopped. patients with covid‐ often present with kidney failure. our simulations indicate that renal impairment (plus lung ph reduction) caused ‐, . ‐ and . ‐fold increases in lung exposures for cq, hcq and az, respectively, with relatively small accompanying increases ( to %) in systemic exposure. although a number of different dosage regimens were assessed, the purpose of our study was not to provide recommendations for a dosing strategy, but to demonstrate the utility of a pbpk modelling approach to estimate lung concentrations. this, used in conjunction with robust in vitro and clinical data, can help in the assessment of covid‐ therapeutics going forward. coronavirus disease of (covid- ) has rapidly become a global pandemic, since the outbreak was initially identified in wuhan, china in december, . the virus, severe acute respiratory syndrome coronavirus (sars-cov- ), can infect the lower respiratory tract causing fevers, cough, and pneumonia. as new drug candidates are being investigated for treatment of covid- , efforts are being made to repurpose existing antimalarial drugs, as they are readily available, and have a known safety profile. specfically, it has been reported that chloroquine (cq) has been successful in treating sars-cov- infections in china ( ) . in vitro studies have since confirmed that hydroxychloroquine (hcq), an analog of cq, is a more potent inhibitor of sars-cov- ( -to -fold) ( ) . given that hcq also has a more favourable safety profile than cq during chronic dosing, a clinical study was conducted in france to determine whether hcq ( mg daily; mg base) could be a more viable option for covid- treatment ( ) . despite its small sample size, the results of the study showed that treatment with hcq alone or in combination with azithromycin (az) shortened the time to resolution of viral shedding. based on the results of this study, clinicians in many countries have already begun using these medications in clinical practice, and multiple randomized trials are being initiated. for effective treatment of viral pneumonia in the lungs, it is expected that the concentration of drug at the site of infection should achieve or exceed ec values for inhibition of sars-cov- . uptake of drugs into the lung is particularly significant for basic amines, such as cq, hcq and az, with pk a values greater than . most basic amines, including cq and hq, are amphophilic, with a large hydrophobic group and a hydrophilic group that is ionized at physiological ph ( ). this makes them susceptible to lysosomal trapping, a mechanism that can lead to significant accumulation of a drug in lungs, or other lysosomal rich organs such as the heart ( ). small changes in the ph of the lung as a consequence of a sars-cov- infection may, therefore, have a significant impact on the accumulation of cq, hcq and az in lung tissue or the epithelial lining fluid (elf) relative to plasma. this article is protected by copyright. all rights reserved most of the published articles on covid- have focused on the lungs as the main organ involved in the disease, while few articles have reported sars-cov- involvement in other organs, including liver and kidneys, both of which are involved in the metabolism and excretion of cq, hcq and az. liver injury and an increased incidence of acute renal injury during sars-cov- infection have been reported ( , ). this is likely to add to the complication of deciding on the correct therapeutic dose of cq, hcq and az in patients with covid- and may lead to an increased risk of adverse drug reactions. physiologically-based pharmacokinetic (pbpk) modelling allows integration of drugrelated data (absorption, metabolism, plasma protein binding and physicochemical) with relevant physiology, to simulate profiles of drug in plasma as well as other organs and tissues, including the site of action ( ). thus, changes in physiology as a consequence of disease can be assessed using pbpk modelling, including renal ( ) and hepatic impairment ( ). two recent publications have applied pbpk modelling to predict the exposure of hcq and/or cq in the lung ( , ) . in the first of these publications, yao et al., ( ), applied a lung to plasma partition coefficient (kp) of for hcq based on rat data and then compared the significantly enhanced lung exposure with an in vitro derived ec . arnold and buckner ( ) reported on the limitations of the former study, mainly focusing on the source of the kp value and the fact that the lung concentrations were referenced against an ec rather than an ec . the authors assessed the impact of a range of kp values (including other rat studies -kp of ) on the lung exposure for a number of different dosing regimens for hcq and concluded that improved pk models were required for hcq in order to have more confidence in the efficacy of hcq against sars- we use previously published pbpk models for cq, hcq and az, and integrate a more mechanistic permeability-limited lung model ( ) , which allows us to investigate the exposure of the drugs in lung tissue and elf, rather than considering the lung as a homogenous perfusion limited organ as in previous publications ( , ) . we also assess the impact of covid- (via potential ph changes in the lung and renal impairment as a covariate) on the accumulation of each of the drugs in the plasma and lung. in addition, we compare the predicted exposure of each drug in plasma and lung with the published ec and ec values. this article is protected by copyright. all rights reserved a literature search was conducted to collate ec values for cq, hcq and az determined in vitro. it was noted that the in vitro viral activity assays differed slightly in their methodology where described in detail. however in essence, vero e cells were maintained in medium supplemented with low levels of serum were infected with sars-cov- virus at a multiplicity of infection (moi) ranging from . - . for several hours at a temperature of °c. these virus cells were then washed with media and treated with medium containing drug over a range of concentrations for or hours. viral rna was extracted and analysed by real-time pcr. in each of the publications, a sigmoidal hill function was used to estimate ec values (half maximal viral inhibition constant) ( , ( ) ( ) ( ) . a survey of literature data was performed to identify parameters, such as plasma binding proteins and ph values for the lung tissue and elf that may influence the predicted systemic and lung drug exposures in a "covid- population". due to the scarcity of data specific for covid- , we expanded our search to other types of viral pneumonia (supplementary information). simulations using full pbpk and the permeability limited lung model the simcyp (v . ) population-based pbpk simulator (simcyp ltd, sheffield, uk) was used to generate the profiles of cq, hcq and az. pbpk models for all compounds were verified and published previously ( , ) . each of these models was refined to include a permeability-limited lung model ( ) . based on its anatomy and physiology, the lung model is described by seven segments representing upper and lower airways ( segments) and the lobes of the lung ( segments). each segment is divided into four compartments representing pulmonary capillary blood, tissue mass, fluid, and alveoli air. the fluid compartment represents mucus and elf (ph= . ), whereas the mass compartment represents the different cell types within the lung (ph= . ). this article is protected by copyright. all rights reserved for each compound, henry's constant was predicted using a quantitative structureactivity relationship (qsar) approach and used to calculate the air:fluid partition coefficient. three different in vitro-in vivo extrapolation approaches are available to predict lung permeability. the preferred method is in vitro permeability data obtained from the calu- cell line ( ) . however, caco- permeability data (p app ) or physicochemical properties, such as log d, pka, hydrogen bond donor count can be used to predict calu- permeability and hence, lung permeability. the in vitro permeability data are corrected by the unionized fraction of compound (calculated at the ph of the in vitro system) as only the unbound, unionized drug is considered to be passively permeable in the lung pbpk model. due to low protein concentrations in elf ( ), the unbound fraction in the elf was assumed to be for all drugs. measured values of unbound fraction in lung tissue homogenate (fu,mass) could not be found for any of the compounds, therefore, fu in lung tissue mass was predicted as reported previously ( , ) . after integrating the permeability-limited lung model, simulations were run to assess the impact of key physiological parameters (ph in the lung) on the exposure of each of the drugs in the lung and plasma using a number of different dosage regimens that are currently being assessed in clinical trials. the effect of severe renal impairment (gfr< ml/min) was assessed using the population (elevated aag, reduced albumin levels, elevated serum creatinine) within the simcyp simulator. unless stated otherwise, subjects aged to years ( % male, % female) were simulated. for all of the above scenarios, the simulated concentrations for total lung, unbound lung, total elf (assumed to be the same for unbound) and total plasma were compared against the lowest and highest reported ec values. in addition, trough lung concentrations (unbound) were compared against a reported or extrapolated ec ( x ec ). the input parameters for the cq model are provided in supplementary supporting material (tables s and s ). an in vitro caco- p app value of x - cm/s was used to predict permeability across the lung ( ) . henry's constant was predicted to be . x - pa m /mol (epi suite). simulations were performed to demonstrate that the updated cq this article is protected by copyright. all rights reserved model was able to generate concentration-time profiles that were consistent with observed data (supplementary information). predicted lung exposures relative to plasma over time were generated for a single mg cq dose and the kp values were compared with observed rat data. the effect of lung ph ( . to . ) on the kp values was also assessed. plasma and lung exposures were simulated for different dosage regimens that have traditionally been used for malaria treatment or are currently being investigated for treatment of covid- . these include: (a) mg cq base on days and , mg on day ( . g over days) -who regimen (b) mg cq base on day at am, followed by mg at pm on day , followed by mg at am on days and ( . g over days) -fda regimen (c) mg cq base on day followed by mg qd for days ( . g over days) (clinicaltrials.gov identifier: nct ) (d) mg cq base bid for days ( g over days) (clinicaltrials.gov identifier: nct ) (e) mg cq base qd for days, followed by mg weekly out to days ( . g over days) (clinicaltrials.gov identifier: nct ) (f) mg cq base qd for days, followed by mg qd out to days ( . g over days) (clinicaltrials.gov identifier: nct ) with the exception of a metabolic intrinsic clearance of . µl/min/mg protein, the input parameters for the hcq model were as cited previously by yao et al, ( ) . an in vitro caco- p app value of . x - cm/s ( -fold lower than that of cq based on ferrari et al, ( ) ) was used to predict permeability across the lung. henry's constant was predicted to be . x - pa m /mol (epi suite) (table s ). plasma and lung exposures were simulated for the dosage regimen used in the french study; mg hcq ( mg base) was given every hours for days ( ). this article is protected by copyright. all rights reserved with the exception of the b:p ratio which was set at . , the input parameters for the az model were as cited previously by johnson et al., ( ) . an in vitro caco- p app value of . x - cm/s calibrated using a p app of . x - cm/s for propranolol ( ) was used to predict permeability across the lung. henry's constant was predicted to be . x - pa m /mol (epi suite) (table s ). simulations were performed to demonstrate that the updated az model was able to generate exposures of az that were consistent with observed data. plasma and lung exposures were simulated for the dosage regimen used in the french study; mg was given on day followed by mg once daily for days ( ). depending on the methodology applied, at h, ec values for cq range between . and . µm (table ) . at h, ec values for hcq range between . and . µm. a single ec value of . µm was available for az. in each case, these represent total ec values as no protein binding corrections have been applied. however, given that the the fu,p values for cq, hc and az are . , . and . , it is expected that the protein binding in these in vitro pharmacology experiments will be minimal. normal elf ph is considered to be acidic, averaging ~ . (range . to . ) in healthy airways compared to a blood and interstitial ph of . . there are indications that in airway diseases, such as cystic fibrosis and pneumonia, elf ph is more acidic, suggesting breakdowns in the ph regulatory mechanisms ( ). using a ph electrode/probe, the most acidic measured ph value in elf of bacterially infected pneumonia patients is . . another study indicated that the ph of exhaled breath condensate (ebc), which reflects the ph on of the lining fluid of the lung, was lower in mechanically ventilated patients compared to spontaneously breathing individuals (mean±standard deviation values of . ± . vs. . ± . ) ( ). in the absence of human data describing the ph value for lung mass, a value of . based on experimental data obtained in dog (( );range of . - . ) is used in the model. based on these data, the range for sensitivity analysis on lung mass ph in the current study was set to . to . . serum albumin is commonly measured as part of the initial evaluation of critically ill patients with infectious disease. several studies show that the frequency of hypoalbuminemia at admission is high in hospitalized patients with community acquired pneumonia ( ). the leading causes of hypoalbuminaemia are malnutrition, reduced hepatic synthesis, renal losses, and inflammation; however, the predominant mechanism by which serum albumin decreases is secondary to increased capillary permeability and redistribution from plasma to the interstitium in critically ill patients ( ). a summary of this article is protected by copyright. all rights reserved serum albumin levels in differing types of viral pneumonia in the literature is presented in table s . the weighted mean albumin levels in severe cases of viral pneumonia is . g/l. concentrations of an inflammation marker, alpha- -acid-glycoprotein (aag), are generally believed to increase during an infection, however, quantitative information is limited in patients with viral pneumonia. a . -fold to -fold increase in aag levels in cap ( ) or sars-cov patients ( ) has been reported. as the above parameters appear to be reasonably consistent with those previously cited for patients with severe renal impairment ( ), they were accounted for in simulations of virtual patients with gfr< ml/min. the predicted kp values for lung after a single oral dose of mg cq base are shown in figure . observed data in humans are not available for comparison and rat data from several different studies are presented. the predicted kp value after days (kp= ) appears to be reasonably consistent with the rat kp values (approximately ) that have been previously published (mcchesney et al., ) . of particular importance, is the finding that the predicted kp, and hence the exposure in the lung, is very sensitive to small changes in ph. indeed, a reduction in ph from . to . or down to . , results in an increase in predicted kp from to and , respectively. the simulated concentration-time profiles for cq in plasma and lung (lung tissue and elf) based on various different dosage regimens are shown in figure . when comparing the unbound lung exposure of cq against the minimum cited ec value for inhibition of sars-cov- , all dosage regimens appear to generate high enough exposures from the first day of dosing. however, when compared against the ec value ( . µm), the only dosage regimen that meets this target on day is mg bid for days ( table ). for of the dosage regimens, the effect of reducing lung ph to (lung tissue and elf) and renal impairment are shown in figure . although the increase in cq lung exposure is more sensitive to changes in ph than the added complication of renal impairment (protein binding and gfr), the combined effect resulted in -to fold increases relative to the control scenario (lung ph= . and normal gfr) ( table ) . this article is protected by copyright. all rights reserved after a single oral dose of mg az, predicted plasma c max and auc values were within - % of observed values (( ); data not shown). predicted c max values in the elf and lung were . and . µg/ml, respectively, which were consistent with observed values of . and . µg/ml, respectively. based on c max values, predicted tissue to plasma ratios were . and . for elf and lung tissue, respectively, which were again similar to observed values of . and . . concentration-time profiles for az in plasma and lung (lung tissue and elf) after a single oral dose of mg followed by mg daily for days are shown in figure . a reduction in lung ph does not lead to a change in plasma exposure but a . -fold increase in c max is expected in the lung ( . versus . µm). a % increase in plasma exposure is predicted in subjects with renal impairment and the c max in lung is further increased to . µm. unbound lung concentrations of az do not attain the cited ec or ec values ( figure ) . in the current study, we sought to demonstrate that the accumulation of cq, hcq and az in the lungs is sensitive to lung ph, a parameter, which could change significantly in patients as a consequence of having covid- . the degree of accumulation is largely this article is protected by copyright. all rights reserved dependent on drug related parameters (log d, pka, fu plasma and fu mass ) and the ph gradients maintained across the blood vessels and lung. this complex interplay of parameters is captured by the mechanistic multi-compartment permeability limited lung model ( ) and allowed us to investigate the effect of covid- and associated comorbidities on the predicted concentrations of cq, hcq and az in the lung and plasma. a change in ph from . to in the lung, which is observed in respiratory disease, led to -, . -and . -fold increases in lung exposure of cq, hcq and az, respectively, due to an increase in ionisation of the drugs. furthermore, the high concentrations of cq and hcq in lung tissue were sustained long after administration of the drugs had stopped. a significant number of patients with covid- present with kidney failure ( ). our simulations indicate that renal impairment (plus lung ph reduction) one of the limitations of the study is that predicted baseline concentrations of the drugs in lung could only be verified for az. clinical data for az were characterised in plasma, elf, alveolar macrophages (am) and lung tissue itself ( ). for cq and hcq, kp values for lung derived from rat data provided some level of verification. the lung model has been applied previously, with reasonable success, to predict the lung exposure of at least drugs used in the treatment of tuberculosis ( ) . however, future studies that characterise cq and hcq concentrations in elf, plasma, am and/or bronchial tissue are warranted. in addition, robust in vitro data relating to the permeability of the drugs across the lung are required to enhance the pbpk models for these drugs. it is assumed that drug efficacy at the site of action is determined by the unbound drug in the tissue. the unbound fraction in lung was not available for these drugs and therefore, had to be predicted based on physicochemical properties ( ) . this article is protected by copyright. all rights reserved dosage regimens that were simulated for cq, hcq and az appeared to attain concentrations that were comparable with the in vitro derived ec values, despite the ec values being highly variable for each compound. when compared against the ec values, it appears that for hcq, the dosage regimen investigated in the french study ( mg base three times a day for days) ( ) was not able to attain lung exposures in this target range, a finding which was in agreement with arnold & buckner( ). yao et al. ( ) reported that based on their pbpk modelling results, hcq (a loading dose of mg base given twice on day followed by mg twice daily for days) should be recommended for treatment of covid- as this dosage regimen was likely to attain high enough concentrations in the lung to inhibit the sars-cov- virus. for cq, the potential -fold increase in lung exposure in covid- patients relative to healthy subjects, led to predicted unbound concentrations in the lung that were higher than the ec from day even for one of the more conservative dosage regimens ( mg for days to , followed by mg qd). given the uncertainty associated with the predicted fu mass values and their impact on dose selection, appropriate in vitro studies to measure tissue binding in lung homogenate should be conducted to lend more weight to the predictions. it may also be pertinent to use ec values derived from measured intracellular concentrations rather than those added to the cell culture media in vitro ( ). as uptake of both cq and hcq is sensitive to ph, it cannot simply be assumed that uptake into cells in vitro reflects accumulation of the drugs in the lung ( ). last, but certainly not least, concerns relating to cq and hcq toxicity should also help inform dose selection. there are an increasing number of reports of associated qt prolongation and it is possible that patients with covid- may be more susceptible to cq and hcq toxicity ( ). going forward, efforts should be made to link the exposure of cq and hcq in the heart with markers of qt prolongation, such as the ec data derived for inhibition of cardiac ion channels ( ) to better understand margins of cardiac safety. whilst it is important to acknowledge the limitations described and collect more clinical data to verify drug exposures in the lung, as highlighted by arnold & buckner, ( ) , our study does show that there is a place for pbpk modelling to inform the advancement of this article is protected by copyright. all rights reserved hq and cq and az as potential candidates for treatment of covid- . furthermore, the strategy taken here may be useful to assess and prioritise other drugs that are potential candidates for repurposing. several publications have recently appeared relating to the application of pbpk models for prediction of the exposure of cq and hcq in lung and plasma. both drugs have been identified as potential candidates for treatment of covid- . we sought to demonstrate that a modelling approach can be used to assess the effects of physiological changes, potentially evoked by covid- , on the plasma and lung exposure of cq, hcq and az. we confirm that the accumulation of cq, hcq and az in the lung is very sensitive to changes in ph, a parameter that could be affected by covid- . we also demonstrate that renal impairment, a comorbid condition of covid- , may lead to increased concentrations of all drugs in the lung. the approach and mechanistic lung model described here could be applied to other drugs being investigated as covid- therapeutics. this article is protected by copyright. all rights reserved mass unbound concentration to ec value ( . µm) against sars-cov- virus in vero e cells. table s . table s . table . figure s . figure s . figure s accepted article this article is protected by copyright. all rights reserved : ratio of the simulated lung mass unbound concentration to ec value ( . µm) against sars-cov- virus in vero e cells . breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) hydroxychloroquine and azithromycin as a treatment of covid hydroxychloroquine for treatment of sars-cov- infection? improving our confidence in a model-based approach to dose selection development of a multicompartment permeability-limited lung pbpk model and its application in predicting pulmonary pharmacokinetics of antituberculosis drugs hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro in vitro screening of a fda approved chemical library reveals potential inhibitors of sars-cov- replication remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro how does in vivo biliary elimination of drugs change with age? evidence from in vitro and clinical data using a systems pharmacology approach interpretation of antibiotic concentration ratios measured in epithelial lining fluid a mechanistic framework for in vitro-in vivo extrapolation of liver membrane transporters: prediction of drug-drug interaction between rosuvastatin and cyclosporine physiologically based pharmacokinetic modeling : predicting the tissue distribution of moderate-to-strong bases using human plasma as an assay medium in caco- studies improves mass balance for lipophilic compounds kinetics and thermodynamics of chloroquine and hydroxychloroquine transport across the human erythrocyte membrane assessment of macrolide transport using pampa, caco- and mdckii-hmdr assays we would like to acknowledge the bill & melinda gates foundation for funding the development of the original chloroquine model. we would like to thank dr ping zhao for his review of the manuscript and eleanor savill for her assistance with manuscript preparation. this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved key: cord- -errwp i authors: ren, l.; xu, w.; overton, j. l.; yu, s.; chiamvimonvat, n.; thai, p. n. title: assessment of hydroxychloroquine and chloroquine safety profiles: a systematic review and meta-analysis date: - - journal: medrxiv : the preprint server for health sciences doi: . / . . . sha: doc_id: cord_uid: errwp i background: recently, chloroquine (cq) and its derivative hydroxychloroquine (hcq) have emerged as potential antiviral and immunomodulatory options for the treatment of coronavirus disease (covid- ). to examine the safety profiles of these medications, we systematically evaluated the adverse events (aes) of these medications from published randomized controlled trials (rcts). methods: we systematically searched pubmed, medline, cochrane, the cochrane central register of controlled trials (central), embase, and the clinicaltrials.gov for all the rcts comparing cq or hcq with placebo or other active agents, published before march , . the random-effects or fixed-effects models were used to pool the risk estimates relative ratio (rr) with % confidence interval (ci) for the outcomes. results: the literature search yielded and studies for cq and hcq, respectively, that satisfied our inclusion criteria. of these studies, we performed meta-analysis on the ones that were placebo-controlled, which included studies for cq and studies for hcq. we did not limit our analysis to published reports involving viral treatment alone; data also included the usage of either cq or hcq for the treatment of other diseases. the trials for the cq consisted of a total of , participants (n= , cq, n= , placebo), while the trials for hcq involved , participants (n= hcq and n= placebo). the overall mild or total aes were statistically higher comparing cq or hcq to placebo. the aes were further categorized into four groups and analyses revealed that neurologic, gastrointestinal, dermatologic, and ophthalmic aes were higher in participants taking cq compared to placebo. although this was not evident in hcq treated groups, further analyses suggested that there were more aes attributed to other organ system that were not included in the categorized meta-analyses. additionally, meta-regression analyses revealed that total aes was affected by dosage for the cq group. conclusions: taken together, we found that participants taking either cq or hcq have more aes than participants taking placebo. precautionary measures should be taken when using these drugs to treat covid- . the coronavirus disease is caused by the novel, highly infectious, severe acute respiratory syndrome coronavirus (sars-cov- ). since its discovery in december of in wuhan, it has now caused a global pandemic. as of march , , there were , confirmed cases and , deaths from the disease, which brings the mortality to approximately . %. thus, scientists, pharmaceutical companies, and medical professionals have united their efforts to develop a vaccine for sars-cov- . although it is estimated that vaccine development will take at least - months, two medications-chloroquine (cq) and hydroxychloroquine (hcq)-have emerged as strong contenders to treat the novel covid- . cq and hcq are antimalarial drugs clinically used to treat inflammatory rheumatic diseases such as rheumatoid arthritis and systemic lupus erythematosus. emerging evidence has suggested that these drugs are effective in treating sars-cov- in vitro. , viral replication begins when the virus attaches to the host cell and penetrates the cell. in the case of sars-cov- , it uses its surface unit (s ) of the s protein to attach to the angiotensinconverting enzyme (ace ) receptor, which facilitates viral entry. when african green monkey kidney veroe cells were pretreated for an hour with cq or hcq prior to four different multiplicities of infection by sars-cov- , both drugs prevented viral entry as well as post-entry stages of sars-cov- infection. inhibition of viral entry may be due to interference of terminal glycosylation of the ace receptor. additionally, cq and hcq can alkalinize the phagolysosome, which disrupts the ph-dependent steps of viral fusion and uncoating-processes that are absolutely essential for viral replication. moreover, both cq and hcq have immunomodulatory properties that may be beneficial in extreme, life-threatening covid- cases. indeed, there has been a recent surge in covid- patients with severe hyper immune activity, known as the cytokine storm syndrome . in this patient population, immunosuppression is likely to be beneficial, since the over-active immune response is paradoxically causing more harm than benefit to the patients. therefore, cq and hcq have recently became appealing due to their antiviral and anti-inflammatory properties, which may help treat covid- , especially under dire circumstances. although the promising findings suggest that cq and hcq are great candidates, much concern exists regarding their mechanisms, effective dosing regimen, clinical efficacy, and adverse effects (aes) with respect to covid- . additionally, precautions must be taken to ensure that the drugs are effective in preventing sars-cov- viral replication, but do not disrupt the body's own immune response in fighting against the infection. indeed, the rise in popularity of these drugs as potential medications to treat covid- and the current, desperate need for better therapeutics have fueled rapid and ongoing research and clinical trials to further elucidate their antiviral and anti-inflammatory properties, pharmacodynamics, and safety profiles with respect to covid- . currently, the safety profiles of these drugs are not entirely known due to the lack of large clinical trials, as well as sparse randomized controlled trials (rcts). moreover, the drugs have a narrow therapeutic range, which presents another challenge when using these drugs. , despite these shortcomings, combining the existing data on these drugs can provide powerful and valuable insight regarding their safety profiles, which will not only drive future clinical trials, but also help health professionals make informed decisions. to this end, our objective for this study is to address the safety profiles of these medications by performing a comprehensive systematic review and meta-analysis of the current published data. we thoroughly searched and screened large databases to acquire rcts for cq and hcq. since there is a paucity of rcts for cq and hcq for antiviral therapy, we analyzed studies that included the use of cq and hcq for non-antiviral treatments. in total, and studies for cq and hcq were identified, respectively, that satisfied our inclusion criteria, which are described in the tables. of these studies, we performed meta-analyses on cq and hcq reports, that included a placebo control. here, we report the results of the meta-analyses to determine the effects of cq or hcq on ae relative to placebo control. the meta-analysis was conducted in accordance with the preferred reporting items for systematic reviews and meta-analyses (prisma) guidelines. a comprehensive search strategy was designed to retrieve relevant clinical data from published literature. our objective was to identify all randomized clinical trials (rcts) that compared the safety profiles of chloroquine or hydroxychloroquine with placebo or other active agents. we searched pubmed, medline, cochrane, the cochrane central register of controlled trials (central), embase, and the clinicaltrials.gov for all the rcts comparing cq or hcq with placebo or other active agents, published before march , . we also searched conferenced proceedings to acquire relevant papers. medical subject headings (mesh terms) and keywords such as "randomized controlled trial," "adverse effects," "tolerability," "toxicity," and "side effects" were used. this review was not restricted to studies conducted in the english language; it includes reports from any countries that compared cq or hcq with placebo or other active agents, since there is a wealth of information in rcts from many different countries. to gather the eligible trials for this systematic review and meta-analysis, we searched through large databases (pubmed, cochrane, and clinicaltrials.gov) and identified those that involved either cq (n= , ) or hcq (n= , ). of the published reports we identified, we initially screened them through the titles and abstracts to examine if they were relevant to our objective of identifying safety profiles for cq and hcq. therefore, and reports were initially excluded for cq and hcq, respectively. of the remaining ones (n= for cq and n= for hcq), we performed a more thorough review using the inclusion and exclusion criteria described in the methods. in total, cq and hcq studies satisfied our requirements. for our meta-analysis, we extracted data from rct that had placebocontrolled, rather than studies that compared cq or hcq with other drugs. therefore, a total of studies and studies were used for data extraction for cq and hcq, respectively. characteristics of trials, patients, and interventions. we collected data for cq and hcq studies. table describes the characteristics of the trials, patients, and interventions of cq, while table describes the same parameters for hcq. the trials indicated with asterisks next to the primary author's last name were the trials used for our meta-analyses. as manifested from the tables, we did not restrict our systematic review to just the united states. additionally, investigators used cq as treatment options for breast cancer ( ), malaria ( ), hepatitis ( ), viral all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . infections ( ) , and lupus erythematosus ( ) . to conduct our meta-analysis for cq, we used double-blinded, placebo-controlled, randomized studies that used cq for the treatment of breast cancer, autoimmune hepatitis, dengue fever, and influenza. age of participants ranged from -to years-old. dosing regimen ranged from approximately mg/day to mg/day. of these studies, general findings reported in the studies noted that cq did not have a significant effect when compared with placebo. however, of the studies that compared cq with other medications, the authors noted that cq was generally more effective. similarly, the hcq studies ( table ) that we examined were conducted from a plethora of countries and used hcq to treat a myriad of disorders, which included dermatologic disorders ( ), rheumatoid arthritis ( ), hiv ( ), primary sjögren's syndrome ( ), graft-versus host disease ( ), diabetes ( ), chronic spontaneous urticaria ( ), dementia ( ), kidney failure ( ), cardiovascular disease ( ), and covid- ( ) . to conduct our meta-analysis for hcq, we used rcts that were pilot studies (one specifically for covid- ), single-blinded, and the rest double-blinded. in the table, these studies have asterisks next to the primary author's last name. for these particular reports, age of participants ranged from to years. dosage schedule ranged from mg/day to mg/day, with a mode of mg/day. general outcomes from about a third of the studies revealed that hcq had no significant effects, while the rest of the studies showed that it was effective for the disorders. mild, severe, total aes, and withdrawals due to aes from trials involving cq and hcq. the cq meta-analyses of mild, serious, total aes, and withdrawals due to aes were based on comparisons between cq and placebo (control), while the hcq meta-analyses of mild, serious, total aes, and withdrawals due to aes were based on comparisons between hcq and placebo (control), as depicted in figure . when assessing mild ae (figure a) , the overall relative risk (rr) of cq compared with placebo was . ( % ci . - . , p< . ), while the overall rr of hcq compared with placebo was . ( % ci . - . ). the rr for severe aes ( figure b) , however, was insignificant for both drug usage when compared with placebo. when assessing total aes of either drug compared with placebo ( figure c) , the combined rr for cq was . ( % ci . - . , p< . ), while for hcq it was . ( % ci . - . , p< . ). there was statistical evidence of overall heterogeneity between cq trials with regards to total aes (i = . %). withdrawals due to aes was only significant with cq compared with placebo. as evident in figure d , the overall rr was . ( % ci . - . , p< . ). there was no evidence of heterogeneity (i = %). taken together, these data suggest that both drugs induced higher mild and total aes as compared to control. additionally, cq had significantly more patients that withdrew from the studies due to aes. based on the reported aes, we divided our analyses to examine four groups: neurologic, gastrointestinal (gi), dermatologic, and ophthalmic aes. neurologic aes reported by participants included headache, dizziness, neuropathy/seizure, or other central nervous system (cns) related aes; gi aes included vomiting, nausea, abdominal pain, diarrhea, liver dysfunction, or non-specific gi aes; dermatologic aes included rash, itchiness, dryness; and ophthalmic aes included blurred vision or pain. with the usage of cq, there was a significant increase in all four groups of aes (figure ). the overall rr was . ( % ci . - . , p< . ) for neurologic aes; . ( % ci . - . , p< . ) for gi aes; . ( % ci . , . , p< . ) for dermatologic aes; and . ( % ci . - . ) for ophthalmic aes. no heterogeneity between the trials were observed. with the usage of hcq, there all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . was no significance increase in the groups that we examined. these data suggest that patients treated with cq experienced more neurologic, dermatologic, ophthalmic, and gi aes relative to placebo control. further analyses on heterogeneity, as well as publication bias, can be seen in supplementary figures - . stratification of all aes. to fully appreciate the wealth of information from the rcts, we constructed a flow chart that contains information on the number of participants who experienced a certain ae, as well as the percentages. four groups (cns, gi, skin, and vision) underwent metaanalyses (figure ) , since they had robust reports in the studies that we examined. in figure , panel a and b show the charts for cq and hcq, respectively. the cq studies contained a total of , participants for cq-treated group and it contained a total of , participants for placebotreated. of these participants, ( . %) and ( . %) ae were reported in the cq and placebo group, respectively. the highest reported aes for the cq group occurred in the cns, with about . % of overall cq participants reporting headache, dizziness, neuropathy, or other cnsrelated aes. in contrast, placebo group had higher reports for respiratory distress, such as coughing, sore throat, or running nose. the hcq studies contained participants for hcq-treated group and participants for placebo-treated group. total aes reported for hcq was ( . %), while total aes reported for placebo was ( . %). gastrointestinal aes, such as diarrhea, nausea, liver damage, abdominal pain, and other non-specific gi aes seemed to be the most dominant for both groups. interestingly, cardiovascular aes was reported in of the studies that we examined; one ae involved hypertension, while the other was acute coronary syndrome. together, these stratified data provide ample information regarding the percentage of participants who experienced specific aes. subgroup meta-analysis for cq and hcq with respect to age, duration, and dosage. since we found a significant increase in total aes when taking either drugs, we tested whether differences in age, duration, or dosage had any bearing on the results. we therefore performed subgroup meta-analysis. first, we examined age ( figure a) . we divided the cq trials into two groups: participants < years-old and participants ≥ years-old. we stratified the hcq trials into two groups: participants < years-old and participants ≥ years-old. these ages were chosen to ensure that there was robust comparison, since the number of rcts was very limited. we found that there was no group difference in either case, which suggests that age (younger vs older) had no bearing on the total aes experienced in participants. next, we assessed whether duration had any relevance to total aes ( figure b ). cq trials were divided into two groups: < week and ≥ week. hcq trials were divided into two groups: < months and ≥ months. there was significance difference between the two groups for cq (p< . ). the overall rr of total aes in trials that lasted < week was . (ci % . - . ), while the overall rr of total aes in trials that lasted ≥ week was . (ci % . - . ). additionally, there was evidence of heterogeneity (i = . %) between the two groups. although overall heterogeneity did exist between the groups, it is important to note that when these studies were separately analyzed, there was statistical significance for either group (p< . ). upon close inspection of hcq trials, we noted that they generally lasted longer. there was no evidence that a higher duration is any different than a lower duration in terms of rr of total aes. all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . finally, to determine if there were significant differences between a low versus a high dosage with respect to total aes for either drug, we stratified the dosages into two groups: < mg/day and ≥ mg/day. this arbitrary grouping ensured that we included enough studies in each group for cq, since the number of rct for cq is limited. additionally, most of the hcq studies used mg/day as the dosage ( hcq studies), so many reports fell below the < mg/day threshold that we set. as evident in our meta-analyses, there was no significant difference in the subgroups when using either dosage for both drugs. taken together, there was no statistical evidence to suggest that (younger vs older) age and (lower vs higher) dosage differentially affected the total aes when using either drug. additionally, there was statistical evidence to suggest that separation according to duration had an impact on total aes in the cq studies. meta-regression analyses were performed to determine the relationship between rr and age, duration of trial, and dosage, as depicted in figure . we examined if age of participants, duration of trial, or dosage has any effects on total aes or withdrawals due to aes. the size of the symbols indicates more weight towards a particular study. in all plots, the predicted regression lines and % confidence-interval lines are displayed. regression of logarithm of rr of total ae with cq and dosage revealed that dosage had an effect on total aes. age and duration of trial did not affect the total aes for cq. for hcq, there was no evidence that age, duration of trial, or dosage affected total aes. further meta-regression analyses can be found in supplementary figure . the current pandemic with sars-cov- has relentlessly claimed thousands of lives and caused significant economic hardship. the current need for viable therapeutic options while vaccine development is in progress has resulted in the proposal of numerous antiviral medications. chloroquine (cq) and its derivative hydroxychloroquine (hcq) have been circulating in the media as potential drugs to treat covid- . however, little is known regarding their safety profiles due to the lack of randomized controlled trials (rcts). to address this urgent issue, we performed a systematic review and meta-analysis by pooling the existing published data of adverse events (aes) for cq and hcq relative to placebo. after comprehensively perusing through the literature, we identified cq and hcq eligible studies that satisfied our inclusion criteria (figure ) . the characteristics of these studies are documented in tables and . for our meta-analyses, we included cq and hcq, since these studies were placebo-controlled. we found that the usage of either drug increased the relative risk (rr) for mild and total aes (figure ) . further system analyses showed that overall participants in the cq trials experienced more neurologic, gastrointestinal, dermatologic, and ophthalmic aes (figure ) . we did not observe a significant increase in the hcq group compared to placebo with regards to these ae categories. therefore, the significant increase in mild and total aes from hcq was most likely attributed to other reported aes, such as respiratory and cardiovascular aes. given the severity of cardiovascular aes, it is critical to note that two studies reported two cardiovascular aes: one was hypertension and the other was acute coronary syndrome. , although there were no cardiovascular aes reported in the cq studies that we analyzed, its cardiotoxicity has also been noted in a plethora of studies. an excellent systematic review article all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . by chatre et al. documented the cardiac complications that are attributed to cq and hcq. in their review, they found that among other cardiovascular complications, conduction bundle or atrioventricular block were reported more frequently. moreover, qt interval prolongation has been noted in numerous studies. [ ] [ ] [ ] [ ] severely prolonged qt interval can lead to lethal arrhythmias and sudden cardiac death. therefore, the prevalence of these cardiovascular aes warrants periodic electrocardiogram (ecg) monitoring when participants are undergoing these therapies, as cardiovascular aes can be fatal. since many of the studies that involved participants who experienced cardiovascular complications were not rcts with placebo controls, we were unable to include them in our meta-analyses. overall, participants who took cq exhibited more aes ( . %) relative to placebo control ( . %, figure ) . in the hcq studies, only . % of total aes were reported versus . % for placebo. the high percentage of total aes occurring with cq participants is concerning, but consistent with the consensus that hcq is a safer alternative to cq. , - when total aes were stratified according to different organ systems, we found that cq had more participants exhibiting cns aes ( . %), while hcq participants had more participants experiencing gi aes ( . %). it is worth noting that only . % of hcq participants exhibited cns aes. the extra hydroxyl group in hcq may decrease the occurrence of cns aes. more mechanistic, controlled studies need to be performed to confirm this finding. furthermore, subgroup analyses ( figure ) revealed no evidence in differences of rr of total aes when studies were divided by age (younger vs older) and dosage (lower vs higher). however, when we performed meta-regression analyses (figure ) , there was a relationship between dosage and total aes in the cq group, which suggests that the subgroup meta-analyses for dosage would be more robust if more cq rcts existed. interestingly, we found that duration of drug had an impact on total aes when using cq, with lower duration (< week) having higher rr. when examining studies with duration < week, - we noticed that cq was used to treat dengue ( ) and chikungunya ( ) infections. cq was used to treat various other conditions in trials with higher duration (≥ week). upon closer inspection of the hcq duration subgroup analysis, we observed that studies with duration < months had an insignificant overall rr of total aes, while studies with duration ≥ months had a very significant overall rr of total aes (p< . ). however, when these two groups were compared (p= . ), there was no significant difference between them. indeed, given the long halflife of hcq, it is plausible that the longer the duration of dosing regimen, the more total aes may be observed. however, due to the small number of rcts for hcq, it is not possible to determine the duration when hcq would produce more aes. taken together, our data show that participants taking either cq or hcq experienced more mild and total aes relative to placebo control. precautionary measures should be taken when giving these medications for their therapeutic impact. lr and pnt designed the study. lr and pnt screened and evaluated studies. lr performed statistical analyses. sy checked studies included. pnt and sy checked statistical analyses. lr, wx, jo, and pnt performed comprehensive characterization of studies. sy and nc provided expertise. lr, pnt, and nc wrote the manuscript. reports were identified through pubmed, cochrane, and clinicaltrials.gov. we used the same process of study collection for both cq and hcq. we performed an initial screening, followed by a more stringent screening using our selection criteria. the studies that remained after all the exclusion were the ones used for data extraction. in total, we identified and studies for cq and hcq, respectively, which are described in tables and . of those studies, cq and hcq reports are placebo-controlled rcts, so we used these studies for our data analysis. figure . mild, severe, total aes, and withdrawals due to ae from trials involving cq and hcq. we performed comparisons between cq and placebo and comparisons between hcq, as evident in the forest plots. aes were divided into a) mild, b) severe, and c) total. d) additionally, we also examined withdrawals from trials due to aes. meta-analyses were performed. we tested heterogeneity between trials, as well as overall effect. statistical data are displayed in the forest plots. shows the data for hcq. both panels begin with the total number of participants in the studies (n= cq, n= hcq), which is then followed by the total number of ae. the ae were then divided into different systems, which is then broken down into specific ae. figure was generated using biorender. figure . subgroup meta-analyses for cq and hcq with respect to age, duration, and dosage. we stratified the dosages used in the studies for both cq and hcq into two subgroups. we then performed subgroup analysis for dosage and trial duration. a) for age, we separated cq trials into < years-old and ≥ years-old, while we separated hcq trials into < years-old and ≥ years-old. b) for drug duration, we divided cq studies into < week and ≥ week, while we divided hcq studies into < months and ≥ months. c) and for dosage, we wanted to investigate if there was a difference in using < mg/day versus using ≥ mg/day for either drug. statistical data are presented in the figures. all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . meta-regression analyses were performed using the log rr of total aes with respect to age, duration, and dosage for both cq and hcq, as shown in panel a). panel b depicts the meta-regression analyses of rr of withdrawals due to aes with respect to age, duration, and dosage for both cq and hcq. statistical data are presented in the figure. all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint figure all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro chloroquine is a potent inhibitor of sars coronavirus infection and spread sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor recycling of chloroquine and its hydroxyl analogue to face bacterial, fungal and viral infections in the st century covid- : consider cytokine storm syndromes and immunosuppression immunosuppression for hyperinflammation in covid- : a double-edged sword? a systematic review on the efficacy and safety of chloroquine for the treatment of covid- of chloroquine and covid- chloroquine intoxication the cochrane collaboration's tool for assessing risk of bias in randomised trials statistical primer: heterogeneity, random-or fixed-effects model analyses? predicting commercially available antiviral drugs that may act on the novel coronavirus (sars-cov- ) through a drug-target interaction deep learning model effects of hydroxychloroquine on symptomatic improvement in primary sjögren syndrome: the joquer randomized clinical trial no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint hydroxychloroquine in cardiovascular disease in patients with chronic kidney disease: a proof of concept study cardiac complications attributed to chloroquine and hydroxychloroquine: a systematic review of the literature the qt interval in patients with sars-cov- infection treated with hydroxychloroquine/azithromycin suspected hydroxychloroquine-associated qt-interval prolongation in a patient with systemic lupus erythematosus prolongamento do intervalo qt do eletrocardiograma em pacientes reumáticos usando antimaláricos chloroquine-induced qtc prolongation in covid- patients comparison of hydroxychloroquine and chloroquine use and the development of retinal toxicity the comparative efficacy and toxicity of secondline drugs in rheumatoid arthritis. results of two metaanalyses animal toxicity and pharmacokinetics of hydroxychloroquine sulfate chloroquine use improves dengue-related symptoms. memórias do instituto oswaldo cruz a randomized controlled trial of chloroquine for the treatment of dengue in vietnamese adults on chikungunya acute infection and chloroquine treatment bioavailability of hydroxychloroquine tablets in healthy volunteers a randomized, double-blind, window of opportunity trial evaluating the effects of chloroquine in breast cancer patients chloroquine as weekly chemoprophylaxis or intermittent treatment to prevent malaria in pregnancy in malawi: a randomised controlled trial chloroquine is effective for maintenance of remission in autoimmune hepatitis: controlled, double-blind comparison of artemether-lumefantrine and chloroquine with and without primaquine for the treatment of plasmodium vivax infection in ethiopia: a randomized controlled trial artemether-lumefantrine versus chloroquine for the treatment of uncomplicated plasmodium knowlesi malaria: an open-label randomized controlled trial can know comparison of the safety and efficacy of fixed-dose combination 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malawi a double-blind, randomized study of azithromycin compared to chloroquine for the treatment of plasmodium vivax malaria in india double-blind, randomized, controlled clinical trial of clofazimine compared with chloroquine in patients with systemic lupus erythematosus atovaquone and proguani hydrochloride compared with chloroquine or pyrimethamine/sulfodaxine for treatment of acute plasmodium falciparum malaria in peru efficacy and safety of cgp (artemether and benflumetol) compared with chloroquine to treat acute falciparum malaria in tanzanian children aged - years a double-blind comparative clinical trial of mefloquine and chloroquine in symptomatic falciparum malaria a pilot study of hydroxychloroquine in treatment of patients with common coronavirus disease- (covid- ) hydroxychloroquine in the treatment of antihistamine refractory chronic spontaneous urticaria, randomized single-blinded placebo-controlled trial and an open label comparison study antidiabetogenic effects of hydroxychloroquine on insulin sensitivity and beta cell function: a randomised trial effect of hydroxychloroquine on insulin sensitivity and lipid parameters in rheumatoid arthritis patients without diabetes mellitus: a randomized, blinded crossover trial effects of hydroxychloroquine on immune activation and disease progression among hiv-infected patients not receiving antiretroviral therapy: a randomized controlled trial a randomized double-blind trial of hydroxychloroquine for the prevention of chronic graft-versus-host disease after allogeneic peripheral blood stem cell transplantation the effectiveness of hydroxychloroquine in patients with type diabetes mellitus who are refractory to sulfonylureas--a randomized trial effect of hydroxychloroquine on progression of dementia in early alzheimer's disease: an -month randomised, double-blind, placebo-controlled study hydroxychloroquine treatment of patients with human immunodeficiency virus type a randomized trial of hydroxychloroquine in early rheumatoid arthritis: the hera study hydroxychloroquine compared with placebo in rheumatoid arthritis. a randomized controlled trial hydroxychloroquine treatment for primary sjögren's syndrome: a two year double blind crossover trial the relative toxicity of disease-modifying antirheumatic drugs hydroxychloroquine and sulphasalazine alone and in combination in rheumatoid arthritis: a randomised double blind trial this work was supported by american heart association predoctoral award pre (lr); postdoctoral fellowship from nih f hl (pnt); nih r hl , hl , hl , and s rr , research award from the rosenfeld foundation, va merit review grant i bx and i cx (nc). key: cord- -ka asw m authors: alsuliman, tamim; alasadi, lugien; alkharat, banan; srour, micha; alrstom, ali title: a review of potential treatments to date in covid- patients according to the stage of the disease date: - - journal: curr res transl med doi: . /j.retram. . . sha: doc_id: cord_uid: ka asw m abstract introduction and motivation: since the end of , the covid- pandemic has affected millions of people worldwide. with the rapid spread of this virus, an immense burden has fallen upon both healthcare and economic systems. as a consequence, there is an unprecedented urgency for researchers and scientific committees from all over the world to find an effective treatment and vaccine. review structure: many potential therapies are currently under investigation, with some, like hydroxychloroquine, being authorized for emergency use in some countries. the crucial issue is now clearly to find the suitable treatment strategy for patients given comorbidities and the timeline of the illness.vaccines are also under development and phase clinical trials are rolling. despite all efforts, no single drug or vaccine has yet been approved. in this review, we aim at presenting the proposed pathophysiological mechanisms of sars-cov- and to provide clinicians with a brief and solid overview of the current potential treatments classified according to their use at the three different currently proposed disease stages. in light of pathogenesis and proposed clinical classification, this review’s purpose is to summarize and simplify the most important updates on the management and the potential treatment of this emergent disease. after an incubation period, sars-cov- invades the mucosal membranes, especially nasal and oralpharyngeal membranes, causing upper respiratory infection with mild symptoms in the first phase. the virus binds to target cells through angiotensin-converting enzyme (ace ). this activates the serine protease tmprss for s protein priming ( figure ) [ ] . ace is expressed by epithelial cells of the intestine, lung, kidney, and blood vessels [ ] . treatment in this phase is based essentially on symptomatic relief, supportive and non-specific therapies, such as acetaminophen. the use of virus-targeting treatments may be beneficial in this period in order to stop viral replication [ ] . although some argue that hydroxychloroquin (hcq) can be used early in stage . this use may have negative effects regarding loss of immunization chances due not only to its reduction of viral load, but also to its mmuno-suppressive characteristics [ ] . isolation remains the cornerstone intervention for containment of covid- as no treatment or vaccination has yet been globally approved. supplementary oxygen, acetaminophen, and antibiotics should be administered as required. non-invasive ventilation, intubation and mechanical ventilation might be required for critically ill patients and in cases of respiratory failure in the advanced stages iib and iii. close monitoring of organ function should be undertaken for early detection of organ failure or septic shock in predisposed patients [ , ] . antiviral therapies: remdesivir is a phosphoramidate prodrug of an adenosine c-nucleoside that targets the viral rnadependent rna polymerase (rdrp) proteins. working as a nucleotide analogue, this drug has proved its broad-spectrum antiviral activity including the effect against several human and zoonotic coronaviruses including sars-cov- . since human safety data for remdesivir are available from several clinical trials that tested remdesivir's efficacy against ebola virus, several clinical trials are already being held in the united states and china to investigate its efficacy treating covid- patients [ , , ] . the dose under investigation for treatment of covid- is mg intravenously (iv) on day followed by mg iv daily for up to days, infused over - minutes [ ] . several trials of remdesivir treatment on few patients in the united states have shown early promising benefits in cases with severe pneumonia [ , ] . in a cohort study that included severe covid- patients from different countries, using remdesivir ( mg iv. on day , then page of j o u r n a l p r e -p r o o f mg daily for days) led to a clinical improvement in % of patients with a median follow-up of days. the total mortality rate in this study was % [ ] . lopinavir (lpv) is a human immunodeficiency virus (hiv- ) protease inhibitor which is administered in combination with the "booster" ritonavir (rtv), a potent cyp a inhibitor that increases lpv half-life. even though lpv/rtv was effective against sars-cov in tissue cultures, its efficacy against mers-cov was controversial. results of a randomized, controlled, open-label trial that evaluated lopinavir-ritonavir vs. standard of care treatment in adults hospitalized adults with severe covid- were recently published [ ] . the two arms of this study included confirmed covid- patients and showed similar -day mortality rates and viral load at varying times since onset of symptoms. this study also showed withdrawal of medication in patients in the lpv group due to adverse events (table ) . however, the relatively long median time to randomization ( days) after onset of symptoms is thought to partially explain lack of effectiveness in the covid- arm [ ] . other clinical trials including lopinavir/ritonavir either as monotherapy or mostly in combination with other drugs have been conducted or are still ongoing. reported data from published investigations are difficult to interpret due to concomitant drug therapies, varying illness severity amongst patients and lack of comparison groups [ , ] . china, however, has included lopinavir/ritonavir in its guidelines at an oral dose of mg/ mg twice a day for no more than days. but more data are needed from ongoing studies, and caution should be taken regarding adverse effects and the real advantage over standard of care management [ , ] . umifenovir is an antiviral agent that has been used for influenza treatment and has shown activity against sars-cov in vitro [ , ] . its activity against sars-cov- was investigated recently at a dose of mg every hours in combination with lopinavir/ritonavir in a retrospective cohort chinese study [ ] . results showed better clinical response in the combination group in comparison with the lopinavir/ritonavir monotherapy group. umifenovir is currently being tested in seven randomized trials for the treatment of covid- [ ] . anti-endocytosis treatment: baricitinib is a selective janus kinase and janus kinase (jak and jak ) inhibitor. the mechanism of action is considered to be by modulation of viral endocytosis. but there are huge concerns that this blockage page of j o u r n a l p r e -p r o o f may potentiate sars-cov infection, and there are other safety issues related to this product's administration [ ] . ace was identified as a key receptor for sars-cov- . data regarding the interaction between covid- severity and angiotensin converting enzyme (ace ) receptor inhibition is conflict, and contrary theories have been proposed so far [ , ] . initially, concerns were raised on the basis that aceis and arbs led to an increase in the number of ace receptors in the cardiopulmonary circulation in experimental animals, and that this might lead to more severe outcomes due to sars-cov- infection [ ] . on the other hand, ace has been found to protect the lungs from injury. and some data suggest that sars-cov- down-regulates ace expression after initial engagement with the receptor. this down-regulation of ace activity in the lungs might facilitate lung injury. in a recent in-vitro study hrsace was shown to significantly block early stages of sars-cov- infections [ , ] . in a recent retrospective study that included patients, hypertensive patients treated with acei/arb were less likely to develop severe pneumonia, and the study concluded that these drugs might have a protective effect [ ] . until more data are available, there is no indication for patients treated with aceis or arbs to withhold their treatment (renin-angiotensin-aldosterone system inhibitors in patients with covid- [ ] . camostat mesylate is a serine protease inhibitor, a drug approved in japan for use in pancreatic inflammation. it usually blocks tmprss , a protease that was recently shown to be responsible for the coronavirus s protein priming, which is crucial for viral entry into target cells and for viral spread in the infected host as published by hoffman et al, in cell, from the german primate center [ ] . several clinical trials are ongoing comparing camostat mesylate as a single agent vs. placebo or in combination with other drugs such as hydroxychloroquin [ , ] . in this stage, pulmonary involvement is well settled, while we note remarkable viral multiplication and pulmonary localized inflammation. viral pneumonia, with cough, fever and hypoxia (stage iib: hypoxia defined as a pao /fio of < mmhg) identify this period [ ] . chest imaging reveals bilateral infiltrates or ground glass opacities [ ] . laboratory tests show increasing lymphopenia while inflammation markers may be normal or slightly elevated [ ] . normally most hospitalizations occur in this phase. with advanced disease, the virus infects the lower respiratory tract leading to pneumonia and worsens symptoms such as dyspnea and hypoxemia [ , , ] . treatment usually relies on supportive measures and the aforementioned anti-viral therapies. in early parts of this stage (iia), the use of corticosteroids in patients with covid- may be avoided. while in stage iib, the j o u r n a l p r e -p r o o f use of anti-inflammatory treatment may be permitted, indeed, the presence of hypoxia may indicate the high probability of unfavorable systemic evolution [ , ] . coagulopathy seems to be another issue that affects the prognosis in more severe cases. importantly, elevated d-dimer levels seem to be a marker to predict severe illness and mortality. other laboratory findings like hrombocytopenia and prolonged prothrombin time also indicate a hyper-coagulation state in covid- patients. the use of anticoagulant therapy has improved mortality rates in hospitalized patients with markedly elevated d-dimer or those who have sepsis-induced coagulopathy (sic) score ≥ . with all the emerging evidence, current guidelines recommend a prophylactic dose of low molecular weight heparin (lmwh) for all hospitalized patients in the absence of contraindications [ ] [ ] [ ] in order to eliminate the virus, a good immune status is essential in this phase [ , ] . even in elderly and immunocompromised patients, these phases tend to be respected, albeit in presenting different severe clinical manifestations [ ] . treatments potentially investigable in this stage are the following: chloroquine (cq) is a -aminoquinoline antimalarial agent that has proven anti-inflammatory and immunomodulatory activities. cq also has anti-viral properties due to the following mechanisms: blocking virus/cell fusion; interfering with the glycosylation of cellular receptors, lysosomes and autophagosomes impairment; inhibiting viral enzymes ( i.e. viral dna and rna polymerase); increasing ph of intracellular vesicles which interferes with ph-dependent viral replication [ ] [ ] [ ] . hydroxychloroquine (hcq) is a derivative of cq with similar characteristics but with better safety profile [ , ] . both drugs have narrow therapeutic range which makes drug-toxicity probable especially with uncontrolled usage. cardiac toxicity (qt interval prolongation) is the major, and even lethal, concern about usage of these drugs [ , ] . in vitro studies have demonstrated cq efficacy against sars-cov- [ ] . with its known safety profile, clinical investigations have been held in different countries; and at least trials evaluating hcq or cq are currently underway worldwide [ , ] . a pilot randomized chinese study [ ] investigated hcq in confirmed covid- cases. fifteen patients received mg of hcq per day for days, while others received the conventional treatment. the primary endpoint was covid- nucleic acid negativity in respiratory pharyngeal swab on day after randomization. the results were comparable between the study and the control group as of day . throat swabs were negative in ( . %) cases in the hcq group and in ( . %) cases in the control group (p ＞ . ). furthermore, the median duration from hospitalization to virus nucleic acid negative conversion and the median time for body temperature normalization were also comparable between the two groups. these results are comparable to another chinese open-label, randomized, controlled trial that included hospitalized covid- patients. hcq dosage in this study was , mg daily for days with a maintenance j o u r n a l p r e -p r o o f dose of mg daily for or weeks depending on illness severity. no difference regarding negative viral conversion was observed between the hcq and the standard of care group, but significant clinical improvement was noticed in the hcq group [ ] . a preliminary brazilian randomized, double-blinded, clinical trial has investigated two different cq dosages ( mg twice daily for days vs. mg for days, twice daily only on the first day) in patients with severe covid- . all patients received ceftriaxone and azithromycin as well. the high dosage cq arm had higher qtc prolongation and mortality rates, with no apparent benefit of cq regarding viral clearance or mortality in either study arms [ ] . of note, only out of the enrolled patients had been confirmed by rt-pcr. on the other hand, data emerging from other ongoing chinese trials have demonstrated that cq phosphate is superior to a control treatment in the following areas: pneumonia exacerbation inhibition, imaging findings improvement, virus negative conversion promoting, and disease course shortening [ ] . additionally, a randomized chinese cohort of in-hospital patients with covid- showed that hcq may help shorten the time to clinical recovery [ ] . gautret, p. et al, reported promising results in two studies in france [ , ] . in these studies, researchers investigated the efficacy of hcq in combination with azithromycin for the treatment of confirmed covid- patients. in the first study, a significant reduction in viral carriage on day post inclusion compared to control group was noticed. meanwhile, the second study reports a rapid fall in nasopharyngeal viral load on day ( %) and day ( %) which was confirmed by pcr. in addition, virus cultures were negative in . % patients on day . this allowed rapid discharge with a mean length of stay of five days. hcq dosage used in these studies was mg per day for days. combining all in-vitro and in-vivo data, some countries have authorized treating hospitalized patients, with some conditions, with cq and hcq [ , , ] . however, there are serious concerns raised regarding its usage. for example, clinical data from reliable randomized controlled studies are still missing, and data published to date lacks homogeneity in terms of recommended dose concentration, treatment duration, and severity of patient illness [ ] . recently, the american thoracic society has released interim guidance on treating covid- . for hospitalized patients with pneumonia, hcq and cq may be used on a case-by-case basis, but clinicians must consider the potential benefit/risk ratio and the patient's condition must be severe. for outpatients with covid- or hospitalized patients without pneumonia there are no specific recommendations [ ] . in contrast, the surviving sepsis campaign announced that there's insufficient evidence to declare a recommendation on the use of cq and hcq in critically ill adults with covid- [ ] . the infectious diseases society of america (idsa), on the other hand, in its recently published guidelines [ ] has recommended hcq/cq (+/-azithromycin) in the context of clinical trials only. clinical data regarding this combination in the treatment of covid- patients is still lacking, but several clinical trials are currently being conducted [ , ] . the who has launched a multinational trial called "solidarity trial". this trial will test the four most promising coronavirus treatments remdesivir, lopinavir/ritonavir, lopinavir/ritonavir plus interferon b, hydroxychloroquin with the aim to end the pandemic [ ] . ivermectin is a broad spectrum anti-parasitic drug, but it has also shown anti-viral activity in vitro with its confirmed ability to inhibit integrase protein (in) nuclear import and hiv- replication. it has also shown its ability to inhibit nuclear import of host and viral proteins [ , ] . a recent in vitro study tested the antiviral activity of ivermectin against sars-cov- [ ] . ivermectin treatment resulted in . % reduction in all viral material within h compared to control samples. these results made the authors propose ivermectin as a possible treatment for covid- since it's an fda approved drug with a known safety profile. nitazoxanide has shown in vitro activity against sars cov- with known broad antiviral activity against other viruses like influenza and rotavirus. the mechanism of action is believed to be due to interference with viral replication by targeting host regulated pathways rather than virus-specific pathways. in addition to its antiviral activity, it inhibits the production of pro-inflammatory cytokines tnf-, il- , il- , i- , il- , il- and il . no clinical information is yet available on the efficacy of nitazoxanide in the treatment of covid- [ , ] . this is the most severe stage in this disease stage classification and luckily it concerns a fewer number of patients with ards and cytokine storm syndrome (css) being the hallmark of the pathogenesis [ , ] . in these severe cases, virus replication and tissue damage continue, especially in the lungs and other ace expressing organs, which leads to an even higher increase in pro-inflammatory cytokines released by macrophages and granulocytes [ , ] . high levels of pro-inflammatory cytokines such as interleukin b (il- among these cytokines, several have been suggested as a potential therapeutic target. il- , for example, is activated after the binding between covid- and toll-like receptors (tlr). released il- mediates lung inflammation, fever and fibrosis. il- is also known for its important role in the progression of pulmonary fibrosis [ ] . il- is also an important cytokine during respiratory viral infections, and it has been reported that, during this covid- pandemic, it has been correlated with pulmonary infection severity in icu patients [ , ] . both il- and il- have been presented as potential therapeutic targets [ , ] . in addition to potential shock, stage iii is also marked by extra-pulmonary involvement, such as vasoplegia, myocarditis, and organ failure. treatment is essentially based on the use of immunomodulatory therapies in order to improve systemic inflammation and to block consequent organ failure. the use of corticosteroids may be helpful in this phase, generally in tandem with the use of cytokine inhibitors. intravenous immune globulin (ivig) may also be considered as an immune system modulator. rapid application of such a treatment plan can potentially enhance patient prognosis, which is basically poor in this stage [ , , ] . using corticosteroids to treat severe pneumonia due to covid- is still controversial. the who recommends against the routine use of corticosteroids in these patients [ ] . data from clinical trials are widely variable in terms of participants included and results reported, that's why clinical judgment should be based on a case-by-case approach. delayed viral clearance and infection susceptibility are two major concerns that come with corticosteroid usage in covid- patients [ , , ] . in a recent release, the surviving sepsis campaign has suggested systemic corticosteroid administration for ards adult patients with covid- who are mechanically ventilated. on the other hand, recommends against corticosteroid use in adults without ards [ ] . five randomized controlled trials investigating methylprednisolone in covid- patients are also currently registered [ ] . a-bio-immune-modulatory treatments: immunoglobulins have been widely used in medical practice, and their use has shown clinical benefits in previous studies of sars and mers. ivig is currently under investigation for treatment of covd- [ , , ] . a case series of severe covid- patients who received high-dose ivig ( . - . g/kg/day) for five days was reported [ ] . all patients experienced clinical improvement shortly after administration. however, other therapeutic agents were administered for these patients including antivirals and a short course of steroids. in a pre-print retrospective study [ ] , authors reported the data of severe covid- patients who didn't respond to a combination of low-dose corticosteroid ( - mg/d) and immunoglobulin ( g/d) but have considering the genetic relation between sars-cov- and sars-cov- some argue that it may be considered for further verification as a potential therapeutic, as of sars patients were reported, in a publication, to have an uneventful recovery after treatment with this product [ , ] . the fda has recently approved convalescent plasma for serious or immediately life-threatening covid- infections under emergency investigational new drug application (einds) [ ] . convalescent plasma has been previously studied during other epidemics including h n influenza virus pandemic, sars-cov- epidemic, and the mers-cov epidemic. recently, a preliminary case series of five intubated covid- patients with ards showed promising results. these patients received ml of convalescent plasma containing neutralizing sars-cov- -specific antibody (igg) from recovered covid- donors. all patients had gradual clinical and radiological improvement within days and four patients no longer required respiratory support by day , viral loads also became negative within days after transfusion. seven clinical trials are currently registered [ , , ] . tocilizumab is a monoclonal antibody (mab) that inhibits the il- receptor. as mentioned earlier, il- plays a key role in respiratory viral infections and in crs which is linked to poorer prognosis and higher mortality rate in covid- patients. tocilizumab is approved for the treatment of rheumatoid arthritis and its safety and efficacy profile has previously been well studied [ , ] . preliminary data [ ] included cases of severe and critical pneumonia in covid- confirmed patients who showed clinical and radiological improvement in . % (severe pneumonia) and . % (critical pneumonia) of participants. lymphocyte count also returned to normal in . % of cases on day of drug administration. all patients received mg of intravenous tocilizumab once, and three patients, due to persistent fever, received another mg dose after hours. despite promising primary data, when it comes to studies regarding optimal timing and il- threshold of tocilizumab administration during the course of covid- , information is still lacking from larger, controlled, long-term studies. moreover, il- monitoring might be an obstacle in some institutions, and its adverse effects bacterial infection susceptibility, neutropenia and thrombocytopenia should be considered when a clinical decision is to be made. two clinical trials are ongoing right now which might provide further needed information [ , ] . leronlimab is a humanized igg monoclonal antibodies that blocks cc chemokine cellular receptor (ccr ) and plays a key role in several immunological processes. leronlimab is being evaluated for hiv and breast cancer treatment and it is believed to have an antiviral activity while mitigating the cytokine storm. some claim that leronlimab has achieved preliminary results in a few severe cases of covid- [ , ] . bevacizumab is an approved treatment for multiple cancers. it is a humanized monoclonal antibody that prevents the association between vascular endothelial growth factor (vegf) and endothelial receptors flt- and kdr, and it may reduce the levels of vegf caused by hypoxia and severe inflammation. eventually, it might suppress the edema in patients with covid- . bevacizumab is currently being tested in a chinese randomized controlled trial [ , ] . c-anti-interleukin : anakinra® is an il- receptor antagonist. as mentioned earlier, il- seems to have a key role in the respiratory viral infections and in progression of pulmonary fibrosis. so anakinra® has been suggested as possible adjunctive treatment. but, to the best of our knowledge, no in-vitro or clinical data are available [ , ] d-cell-based therapy: the italian college of anesthesia, analgesia resuscitation and intensive care has reported recent guidelines to treat coronavirus patients. it includes a statement that stem cells could have a serious potential to treat covid- by decreasing the number of patients admitted to the icu and discharging them quickly [ ] . msc can theoretically inhibit immune system overreaction and improve the microenvironment that promotes endogenous repair which could potentially protect alveolar epithelial cells. this would, in theory, prevent pulmonary fibrosis and also improve lung function [ , ] . the published studies are from china, including a small cohort in the treatment group and in the control group [ ] and a case report [ ] . some expect that clinical trials may be, nowadays or very soon, recruiting. the available data shows some promise in managing covid- illness, especially in critically ill patients that could be treated under a compassionate-use protocol [ ] . cynk- (intravenous infusion of natural killer (nk) cells) is the name of yet another trial that has enrolled covid- patients. it aims to boost immune systems of patients at risk of more severe disease and those starting to show symptoms by applying cynk- . the mechanism has been slowing virus replication [ ] . prevention: an urgent response to the brisk expansion of covid- has been evoked throughout scientific communities in order to develop an effective vaccine. rapid genome sequencing and potential molecular targets have been identified, and more than vaccines are currently under investigation. some of these vaccines have shown effectiveness in preclinical studies, while still others are already in phase i clinical trials [ ] [ ] [ ] . until the final approval of a given vaccine and its effective distribution, an approach based on early case detection and isolating, laboratory testing, contact tracing and quarantining (as recommended by the who) seem the most effective way to prevent further spread of sars-cov- [ , ] . growing evidence suggests a link between universal bcg vaccination policy and reduced morbidity and mortality for covid- . comparing italy to japan, for example, the first case of covid- appeared in japan earlier than in italy, but japan has maintained a low mortality rate despite not implementing restrictive social isolation measures [ ] . another study found that the mortality rate has been . /million in countries with a bcg program compared to /million in countries without such a program [ ] . to date, the efficacy of bcg vaccine on preventing covid- is controversial due to many limitations, one being the reality of comparing countries in different phases of the pandemic. this association might be clearer with upcoming data. tuberculosis vaccine (vpm ), on the other hand, is a new vaccine based on the old bcg vaccine. the idea is that in many studies conducted on mice, the vaccinated mice had lower influenza, lower serum viral load, and less lung damage. it is claimed that the bcg vaccine may activate the immune system against viruses and possibly decrease covid- mortality rates [ ] . while the complete pathophysiology of covid- needs to be better understood, urgent research for rapid solutions based on already established knowledge is still ongoing. page of j o u r n a l p r e -p r o o f for many physicians, these new potential strategies may be seductive. although the disastrous situation currently faced by many countries may explain the attractiveness of such treatments, the urgent need for a cure does not justify any use which is unauthorized by national health regulatory authorities. meanwhile, as the world waits for a widely approved treatment, preventive interventions coupled with clear local and international management guidelines must be always respected in order to lessen the damage and permit more exhaustive and conclusive research to be conducted. ta has received honorarium from biotest france sas. biotest ag commercializes pentaglobin®. symptomatic treatment is a basic step in all clinical stages of the disease, with oxygen therapy here reflecting all interventions that may be needed from nasal cannula to mechanical ventilation. while some argue that antiviral therapy (especially remdesivir) could be considered for some patients in stage i of the disease, other preserve it for the stage ii patients. ribavirin and ifnα- b in particular, in our opinion shouldn't be considered in the management of stage i. more severe cases (stage iii) need more extensive interventions by adding immunomodulatory treatments to the previous management steps in the attempt to contain the hyperinflammatory response. *these treatments might also be investigated in both stage ii and iii. **these treatments might also be investigated in stage iii. ***these treatments are mainly investigated in stage iii. hydroxychloroquine (hcq) targets several levels of viral infection. it changes the glycosylation of ace to inhibit virus entry. being a weak base, it also affects endosomal activity by increasing, the ph of acidic intracellular organelles. finally, it interferes with rna replication by targeting the polymerase. in severe and critical cases, macrophage activation leads to an uncontrolled and lethal inflammatory process known as "cytokine storm syndromes" driven by the release of multiple cytokines; il- plays a key role in this process. tocilizumab is a monoclonal antibody that inhibits the il- receptor which might mitigate the inflammatory response. clinical characteristics of hospitalized patients 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trained immunity, and immune ontogeny the authors thank rachel tipton, hasan iessa, phd., and danah el-refaie for the proof reading of this manuscript and layth sliman for the technical support. key: cord- -fspmgg s authors: sehailia, moussa; chemat, smain title: antimalarial-agent artemisinin and derivatives portray more potent binding to lys and lys -binding hotspots of sars-cov- spike protein than hydroxychloroquine: potential repurposing of artenimol for covid- date: - - journal: journal of biomolecular structure & dynamics doi: . / . . sha: doc_id: cord_uid: fspmgg s medicinal herbs have proved along history to be a source of multiple cures. in this paper, we demonstrate how hydroxychloroquine can act as a good inhibitor of sars-cov- spike protein receptor-binding-domain using molecular docking studies. we also unveil how hydroxychloroquine can interfere in the prevention of lys in hace from interacting with the corresponding binding hotspot present on the spike protein. further screening of artemisinin & derived compounds produced better vina docking score than hydroxychloroquine (- . kcal mol(− ) for artelinic acid vs. − . kcal mol(− ) for hydroxychloroquine). artesunate, artemisinin and artenimol, showed two mode of interactions with lys and lys binding hotspots of the spike protein. molecular dynamics analysis confirmed that the formed complexes are able to interact and remain stable in the active site of their respective targets. given that these molecules are effective antivirals with excellent safety track records in humans against various ailment, we recommend their potential repurposing for the treatment of sars-cov- patients after successful clinical studies. in addition, an extraction protocol for artemisinin from artemisia annua l. is proposed in order to cope with the potential urgent global demand. communicated by ramaswamy h. sarma the spread of covid- pandemic has triggered a race to unveil the secrets of severe sars-cov- and its underlying acute respiratory syndrome. although, functional importance of different targets has been linked to the viral replication and maturation of coronaviruses' family such as chymotrypsin-like protease( clpro) or known as mpro (khan et al., ; muralidharan et al., ) or envelope protein (e) (gupta et al., ; boopathi et al., ) but it has been confirmed that the binding of the viral trimeric surface spike glycoprotein (sprotein) of sars-cov- to the human receptor angiotensin-converting enzyme (hace ) is the first step in host infection . in fact, vankadari and wilce ( ) revealed that s domains of the spike protein has an open conformation, enabling it to interact with target host proteins. yan et al. ( ) identified the structure of hace as a dimer in complex with membrane protein, they also showed that the two trimeric sproteins of the receptor binding domain (rbd) of sars-cov- bind very tightly to the hace dimer. the latter is activated by a specific cellular enzyme called furin (hasan et al., ) . medicinal plants reap an important source of complex active molecules that have been proposed, and sometimes traditionally used for the treatment of several pathologies, such as cancer, autoimmune diseases, and infectious diseases. the scientific community are relying on different modes and various mechanisms of action these molecules hold to halt sars-cov- severity. recently, remdesivir and chloroquine have been proved to inhibit in vitro vero e cells of emerged novel coronavirus ( -ncov) . chloroquine has been envisaged for sars-cov- (de clercq, ) , and has long been used for malaria therapy but has been replaced with hydroxychloroquine (hcq) due to the increased plasmodium falciparium parasite resistance, whereas an overdose of cq can cause acute poisoning and death (weniger, ) . they have shown similar activities in in-vivo essays against malaria parasite but accompanied by an increased risk of retinopathy (schrezenmeier & d€ orner, ) . nonetheless, hcq is considered safer than cq as the later has mediated cardiotoxic effects including rhythm disorders, and propels the development of cardiomyopathy in patients with rheumatic diseases (schrezenmeier & d€ orner, ) . for its turn, hcq has been found to be effective in inhibiting sars-cov- infection in vitro and attenuate inflammatory response . organic extracts of artemisia annua l. have been found to be more effective, faster, and less toxic than cq and hcq in treating malaria. a. annua contains a vital compound known as artemisinin, a sesquiterpene lactone with a peroxide linkage exhibiting low toxicity (table ) , also the parent compound for semisynthetic derivatives chemically modified at the c- position to produce artesunate, artemether, arteether, artenimol (dihydroartemisinin), and artelinic acid (table ) . these compounds, and in some cases their sodium salts, have been formulated as antimalarials for oral, rectal, and parenteral administration (woodrow et al., ) . several reports proved the efficiency of artemisinin derivatives as potent antivirals for hpv bovine viral diarrhea virus (bvdv) for the treatment of anal and cervical intraepithelial high-grade neoplasia, human herpes virus- (hhv- ), human immunodeficiency virus (hiv) and more particularly, artesunate, against human cytomegalovirus (hcmv) (d'alessandro et al., ) . this antiviral potency put artemisinin class of compounds as promising candidates for the treatment of patients suffering from sars-cov- virus. the encouraging results generated from the utilization of hcq to treat patients suffering from covid- pandemic further raises many questions surrounding its mode of action (million et al., ) . at the cellular level, direct and indirect mechanisms of cq and hcq are believed to inhibit immune activation by reducing toll-like receptor signaling and cytokine production and, in t cells, reducing cd expression (schrezenmeier & d€ orner, ) ; however, the absence of binding assay studies between the sprotein and hace protein in the presence of hcq opens the door to two main possibilities (vincent et al., ) : the first possibility revolves around hcq prevention of terminal glycosylation of hace protein which consequently impacts the final attachment between the sprotein and hace protein, whereas the second possibility revolves around hcq interaction with the receptor binding domain (rbd) of sprotein, thus preventing its docking on hace receptor. to further expand on the second possibility, we elected to perform computational studies based on molecular docking to help us understand more about the mode of interaction between hcq and the rbd of sars-cov- sprotein, and eventually, how such interaction prevents the sprotein from docking on the hace . another class of antimalarial and antiviral molecules comprised of artemisinin and artemisinin derived compounds are investigated to reveal how effective these molecules act with binding sites of sprotein, then ultimately how their mode of interaction occur. this study aims to propose also an extraction protocol for artemisinin from artemesia annua l. in order to cope with the potential urgent global demand. the pdb file of sars-cov- sprotein rbd-hace complex (pdb ref. lzg, version . ) was obtained from the research collaboratory for structural bioinformatics (rcsb) protein data bank (pdb) (http://www.rcsb.org/structure/ lzg). ucsf chimera . was used to visualise the structure of the ligand and/or protein-complex structure, to perform the various functions associated with ligand and protein preparations, and acting as an interface to enable molecular docking calculations using locally hosted autodock vina software (pettersen et al., ; trott & olson, ) . prior to molecular docking, the hace protein (part a, protein section coloured in green- figure ) was deleted from the pdb file of the complex. in addition, all non-standard residues including that of water were also removed. the structure of each ligand was incorporated into ucsf chimera using smiles string followed by structure minimisation. the pdbqt files of the sprotein rbd and each ligand was generated after adding all hydrogens and charges to each structure. the number of binding modes was set to with exhaustiveness of search set to . the maximum energy difference was set to kcal mol À . the best scoring pose for each molecule was analysed in terms of its interaction with the receptor binding motif (rbm). the x, y, z coordinates of the grid box covered the full area of the receptor binding motif of the sprotein, in our case, for center (x ¼ À . , y ¼ . , z ¼ À . ) whereas for size (x ¼ . , y ¼ . , z ¼ . ). in the case of the receptor, all hydrogen atoms were added to the structure, charges were merged and nonpolar hydrogen were removed; water molecules and side chains of non-standard residues were also ignored. for the studied ligands, charges were merged and non-polar hydrogens were also removed. the obtained molecular docking results were then aligned with the pdbqt file of the sprotein rbd-hace complex in order to visualise the type of interactions of each docked molecule in the sprotein-hace binding interface. computations were performed at the al-farabi cluster computer of the ecole nationale polytechnique oran -maurice audin (algeria). all molecular dynamics (md) studies are performed using groningen machine for chemical simulations (gromacs) which is a software package designed to perform md simulations of proteins, lipids and nucleic acids. amber force field was utilised during the parameterisation of each protein complex system followed by solvation with tip p water molecules; water.tip p force field in a cubic periodic box with Å spacing protein-box edge was applied. na þ ions were introduced to neutralise the overall charge. further energy minimisation was performed using steepest descent and conjugate gradient algorithms. the system was subjected to ns md at k and pressure of bar, the latter value was maintained using berendsen barostat. the generated trajectory files were analysed using visual molecular dynamics (vmd) software. most explanations associated with hcq mode of action are based on findings revolving around the mode of action of cq on sars-cov infection (simmons et al., ; yang et al., ) . amongst the cited reasons are: (i) cq can increase the value of endosomal ph which can reduce the transduction of sars-cov pseudo-type viruses (simmons et al., ; yang et al., ) , (ii) cq can raise the possibility of affecting the endosome-mediated fusion if added at the initial stage of the infection (vincent et al., ) , and (iii) once the virus is inside the cell, cq can inhibit the production of glycoproteins in vesicular stomatitis, thus preventing virus replication (dille & johnson, ) . previously, vincent et al. ( ) showed that introduction of cq prevents terminal glycosylation of ace receptor protein of the host cell, thus destabilizing the recognition mode of the sprotein on the surface of the virus, albeit such action did not impact the level of expression of surface hace proteins of the host cell. therefore, we elected to perform computational docking studies of hcq as safe cq substitute against sprotein rbd of sars-cov- to further study the nature of such interaction and explore its inhibition potential against sprotein. our molecular docking studies of hcq on the rbd of sars-cov- sprotein produced a vina score of À . kcal mol À (table ). the obtained scoring is relatively moderate given that vina score of the best molecule, i.e. physalin b, previously docked on a homology model of sars-cov- sprotein rbd was À . kcal mol À (micholas & jeremy, ) . analysis of our docking results showed that the hydroxyl group (oh) of hcq molecule formed strong hydrogen bonding with asn side chain residing on one part of the receptor binding motif (rbm) of the sprotein (figure ). in the sprtoein-hace complex, asn of sars-cov- sprotein forms favourable hydrogen bonding with tyr of hace while at the same time helps neutralising the charge of lys (lan et al., ) . therefore, the resulting hydrogen bonding between the oh group of hcq and asn can play a role in destabilising other interactions with hace residues, e.g. tyr , lys , gly and asp , which were already found to play key roles in the interaction with the sprotein furthermore, when our initial docking results were aligned with the structure of sprotein-hace complex, we successfully observed significant clash between the aminoalkyl side-chain of hcq and the lys residue side-chain of hace ( figure ). equally, lys (o) was found to form one hydrogen bonding with gln of the sprotein in the complex as reported by lan et al. ( ) ; at the same time, lys (n) forms important hydrogen bonding with gln while maintaining a salt bridge with asp (lan et al., ; yan et al., ) . similar to lan et al. ( ) findings, lys and lys were both found to be important hotpots in hace responsible for binding to the sprotein of sar-cov- . it is believed that the latter specie developed key mutations to help stabilise and/or neutralise both lysine hotspots via introduction of asn (to neutralise lys ) and gln & leu (to neutralise lys ) of hace protein, thus ensuring tighter incorporation of both hotspots deep into the hydrophobic pockets of the sprotein, which would explain the main reason behind the higher affinity of sars-cov- sprotein to hace compared to that of sars-cov (lan et al., ) . therefore, it is very likely that selective interaction of hcq with the surface of sars-cov- sprotein through the formation of an inclined tape over the hydrophobic pocket responsible for hosting the lys hotspot (the oh group in this case is acting like a hook by forming a hydrogen bond with asn ), can be responsible for the prevention of tighter binding with hace protein via restricting penetration of lys into its finally assigned destination on the sprotein rbd (figure ). similar to asn in sars-cov- , thr in sars-cov sprotein was previously reported by shang et al. ( ) to interact with tyr , lys , gly and asp in hace protein. we advocate here that similarity in the hydrogen bond networking system between both types of sproteins (i.e. that of sars-cov and sars-cov- ) and that of hace may be used to explain the supposed efficacy of hcq in inhibiting sars-cov and sars-cov- interaction with hace . on the other hand, our molecular docking results also showed good interaction between the quinoline aromatic ring in hcq and his in hace ; in normal circumstances, the latter residue interacts well with leu and gln of the sprotein. by doing that, hcq can also disturb interaction in the middle region of the binding interface between the sprotein and hace (figure ) . previously, samarth and kirk ( ) studied the interaction of hydroxychloroquine/azithromycin with sars-cov- sprotein-hace complex using a virtualised quantum mechanical modelling approach. in agreement with our study, the authors found that hcq had low potency of interaction with the studied complex compared to azithromycin; they also recommended molecular docking studies to further strengthen their rationale. our approach to study the interaction of molecules with the receptor binding motif (rbm) of the sprotein prior to complexation with hace has helped properly analyse the type of interaction with the sprotein, and how such interaction could prevent hace from docking onto the rbm of sprotein. in addition, we successfully addressed some of the aspects surrounding the mechanism of action of hcq and artemisinin derivatives in preventing the interaction between the virus' sprotein and hace receptor via selectively interacting with the lys binding hotspot of sprotein. with this information on hand, we then elected to perform in-silico screening of other potent antimalarial compounds derived from the core structure of artemisinin; by doing so, we believe we can gain more insight about the potential use of such class of compounds as safer and more potent substitutes to hcq. in this regard, a total number of compounds were successfully screened against sprotein rbd using the same molecular docking approach previously followed with hcq. the obtained results are shown in table . analyses of the data show that artemisinin and its derivative compounds have scored better than hcq, with compounds in entry & of table producing the least and closest vina score (- . kcal mol À ) to hcq. on the other hand, artelinic acid (table , entry ) gave the best vina score of À . kcal mol À ; however, this compound was discarded due to its high toxicity levels (li et al., ) . although artemisinin and its derivative compounds resulted in good vina scoring (- . score . kcal mol À ), only those approved and prescribed as antimalarials were selected namely, artesunate (table , entry ), artemisinin (table , entry ) and artenimol (table , entry ). these were found to possess good clinical records with very promising antiviral properties, thus enhancing their potential to be repurposed for the treatments of sars-cov- . artesunate (table , entry ) with vina score of À . kcal mol À . the calculated inhibition constant (ki) of each top scoring pose is also reported in table . analysis of the data shows that artemisinin class of compounds possess much lower ki than hcq, thus enabling them to become good antiviral candidates against sars-cov- . the elevated ki value of hcq also reflects the high cytotoxic concentration of hcq (cc > mm at multiplicity of infection (moi) ¼ . ) required to eradicate the virus during in-vitro assay studies as previously reported by liu et al. ( ) . besides its antiviral activity, artemisinin derivatives hold immunoregulatory properties and modulate neutrophils, t-cell and b-cell components of the immune system (yao et al., ) , thus enhancing system immunity and touting themselves as promising candidates to synergistically enhance their antiviral effect in vivo and treat inflammation-associated diseases (li et al., ) . the nature of interaction between the aforementioned three compounds and the rbd of sars-cov- sprotein was also analysed in order to see which molecule best influence the repulsion of hace lys and lys from binding to the inner hydrophobic pockets of the sprotein. by analysing the top scoring pose of artesunate (table , entry ), we observe that the molecule binds far away from the hydrophobic regions of lys and lys hotspot binding sites (figure ) . furthermore, upon alignment of artesunate docking results with the structure of sprotein hace complex, no clashes were observed between artesunate structure and the other side-chains present in the hace protein. however, the carboxylic acid moiety of the artesunate side chain was observed to form hydrogen bonding with lys (n), which can further neutralise the overall charge through the formation of a salt bridge, this can adversely lead to tighter interaction between hace and sprotein. therefore, in spite of the high vina scoring associated with artesunate, we predict that this molecule is unlikely to act as a good inhibitor, in its current form, to sars-cov- sprotein (figure ) . in the case of artemisinin's (table , entry ) top pose, despite no hydrogen bonding is observed with the sprotein rbd, we notice a lateral incorporation of the six-membered ring cyclohexane group of artemisinin into the lys hotspot binding pocket, with the peroxy-bridge facing the peripheral hydrophilic surface of the binding region( figure (a) , region coloured in blue next to the peroxy-bridge). such mode of interaction could well be used to prevent the penetration of lys side-chain into the hydrophobic pocket ( figure ). artemisinin lateral penetration into these binding hotspots may reduce the random motion of a-helix and loops present in sprotein and its capability to attach with hac as hypothesized by gupta et al. ( ) where binding with phytochemicals reduces their motion present in the envelope (e) protein of sars-cov , therefore inhibiting a modulation of ion channel activity and stop the pathogenesis caused via sars-cov . artemisinin was also found to interact with lys hotspot binding pocket, although at slightly lower vina score (- . kcal mol À ). the average score is perhaps attributed to the absence of hydrophilic surfaces close to the binding pocket ( figure ) . therefore, the selective interaction of artemisinin with both lys and lys hotspot binding regions raises its possibility to be repurposed for the treatment of sars-cov- patients following successful clinical trials. artemisinin has better tolerance on human hepatoma cell line hepg with cc of mm than artesunate cc of mm (romero et al., ) . additionally, in vitro cytoxicity levels of artesunate against human epithelial cells (hela cells) and human foreskin fibroblasts (hff cells) report very low tolerance with cc of . mm and . mm respectively (he et al., ) . artenimol on the other hand showed a similar mode of interaction to that of artemisinin with both lys and lys hotspot binding sites, although at slightly lower vina scores of À . and À . kcal mol À , respectively (figure ) . both artemisinin and artesunate are susceptible to cyp reduction to generate artenimol once incorporated into the human body, albeit at different conversion rates. we therefore recommend that artenimol can be prioritised for clinical trials to achieve the repurposing of such class of molecules for covid- , however, careful considerations need to be taken into account given the water solubility characteristics of each compound (woodrow et al., ) . successful completion of md calculations permitted us to obtain two crucial graphs, i.e. that of root-mean square deviation (rmsd) vs. time (figure ) and the root-mean square fluctuation (rmsf) of each residue in the three protein complexes, i.e. that of the sprotein rbm in complexation with hcq, artemisinin or artenimol (figure ). rmsd represents a measure of the average change in the displacement of an atom for a particular frame compared to a reference frame. on the other hand, rmsf measures the local changes of each residue in the protein backbone. figure indicates a good protein-ligand stability for all three complexes, with hcq protein complex showing the lowest rmsd value ( . Å) followed by artenimol-protein complex ( . Å) and artemisinin-protein complex ( . Å). similarly, artemisinin-protein complex showed the highest deviation . Å followed by artenimol-protein complex . Å and hcq-protein complex . Å. those values imply the currently, major production of artemisinin is based on solvent extraction from a. annua despite modest but not scalable enough trials to produce it chemically or semisynthetically via its precursor artemisinic acid in engineered bacteria (hale et al., ; dietrich et al., ). artemisinin is abundant in a. annua leaves ( . - . %) and production includes several steps starting with screening and drying before biomass being processed generally via solvent leaching or percolation at - c using low polarity solvents like hexane, toluene or petroleum (chemat et al., ) . this operation is not selective for artemisinin, therefore terpenes, fatty acids and some pigments are inevitably co-extracted which calls for secondary refinery steps including adsorption, flash chromatography and sequential crystallization. herein, an indicative facile production setup is proposed to enhance worldwide production capacity ( figure ). however, a fireproof equipment and facility is a pre-requisite to ensure safety and security measures are met. the plant comprises an extraction step ( ) in which biomass is placed in hollow-fibre bags and processed at c for min using a solvent mixture of hexane/ethyl acetate ( : v/v) with solvent/biomass ratio of to ( l for each kg) (chemat et al., ) . this step can be conducted by means of a stirring tank or a percolation type reactor to : extraction reactor; : frame and plate filter-press (or vibrating-screener/decanter); : adsorption column bed; : clarification column bed; : crystallization stirred reactor; : spray dryer; : distillation column; : solvent storage tank ensure up to % extraction yield is achieved. then, the mixture passes through a cloth or diaphragm plate and frame filter-press ( ) in order to discard fine biomass particles and recover the extract. the latter is submitted to an adsorption bed column ( ) filled with activated carbon aiming at the removal of pigments and tannins. another clarification stage is required to remove other impurities such as free fatty acids and pigments; for instance, an adsorption bed column ( ) filled with celite (merck) is recommended (chemat et al., ) . due to some affinity with activated carbon and celite , an artemisinin loss of - % is expected. after that, the purified extract should be concentrated to at least / th its initial volume using an evaporator. the concentrated extract is submitted into a jacketed crystallization reactor ( ) equipped with a stirring shaft set at tip-speed in the range of - rpm to control breakage effects and to generate a narrow particle size distribution (huter et al., ) . the cooling rate is set to . c/min to reach c and is kept at this temperature for min to let artemisinin crystals settle down. the crystals are sent into a spray drying system ( ) to recover high purity crystals of - % as a final product. the overall yield of artemisinin is expected to reach % from the initial content of artemisinin in a. annua leaves. the spent mother liquor is guided for another row of crystallization with longer residence time. the recovered crude crystals are washed with cold ethanol to recover purer artemisinin and increase the final yield. the inhibition of sars-cov- sprotein rbd with hcq was successfully studied using molecular docking techniques. hcq was found to selectively interact with the lys hotspot binding pocket via the formation of an inclined tape over the binding site with the oh group of hcq acting like a hook. artemisinin class of compounds were also found to interact the same binding pocket. in addition, artemisinin & derived molecules showed extra mode of interaction with the lys binding hotspot, although at slightly lower vina score. molecular dynamics studies confirmed that the formed complexes are able to interact and remain stable in the active site of their respective targets. these results demonstrate the likelihood of repurposing artemisinin as a less toxic substitute of hcq to block the sprotein rbd of the virus from docking onto hace , while at the same time enhancing the immune system of the patient. more focus should be intended to study the in-vivo mode of action of artenimol as most artemisinin derivatives are converted to this compound once incorporated to the body. novel coronavirus structure, mechanism of action, antiviral drug promises and rule out against its treatment extraction mechanism of ultrasound assisted extraction and its effect on higher yielding and purity of artemisinin crystals from artemisia annua l. leaves biosynthesis of spathulenol and camphor stand as a competitive route to artemisinin production as revealed by a new chemometric convergence approach based on nine locations' field-grown artemisia annua l the use of antimalarial drugs against viral infection. microorganisms potential antivirals and antiviral strategies against sars coronavirus infections a novel semi-biosynthetic route for artemisinin production using engineered substrate-promiscuous p (bm ) inhibition of vesicular stomatitis virus glycoprotein expression by chloroquine in-silico approaches to detect inhibitors of the human severe acute respiratory syndrome coronavirus envelope protein ion channel microbially derived artemisinin: a biotechnology solution to the global problem of access to affordable antimalarial drugs a review on the cleavage priming of the spike protein on coronavirus by angiotensin-converting enzyme- and furin unique and highly selective anticytomegalovirus activities of artemisinin-derived dimer diphenyl phosphate stem from combination of dimer unit and a diphenyl phosphate moiety systematic and model-assisted process design for the extraction and purification of artemisinin from artemisia annua l identification of chymotrypsin-like protease inhibitors of sars-cov- via integrated computational approach structure of the sars-cov- spike receptor-binding domain bound to the ace receptor toxicity evaluation of artesunate and artelinate in plasmodium berghei-infected and uninfected rats hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro repurposing therapeutics for covid- : supercomputer-based docking to the sars-cov- viral spike protein and viral spike protein-human ace interface early treatment of covid- patients with hydroxychloroquine and azithromycin: a retrospective analysis of cases in marseille computational studies of drug repurposing and synergism of lopinavir, oseltamivir and ritonavir binding with sars-cov- protease against covid- ucsf chimera-a visualization system for exploratory research and analysis potential inhibitors of the enzyme acetylcholinesterase and juvenile hormone with insecticidal activity: study of the binding mode via docking and molecular dynamics simulations effect of artemisinin/artesunate as inhibitors of hepatitis b virus production in an "in vitro" replicative system energetics based modeling of hydroxychloroquine and azithromycin binding to the sars-cov- spike (s)protein -ace complex mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology structural basis of receptor recognition by sars-cov- characterization of severe acute respiratory syndrome-associated coronavirus (sars-cov) spike glycoprotein-mediated viral entry autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading emerging wuhan (covid- ) coronavirus: glycan shield and structure prediction of spike glycoprotein and its interaction with human cd . emerging microbes & infections chloroquine is a potent inhibitor of sars coronavirus infection and spread remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro review of side effects and toxicity of chloroquine. the bulletin of the world health organization structural basis for the recognition of sars-cov- by full-length human ace ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign immunomodulation of artemisinin and its derivatives a pneumonia outbreak associated with a new coronavirus of probable bat origin the authors would like to acknowledge the support of the directorate general of scientific research and technological development of the ministry of high education and scientific research in algeria. we are thankful to dr. djamila benrezkallah (djillali liabes university of sidi bel abbes, algeria) and al-farabi cluster computer of the ecole nationale polytechnique oran -maurice audin for running the md computations and insightful analysis of the molecular dynamics calculation. no potential conflict of interest was reported by the author(s). http://orcid.org/ - - - key: cord- - o zj v authors: davoodi, lotfollah; jafarpour, hamed; kazeminejad, armaghan; soleymani, eissa; akbari, zahra; razavi, alireza title: hydroxychloroquine-induced stevens–johnson syndrome in covid- : a rare case report date: - - journal: oxf med case reports doi: . /omcr/omaa sha: doc_id: cord_uid: o zj v the international outbreak of respiratory illness termed coronavirus disease (covid- ) began in december that has affected > . million individuals. self-limiting respiratory tract involvement, severe pneumonia, multiorgan failure and death are the spectrum of covid- . to date, there are no especial therapeutic agents for covid- infections. one such medication includes the antimalarial hydroxychloroquine (hcq), which recently reported as a possible therapy for shortening the duration of covid- symptoms, reducing inflammatory reactions to infection, impairing the exacerbation of pneumonia and boosting lung imaging findings. like all medications, hcq has side effects and may occur in covid- patients. here, we report on the case of a -year-old woman, presented with fever and dry cough, who had covid- and days later presented with a pruritic erythematous maculopapular rash, which started from the distal of upper extremities and rapidly, involved the entire body. the international outbreak of respiratory illness termed coronavirus disease (covid- ) began in december , has affected > . million individuals and has been leaded to deaths until april [ ] . the disease is highly contagious, with the pandemic reported in the st world health organization status report on march [ ] . self-limiting respiratory tract involvement, severe pneumonia, multiorgan failure and death are the spectrum of covid- . to date, there are no specific and definitive therapeutic agents for covid- infection. one of the drugs prescribed to improve the condition of covid- patients is the antimalarial hydroxychloroquine (hcq), which recently reported as a supportive drug for shortening the duration of covid- symptoms, reducing inflammatory reactions to infection, impairing the exacerbation of pneumonia and boosting lung imaging findings [ ] . like all medications, hcq has side effects and may occur in covid- patients. despite its positive effect, its side effects should be taken into consideration and replaced with other effective medications if possible. we decided to report a case of the hcq side effects and the appropriate management to it. l. davood et al. a -year-old woman, presented with fever and dry cough in the past days to her family physician. she had a history of contacting someone with similar symptoms and no underlying problems. no abnormality was found in the physical examination, her temperature was • c and other vital signs were normal. oxygen saturation was %. imaging and laboratory tests were requested. lab tests revealed elevated lactate dehydrogenase ( units/liter (u/l), normal: - u/l), c-reactive protein level ( milligrams/liter (mg/l), normal: < mg/l), aspartate aminotransferase ( u/l, normal: - u/l), thrombocytopenia, and leukopenia (white blood cells = /microliter, % lymphocyte cells). serology for rheumatoid factor, antinuclear factor, antideoxyribonucleic acid, antismooth muscle and antimitochondrial antibodies were negative as well as hepatitis b surface antigen and anti-hepatitis c virus antibodies. mild bilateral patchy ground-glass opacification/opacity (ggo) was seen in lung computed tomography (ct) scan (fig. ). due to covid- pandemic and clinical findings, the nasopharyngeal swab test was done, and severe acute respiratory syndrome coronavirus nucleic acid was detected by reverse transcriptionpolymerase chain reaction. hcq mg twice daily was started, and mg of acetaminophen every h. after two days, the patient presented with a pruritic erythematous maculopapular rash and flat atypical targets that started from the distal of upper extremities (fig. ) , rapidly involved the entire body and torn blisters that were only be seen as ulcers on orolabial area (fig. ) . she had genital mucosal involvement but did not allow us to take pictures. the nikolsky sign was positive (fig. ) . finally, a diagnosis of stevens-johnson syndrome (sjs) was made. due to the likelihood of a drug reaction, hcq was discontinued, and covid- treatment was changed to lopinavir/ritonavir mg twice daily in the hospital. loratadine mg twice daily and diphenhydramine mg three times daily were given. she was discharged after five days with nonpruritic scalded skin on the distal of upper extremities (fig. ) . covid- is an emerging disease that currently has no specific treatment, and the treatments are purely supportive. chloroquine and its derivatives, such as hcq, have a long history of being used as prophylactic drugs in malaria areas. in some studies, hcq has been used to improve the symptoms of patients with covid- . this drug is used to support patients with covid- and is not known as a definitive treatment for the disease [ , ] . the mechanism of action of hcq is to affect intracellular components such as endosomes, lysosomes and golgi vesicles and to increase their ph, which interferes with the steps of virus replication, including fusion and uncoating. the side effects of taking this drug occur when it is not sufficiently dispersed through the relatively small central portion. therefore, the amount of drug entering the central part is an important factor in causing skin reactions [ ] . the most common hcq usage complications are headache, dizziness and gastrointestinal complications. some studies of covid- patients have reported complications such as gastrointestinal distress, headache, blurred vision, insomnia and prolonged qt interval [ , ] . in our case, erythema multiforme with maculopapular rash, and flat atypical targets were seen in the distal part of the limb. the skin complications caused by hcq use are infrequent. these include acute generalized exanthematous pustulosis, sjs, toxic epidermal necrolysis and rashes. hcq is a very rare cause of drug-induced sjs. this condition begins as itchy papular erythematous eruptions in the trunk and then affects the face, organs and mucous membranes of the mouth. it also had purulent rashes on the trunk, limbs and face [ ] . in a study by volpe et al. [ ] , a patient with rheumatoid arthritis after taking hcq developed diffuse, erythematous exfoliative rash involving trunk and limbs, which diagnosed as a drug rash with eosinophilia and systemic symptoms syndrome. in another study, after taking hcq, the patient developed skin symptoms of sjs such as a pruritic rash over her abdomen, which described as 'targets' with a persisting exfoliating rash and eczematous patches [ ] . numerous studies have shown that the use of hcq in some people with covid- causes gastrointestinal symptoms and heart problems [ , ] , but so far there have been no reports that taking hcq leads to the symptoms of sjs, which develops skin rashes in different parts of the body, especially in the oropharynx. skin manifestations of covid- included erythematous rash, urticaria and chickenpox-like vesicles [ ] . in our case, while the rash was most likely caused by hcq, the increasing skin manifestations seen with covid- raise the intriguing hypothesis that the rash in our patient might have also been a covid- manifestation. it is worth noting that although hcq appears to be safe, it does have known side effects. the boundary between therapeutic and toxic doses is narrow while any severe disorders of their use can be life-threatening. one of the side effects of hcq is sjs caused by the drug, and given the worldwide pandemic of covid- and the increasing need for this drug, we need to be careful about its use in order to control and manage the side effects of this drug. world health organization world health organization covid- : a recommendation to examine the effect of hydroxychloroquine in preventing infection and progression interim guidance for the use of antiviral therapy in the clinical management of acute respiratory infection with sars-cov- (covid- ) mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology chloroquine and hydroxychloroquine in covid- hydroxychloroquineassociated, photo-induced toxic epidermal necrolysis hydroxychloroquine-induced dress syndrome stevens-johnson syndrome in association with hydroxychloroquine treatment for rheumatoid arthritis cutaneous manifestations in covid- : a first perspective special thanks to the student research committee of mazandaran university of medical sciences for supporting us in this project. no conflicts of interest. none. written informed consent was obtained from the patient for the publication of this case report as well as accompanying images. a copy of the written consent is available for review by the editorin-chief of this journal. alireza razavi. key: cord- -fe u oi authors: nirk, eliise laura; reggiori, fulvio; mauthe, mario title: hydroxychloroquine in rheumatic autoimmune disorders and beyond date: - - journal: embo mol med doi: . /emmm. sha: doc_id: cord_uid: fe u oi initially used as antimalarial drugs, hydroxychloroquine (hcq) and, to a lesser extent, chloroquine (cq) are currently being used to treat several diseases. due to its cost‐effectiveness, safety and efficacy, hcq is especially used in rheumatic autoimmune disorders (rads), such as systemic lupus erythematosus, primary sjögren's syndrome and rheumatoid arthritis. despite this widespread use in the clinic, hcq molecular modes of action are still not completely understood. by influencing several cellular pathways through different mechanisms, cq and hcq inhibit multiple endolysosomal functions, including autophagy, as well as endosomal toll‐like receptor activation and calcium signalling. these effects alter several aspects of the immune system with the synergistic consequence of reducing pro‐inflammatory cytokine production and release, one of the most marked symptoms of rads. here, we review the current knowledge on the molecular modes of action of these drugs and the circumstances under which they trigger side effects. this is of particular importance as the therapeutic use of hcq is expanding beyond the treatment of malaria and rads. antimalarial drugs have a long history, starting around years ago when quinine, a substance in the bark of the cinchona tree, was first used to fight plasmodium falciparum infections (woodward & doering, ; haładyj et al, ) . cq was the first potent and massproducible drug against malaria and was synthesized as an analogue of quinine (shanks, ) . despite its remarkable antimalarial efficiency, cq was deemed too toxic due to its side effects such as gastrointestinal and skin complications, retinopathy, cardiotoxicity or myopathy (kalia & dutz, ; haładyj et al, ) . the discovery of hcq mitigated this issue, and hcq is now regularly used in clinics under the brand name plaquenil (furst, ; aviña-zubieta et al, ; al-bari, ; haładyj et al, ) . already during the second world war, the positive effects of these two antimalarial drugs on rads were observed. soldiers taking cq and hcq as prophylaxis reported improvement of rashes and inflammatory arthritis. today, cq and particularly hcq are commonly used to treat rheumatic and dermatological diseases, and are further being tested in clinical trials as potential drug candidates for covid- , several types of cancer, diabetes type i and ii, multiple sclerosis, recurrent miscarriages and myocardial infarction (al-bari, ; clinicaltrials.gov) . rads, such as systemic lupus erythematosus (sle) (ruiz-irastorza et al, ; willis et al, ; , rheumatoid arthritis (ra) (khraishi & singh, ) and primary sjögren's syndrome (pss) (oxholm et al, ; rihl et al, ; kumar & clark, ; demarchi et al, ) , are caused by a malfunctioning immune system that targets healthy tissues (smith & germolec, ) such as joints (kumar & clark, ) . cqs and hcqs therapeutic role in rads is linked to its anti-inflammatory and immunomodulatory effects (plantone & koudriavtseva, ) . these effects are achieved through the modulation of the autoimmune response by (i) impairing functions of the endolysosomal system through its lysosomotropic effects (ziegler & unanue, ; kaufmann & krise, ; yoon et al, ) , (ii) decreasing the levels of circulating pro-inflammatory cytokines (sperber et al, ; van den borne et al, ) , (iii) inhibiting t-cell proliferation (landewe et al, ; costedoat-chalumeau et al, ) , (iv) blocking tolllike receptors (tlrs) (kyburz et al, ) and (v) autophagy inhibition (an et al, c) . however, numerous questions remain regarding both the mechanism of action of cq and hcq in rads and the side effects caused by this compound. in this review, we report on hcq and cq modes of action at the molecular and cellular levels in the context of rads. additionally, we discuss the relevance of these drugs in the treatment of cancer and infectious diseases. finally, we summarize the side effects reported in patients taking hcq for rads and discuss how some of those can be explained by the current knowledge on cq and hcq. mobilization from the endoplasmic reticulum (er). they might further modulate other cellular and organismal processes, e.g. golgi trafficking (mauthe et al, ) , but the underlying mechanisms remain to be identified. inhibition of lysosomal activity and autophagy cq and hcq are weak bases that easily cross cell membranes and accumulate in acidic subcellular compartments such as lysosomes and endosomes, where they remain trapped in a protonated state (ohkuma & poole, ) . this leads to a ph increase in lysosomes from to , causing inhibition of acidic proteases and other enzymes within the endolysosomal compartments ( fig a) (ohkuma & poole, ; poole & ohkuma, ; ziegler & unanue, ; haładyj et al, ) . as a result, antigen processing and subsequent presentation by mhc-ii complex on the cell surface of both macrophages and lymphoid dendritic cells are impaired (guidos et al, ; chesnut & grey, ; fox, ) , dampening the adaptive immune response (fig ) (fox, ) . cq and hcq also increase ph levels within the golgi stacks. this causes functional alterations of this organelle that possibly contribute to the cellular effects of these two drugs, e.g. by impairing transforming growth factor beta (tgf-b) activity (perkett et al, ; rivinoja et al, ; mauthe et al, ) . the ability to block lysosomal degradation also makes cq and hcq potent macroautophagy inhibitors ( fig a) . macroautophagy, hereafter called autophagy, is a conserved intracellular degradation pathway that is required to maintain cellular homeostasis by recycling damaged or unwanted cytoplasmic proteins, complexes and organelles (eskelinen & saftig, ). autophagy plays a role in many physiological processes, and its misregulation is linked to pathologies such as cancer, neurodegeneration and inflammatory diseases (mizushima et al, ; levine et al, ; dikic & elazar, ; levine & kroemer, ) . during autophagy, cytoplasmic cargoes are sequestered by double-membrane vesicles called autophagosomes, which fuse with lysosomes to generate autolysosomes (eskelinen & saftig, ) . fusion with lysosomes and activity of the lysosomal enzymes are required to break down the autophagosomal cargoes and recycle the resulting metabolites. impairment of both autophagosome-lysosome fusion and lysosomal degradative activity blocks autophagy (klionsky et al, ) . although cq and hcq decrease the acidity of lysosomes (seglen et al, ; poole & ohkuma, ; mizushima et al, ) , the glossary antigen-presenting cells (apc) cells that process proteins derived from pathogens or from dying/ dead cells, into peptides that get presented on their surface, thereby activating t cells and initiating an immune response. autophagy an intracellular process that delivers unwanted cytoplasmic material into lysosome for degradation. b cells a type of lymphocytes (white blood cells) that plays a crucial role in the adaptive immune response by producing antigen-specific antibodies. calcium (ca + ) is the most abundant mineral in the human body and is vital for a multitude of cellular and physiological function. it is also an important second messenger in numerous signal transduction pathways. chloroquine (cq)/hydroxychloroquine (hcq) originally developed to fight malaria, these drugs are used to treat rheumatic autoimmune diseases and are currently tested in clinical trials as therapies for other conditions. cytokines small secreted proteins that mediate communication and modulate interactions between cells, including immune cells. endosomes intracellular organelles that mainly function as a sorting and recycling hub for endocytosed and biosynthetic components, on their route to lysosomes. immune system a network consisting of a variety of different cell types that defend the body against infections and other potentially harmful anomalies, and which, when misregulated, contributes or causes the development of an inflammatory disease. lysosome intracellular organelles containing a large battery of digestive enzymes that degrade extracellular and cytoplasmic material delivered to their interior by endocytosis and autophagy, respectively. a membrane-bound multi-subunit enzymatic complex at either the plasma or endosomal membrane, which participates in a variety of cellular functions, ranging from cellular signalling and gene expression to host defence mechanisms. primary sjögren's syndrome an autoimmune disease that belongs to the group of rheumatic autoimmune diseases, which affect saliva-producing glands leading to symptoms such as dry mouth and dry eyes. retinopathy condition characterized by a damaged retina, which causes vision impairment, and is a documented adverse effect that can occur when taking hcq and cq. rheumatic autoimmune diseases a group of conditions characterized by a dysregulated immune system, which primarily affect the muscles, joints, connective tissue and bones. systemic lupus erythematosus an autoimmune disease that belongs to the group of rheumatic autoimmune diseases, which is the most common form of lupus and is associated with symptoms such as severe fatigue, joint pain and joint swelling. t cells a type of lymphocytes (white blood cells) that is a key component of the adaptive immune system and that orchestrates other cell types in response to antigens. toll-like receptors (tlr) transmembrane proteins that recognize specific molecules at either the plasma membrane or endosomes, and subsequently initiate signalling pathways that are crucial for the innate immune response. primary inhibitory effect of these drugs on autophagy is blocking the fusion of autophagosomes and lysosomes, which is at least in part mediated by the dysregulation of the recruitment of specific snare proteins onto autophagosomes (mauthe et al, ) . this block results in an accumulation of autophagosomes in the cytoplasm (mauthe et al, ) , which can contribute to an enhanced autophagosome-mediated signalling output (martinez-lopez et al, ; barrow-mcgee et al, ) and even compromise tumour cell viability (button et al, ) . although hcq and cq have been extensively described as autophagy inhibitors, there is emerging evidence that these drugs induce a non-canonical form of endocytosis (florey et al, ; jacquin et al, ) . activation of tlrs, especially in macrophages, monocytes and t helper cells, but also in neutrophils and endothelial cells, induces the production and secretion of pro-inflammatory cytokines, a hallmark of rads (beutler & cerami, (a) cq and hcq are weak bases that accumulate inside acidic subcellular compartments, e.g. endosomes and lysosomes. they remain trapped in a protonated state, causing an increase of ph and thereby inhibiting the functions of these cellular compartments. impairment of the autophagosome-lysosome fusion leads to autophagy inhibition. (b) cq and hcq alter endosomal tlr activation by increasing endosomal ph, by blocking the interaction between nucleic acids and endosomal tlrs (trl , tlr and tlr ) and by preventing translocation of tlr to endosomes. hcq also blocks the correct assembly of the nox complex by preventing the translocation of the nox subunit gp phox onto endosomes and consequently the formation of an active nox . (c) cq and hcq impair the release of ca + from the er, resulting in inhibition of ca + -dependent signalling pathways. hcq further inhibits the replenishing of intracellular ca + stores from the extracellular space. kim & moudgil, ). hence, inhibition of endosomal tlrs by hcq or cq is a powerful therapy approach for these diseases (lafyatis et al, ) . tlr , activated by dna in immune cells, can thus be inhibited by hcq and cq (yi et al, ; ahmad-nejad et al, ) . tlr , activated by guanosine analogues, can also be inhibited by cq, but to a lesser extent than tlr (lee et al, ) , indicating different inhibitory mechanisms. tlr is mainly activated by poly(i-c), but also by debris originating from necrotic synovial fluid cells in ra patients, and both modes of activation are hampered by hcq and cq (brentano et al, ; jolly et al, ; imaizumi et al, ) . in general, inhibition of tlr , tlr and tlr by hcq and cq has been attributed to their ability to impair endosomal acidification (macfarlane & manzel, ; lafyatis et al, ; schrezenmeier & dörner, ) , as activation of endosomal tlrs and subsequent downstream signalling only takes place within acidified compartments ( fig b) (blasius & beutler, ) . beside endosomal acidification, kuznik and colleagues discovered a second mechanism by which cq impairs tlr signalling. they showed that cq could inhibit endosomal tlr signalling after stimulation with nucleic acids at concentration too low to influence the endosomal ph. under those conditions, cq blocks endosomal tlr activation by directly interacting with tlr ligands, such as nucleic acids, which changes the nucleic acid secondary structure and prevents their binding to endosomal tlrs (macfarlane & manzel, ; ku znik et al, ) . this notion is further supported by the observation that hcq specifically blocks activation of dendritic cells and macrophages by dna but not by lps, although lps also stimulates these cells via a signalling cascade emanating from endosomes (häcker et al, ) . a third mechanism that interferes with inflammatory cytokine production is the ability to disrupt gmp-amp synthase (cgas) signalling . cgas is a crucial component of the cgas-stimulator of interferon gamma (ifn) genes (sting) signalling cascade that is required for the ifn type i response in immune cells (sun et al, ) , making it an important player in activation of pro-inflammatory response in autoimmune diseases (gao et al, ; kato et al, ) . cgas is also upregulated in a portion of sle patients (an et al, a,b) , and interestingly, hcq and cq can inhibit cgas binding to its ligands, e.g. dna, in vitro and in a t-cell line . importantly, inhibition of cgas activation results in reduced ifnb expression ( fig c) . inhibition of nadph oxidase nox is a protein complex involved in numerous pro-inflammatory signalling cascades, such as tumour necrosis factor alpha (tnfa)and interleukin (il)- b-induced cascades. activation of endosomal nox, which leads to the generation of reactive oxygen species (ros), requires the endocytic internalization and delivery to endosomes of cell surface ligand-receptor complexes (müller-calleja ) . hcq blocks the nox-mediated signalling cascades triggered by tnfa and il- b in monocytes by blocking translocation of gp phox, the catalytic subunit of nox, from the cytosol onto endosomal membranes without changing the endosomal ph (müller-calleja et al, ) . this inhibition prevents the correct assembly and activation of nox, hindering the downstream cellular events and the production of the pro-inflammatory cytokines tnfa and il- . hcq also prevents the redistribution of tlr from the er to endosomes, which is necessary to mediate the inflammatory response (müller-calleja et al, ) ( fig b) . inhibition of ca + signalling ca + mobilization from both the er and extracellular space into the cytoplasm and subsequent ca + -dependent signalling is an important mechanism to activate cells of the immune system, such as t and b cells (feske, ) . high cytoplasmic levels of ca + act as a second messenger for the activation of signalling pathways and transcription factors that regulate the expression and secretion of cytokines and other immune regulatory factors (izquierdo et al, ) . ca + release from the er can be impaired by hcq (goldman et al, ; xu et al, ; , leading to the inhibition of intracellular signals. in particular, t-cell and b-cell receptormediated intracellular ca + mobilization from both intracellular stores and the extracellular milieu is inhibited by hcq in a dosedependent manner (goldman et al, ) . this impairment of ca + mobilization is at least partially caused by the reduction of the ca + stored intracellularly and the inability to replenish these intracellular stores with extracellular ca + (goldman et al, ) . this further enhances its negative impact on the ca + -dependent signalling pathways ( fig c) (feske, ) . the precise mechanism of hcq-induced reduction of internal ca + mobilization remains unknown. however, it has been shown that hcq does not reduce the availability of inositol , , -trisphosphate, but rather the binding to its intracellular receptors that promotes ca + release (misra et al, ) . autoimmunity is characterized by an overreaction of the immune system (smith & germolec, ) , which is linked to both innate and adaptive immunity (mescher, ) . the innate immune system is responsible for the initial recognition of pathogens, which is mostly carried out by antigen-presenting cells (apcs), e.g. dendritic cells, and eventually triggers the activation of the adaptive immune system (mescher, ) . in particular, when apcs get directly activated through exposure to pathogen-associated molecular patterns, they initiate both cell-and antibody-mediated immune responses, which are mediated by the t and b cells, respectively (christmas, ) . the cell-mediated response is executed by t cells that get activated by apcs through antigen presentation at their surface via mhc molecules. in contrast, b cells are activated through t helper (th) cells and cytokines that are secreted by apcs (mescher, ) . activated b cells produce and secrete additional pro-inflammatory cytokines and antibodies to further stimulate the immune reaction (mescher, ) . hcq and cq negatively regulate many aspects of these innate and adaptive immune responses by reducing inflammation, and ultimately the severity of autoimmune diseases (fig ) . through the inhibition of endosomal tlr signalling, hcq and cq treatment decreases the levels of pro-inflammatory cytokines produced by peripheral mononuclear cells in the blood, including ifnc (van den borne et al, ), tnfa (picot et al, ; van den borne et al, ; jang et al, ) , il- (picot et al, ; sperber et al, ; jang et al, ) , il- (sperber et al, ; van den borne et al, ; jang et al, ) and il- (landewe et al, ) . the reduction of tlr signalling-mediated activation of immune cells by both drugs consequently decreases the aberrant immune response and diminishes inflammation symptoms observed in rheumatic patients (da silva et al, ) . in addition to directly inhibiting endosomal tlr signalling, cq and hcq can interfere with the intracellular signals that lead to both the release of phorbol esterinduced arachidonic acid and the block of pro-inflammatory cytokines secretion (e.g. tnfa and il- ) in mouse macrophages (bondeson & sundler, ) . in particular, activation of phospholipase a by phorbol esters, but not by ca + , is inhibited by hcq and cq, which blocks the synthesis of arachidonic acid. furthermore, these compounds negatively impact the generation of zymosan-induced formation of inositol phosphates, a product of phospholipase c activity (matsuzawa & hostetler, ) , suggesting that they have an inhibitory effect on this enzyme as well (bondeson & sundler, ) . hcq also inhibits ca + -activated k + channels in macrophages, and consequently k + efflux, which could result in impaired inflammasome activation and pro-inflammatory cytokine release (eugenia schroeder et al, ) . high levels of pro-inflammatory cytokines are a central characteristic of the ra pathogenesis (mcinnes & schett, ; blasius & beutler, ; pollard et al, ; schinnerling et al, ; muskardin & niewold, ) . in particular, stimulatory cytokines (i.e. il- , il- , il- , il- , il- , il- and type i and ii ifn for t cells, and b-cell activating factor (baff) for b cells) activate t and b cells, which in turn produce pro-inflammatory cytokines and autoantibodies, respectively. pro-inflammatory cytokines contribute to ra pathogenesis by promoting autoimmunity, maintaining chronic inflammatory synovitis and stimulating the destruction of joint tissues. they also play a role in the maturation and activation of osteoclasts, the cells responsible for breaking down bone tissue (mcinnes & schett, ) . excessive production of baff, a cytokine essential for b-cell physiology, alters the immune tolerance by contributing to the maturation and survival of self-reactive b cells, the major source for autoantibodies contributing to joint inflammation (mahdy et al, ) . reduction of the high baff levels in the serum from ra patients by hcq (mahdy et al, ) improves symptoms of rads, both in animal models and in clinical trials (sun et al, ) . cytokines like baff, tnfa, ifna and ifnc are also major contributors to sle severity, by promoting b-cell survival and autoantibody production, and contributing to organ inflammation (rönnblom & elkon, ) . thus, the modulation of their levels represents a potential therapeutic avenue (rönnblom & elkon, ) . this is supported by a cohort study showing that treatment of sle patients with hcq results in a decrease of type i ifn levels and concomitant reduction of disease severity (willis et al, ) . hcq can also directly affect the production of autoantibodies by b cells through tlr inhibition. particularly, hcq interferes with the differentiation of memory b cells into antibody-producing plasmablasts, a subset of b cells, by inhibiting tlr activation (torigoe et al, ) . although the pathogenesis of pss is not fully understood yet, activation of exocrine gland epithelium cells is thought to lead to the release of pro-inflammatory cytokines such as ifna and ifnb (both type i ifn), il- and baff, and chemokines (retamozo et al, ) . these factors stimulate further activation of apcs, but also of t and b cells, which promotes inflammation and autoimmunity (retamozo et al, ) . only a few studies investigated hcq administration in pss patients. nonetheless, pss patients treated with hcq have a significant lower baff levels in the serum, and an improvement in saliva production (mumcu et al, ) , indicating that this drug might be a promising therapy for pss as well. through t-cell receptors (tcrs) on their surface, t cells recognize antigens that are presented by apcs and get activated (goldman et al, ) . this results in both their proliferation and the release of various cytokines, including il- and tnfa (sperber et al, ) . one important step in the signalling cascade downstream of tcrs is the increase of intracellular ca + levels, which is released from internal ca + storages such as the er. as previously mentioned, hcq can impair the release of ca + from the er, which consequently inhibits t-cell activation (goldman et al, ; xu et al, ; schmidt et al, ) . hcq also negatively influences the expression and activity of cd on t cells, which is needed for b-cell activation dewitte et al, ) . cd expression is controlled by the nuclear factor of activated t cells (nfat), a transcription factor that relies on ca + release from the er . by impairing this event, hcq inhibits nfat nuclear translocation, resulting in decreased gene expression of cd . altogether, these studies show that blocking ca + release from the er by hcq leads to a multilevel inhibition of t-and b-cell activation, thereby hindering the immune response (fig ) . alterations in autophagic activity play an important role in the pathophysiology of t-and b-cell-mediated autoimmunity (weindel et al, ; van loosdregt et al, ; alessandri et al, ; mocholi et al, ; zhang et al, ) . in this context, autophagy is required to maintain cellular homeostasis in t cells (an et al, c) and autophagy deficiency impairs mhc class ii presentation and contributes to the generation of autoreactive t cells by thymic epithelial cells (levine et al, ) . moreover, plasma cells require autophagy to sustain immunoglobulin production and b-cell development (wu & adamopoulos, ). an imbalance within the t-cell populations, more specifically an increase in the number of th cells and a decrease in that of t reg cells, has been linked to pathogenesis of autoimmune diseases (yang et al, a; jadidi-niaragh & mirshafiey, ; Á lvarez-rodríguez et al, ) , including sle (an et al, c; Á lvarez-rodríguez et al, ) . this imbalance leads to an increased secretion of pro-inflammatory cytokines such as il- and il- , and a reduction of the levels of circulating factors like tgf-b, which suppresses inflammation and autoimmunity (an et al, c; geng et al, ) . this latter effect can be dampened with hcq and cq, as those drugs rebalance the th /t reg ratio ( geng et al, ) . mechanistically, this could be caused by an alteration of autophagy, as an induction of this process is observed in sle patients (an et al, c) . thus, an and colleagues thought to suppress hyperactivated autophagy by administrating hcq to lupus mlr/pr mice, an animal model for sle. in addition to lowering autophagic activity in this model, hcq rebalanced th and t reg cell numbers, which led to a decrease in pro-inflammatory cytokine levels (fig ) and a concomitant augmentation of antiinflammatory cytokines, resulting in the suppression of the autoimmune response (an et al, c) impact of nox inhibition on the immune system nox inhibition by hcq impairs the production of pro-inflammatory cytokines and the correct distribution of tlr , thereby dampening the immune response (müller-calleja et al, ) . this inhibition also positively affects nitric oxide (no) bioavailability (gómez-guzmán et al, ) . no is involved in a multitude of physiologic functions, including the regulation of blood vessel tone and vasodilation, and is rapidly inactivated by ros (nagy et al, ) . in sle patients, no bioavailability is severely lowered by high ros levels, particularly o À , resulting in endothelial dysfunction (griendling & alexander, ; landmesser & harrison, ; gómez-guzmán et al, ) . by blocking nox, the major producer of o À in the vascular wall, hcq treatment reduces ros levels and helps to prevent endothelial dysfunction in a mouse model for sle (gómez-guzmán et al, ) . in agreement with this concept, nox inhibition by hcq reduces thrombus formation, which is a well-known clinical manifestation in sle, in a venous thrombus mouse model (mü ller-calleja et al, ; miranda et al, ) (fig ) . thus, at the cellular level, hcq and cq inhibit antigen presentation, nox signalling, b-and t-cell activation, and rebalance t reg / th cell ratio. these multifaceted effects on different immune cells synergistically result in a decreased production and release of proinflammatory cytokines, a common hallmark of rads (fig ) . hcq is administered orally in tablet form as hydroxychloroquine sulphate . it is absorbed in the gastrointestinal tract (mclachlan et al, ) before being widely distributed throughout the body to muscles, liver, spleen, lungs, kidneys, pituitary and adrenal glands, and tissues that contain melanin (haładyj et al, ) . daily dosage of hcq ranges from to mg for rads, from to mg for dermatological disorders (ben-zvi et al, ), from to , mg in cancers (chude & amaravadi, ) and from to mg for various infectious diseases. its half-life in the body ranges between and days (mclachlan et al, ) , and - % of hcq is protein-bound (furst, ) , resulting in - % unbound, pharmacologically active drug (rang et al, ) . the majority of hcq is excreted through the kidneys, while the rest is metabolized by the liver or excreted through faeces (furst et al, ; haładyj et al, ) . contraindications for taking hcq are a history of retinopathy or visual field changes, hypersensitivity to -aminoquinoline compounds and long-term therapies in children (https://www.fda.gov/). hcq is, however, considered safe during pregnancy (kaplan et al, ; haładyj et al, ) . hcq ameliorates classical rad symptoms, such as skin problems and joint pain, predominantly by decreasing the inflammation reaction in patients (fig ) . in sle, hcq is given to patients as either a single or a combinatorial therapy together with steroids and immunosuppressive drugs, to improve patients' life expectancy by reducing lupus flares and accrual of organ damage (ponticelli & moroni, similarly, hcq treatment produces significant clinical improvement and functional capacity in ra patients (smolen et al, ; haładyj et al, ) . in ra, prevention of cartilage degradation, which causes joint destruction, is an important aspect of the therapeutic approach (kumar & clark, ) . cartilage degradation is mostly caused by pro-inflammatory cytokines, such as il- , il- and tnfa, and their production can be repressed by hcq treatment (picot et al, ; sperber et al, ; van den borne et al, ; jang et al, ; mcinnes & schett, ; da silva et al, ) . in vitro experiments have also established that cq inhibits proteoglycan turnover (fulkerson et al, ; ackerman et al, ; schug & kalbhen, ; rainsford et al, ) , and early autoradiographic studies following tritium-labelled hcq have revealed that this drug accumulates in the cartilage of mice (cecchi & porzio, ) . these findings and its water-soluble properties led to the proposition that hcq accumulates in the cartilage by binding acidic proteoglycans and protecting them from degradation by proteolytic enzymes (rainsford et al, ) . although an early study pointed out that cq and hcq can indeed inhibit cartilage breakdown, slowing down the disease progression and preventing further joint damage in ra patients (julkunen et al, ) , more recent investigations could not confirm a positive effects on joint damage (sanders, ; smolen et al, ; haładyj et al, ) . the therapeutic benefits of hcq administration on pss classical symptoms, e.g. sicca symptoms, remain controversial; some studies documented beneficial effects (tishler et al, ; rihl et al, ; yavuz et al, ; mumcu et al, ) , while others reported none (gottenberg et al, ; yoon et al, ; wang et al, ) . hcq treatment, however, ameliorates extraglandular symptoms (fox et al, ; demarchi et al, ) , and according to the sjögren's syndrome foundation's clinical practice guidelines (https://www.sjo grens.org/), disease-modifying anti-rheumatic drugs are recommended to treat musculoskeletal pain, with hcq being the therapeutic approach of choice (carsons et al, ) . hcq also reduces immunological alterations of pss, such as decreased levels of immunoglobulins, erythrocyte sedimentation rate, serology and il- production (tishler et al, ; yavuz et al, ; mumcu et al, ) . furthermore, in a retrospective analysis, hcq administration to pss patients significantly improved saliva production (rihl et al, ) . this improvement was more pronounced in patients who were positive for autoantibodies against anti-a-fodrin, an intracellular filamentous cytoskeleton protein. while the cause for this difference remains unknown, a possible explanation is that hcq could improve saliva production by decreasing elevated levels of cholinesterase, an enzyme that counteracts saliva production (dawson et al, ) . anti-viral effects the anti-viral function of hcq and cq has mainly been linked to their ability to increase the ph of the endosomal system and the trans-golgi network (tgn) (savarino et al, ) . thus, these drugs are able to inhibit cell entry of numerous viruses, as a low endosomal ph is required for the fusion of endocytosed virions with the limiting membrane of endosomes. in this context, cq and hcq decrease replication of viruses such as dengue virus (denv ), chikungunya virus, hepatitis a and c virus, influenza a virus, zika virus, severe acute respiratory syndrome coronavirus (sars-cov) and borna disease virus in cellular models (bishop, ; gonzalez-dunia et al, ; keyaerts et al, ; vincent et al, ; blanchard et al, ; de clercq, ; eng et al, ; di trani et al, ; sourisseau et al, ; khan et al, ; ashfaq et al, ; boonyasuppayakorn et al, ; farias et al, ; delvecchio et al, ; shiryaev et al, ) . for some viral structural proteins, a maturation step involving post-translational modification and/or processing in the tgn is crucial for their function and ultimately for the assembly of infectious viral particles, e.g. glycosylation of hiv gp (tsai et al, ; savarino et al, ) or cleavage of the denv prm protein (randolph et al, ) . glycosylation in the tgn is also required for the correct assembly of ace , the entry receptor for sars-cov (vincent et al, ) . thus, hcq and cq contribute to inhibit viral infections by neutralizing the ph of intracellular organelles, interfering with important processes required for viral life cycle. although hcq and cq have shown beneficial therapeutic effects in animal models for denv , hepatitis c virus, avian influenza a virus, zika virus and sars-cov infections, clinical trials have so far failed to conclusively prove their anti-viral potential in humans (rodrigo et al, ; fragkou et al, ; mckee et al, ) . this might be due to the fact that drug concentrations required to deacidify intracellular compartments cannot easily be reached in humans (al-bari, ) . therefore, neither hcq nor cq is currently recommended as anti-viral drugs (rodrigo et al, ) . during the sars-cov- pandemic in , the need to find an effective medication has brought major attention to hcq and cq due to their ability to both inhibit viral infections and dampen the massive cytokine response that is observed in sars-cov- -infected patients (badgujar et al, ; ibáñ ez et al, ; moore & june, ) . the effectiveness of hcq and cq against sars-cov- , however, has so far not been proven in humans, and the results at the time that this review was completed were still controversial (boulware et al, ; fragkou et al, ) . ª the authors embo molecular medicine : e | anti-cancer therapy cq and hcq are being increasingly used in clinical trials to treat cancer (https://clinicaltrials.gov/). because high doses are required to achieve anti-tumoural effects in monotherapies, they are often used in combination with radiotherapy and/or other chemotherapeutical drugs (plantone & koudriavtseva, ). we briefly discuss here possible mechanisms of action for hcq and cq in cancer. for a more detailed discussion on this topic, more specific reviews are available (manic et al, ; pascolo, ; levy et al, ; shi et al, ; verbaanderd et al, ) . elevated autophagic activity is crucial for tumour cell survival and growth as it supplies the high demand of nutrients within a developed tumour (amaravadi et al, ) . this is especially relevant for autophagy-dependent cancers that rely on this pathway when faced with metabolic stress. consequently, hcq or cq treatment has been successful in regressing the growth of some of those cancers in preclinical studies (e.g. with ras pathway mutations (guo et al, ; lock et al, ) , such as specific pancreatic cancers (mancias & kimmelman, ; yang et al, b; sousa et al, ) , or braf-driven tumours (levy et al, ; strohecker et al, ; xie et al, ) . the effectiveness of hcq and cq in cancer therapy is, however, controversial. in animal models, hcq dosages are often mg/kg/day or higher, which is too high to be administered in humans (pascolo, ) , and with lower dosages, autophagy is not sufficiently inhibited to achieve tumour regression (pascolo, ) . moreover, some cancer cells (e.g. derived from breast tumours or melanomas or kras-driven cancer cell lines) have shown cq-mediated cell growth inhibition that was independent of autophagy (maycotte et al, ; maes et al, ; eng et al, ) . various cancer cells express high levels of tlr , e.g. breast and prostate cancer cells (merrell et al, ; verbaanderd et al, ) , which is linked to cancer invasiveness in vitro and associated with poor prognosis (väisänen et al, ; verbaanderd et al, ) . tlr -mediated nf-jb signalling is required for cancer cell migration and proliferation in gastric cancer cell models, which is inhibited by cq (zhang et al, ) . the exact molecular mechanism of tlr signalling inhibition in cancer cells remains unknown. another mechanism by which hcq affects cancer growth is by modulating the immune system. tumour-associated macrophages (tams), which are phenotypically described as m macrophages, play a role in promoting tumour growth and immune escape, angiogenesis and metastasis (mantovani et al, ; li et al, ) . in contrast, tumour killing macrophages (m macrophages) have an opposite effect and are activated by cytokines such as ifnc, which are released from t cells (de palma & lewis, ; ostuni et al, ) . interestingly, in a melanoma-bearing mouse model, intraperitoneal injection of mg/kg cq effectively inhibited melanoma growth in a t-cell-dependent manner, and prolonged animal survival (chen et al, ) . mechanistically, cq can switch tams into m macrophages by raising lysosomal ph, and thereby mobilizing lysosomal ca + through upregulation of the lysosomal ca + channel mucolipin . the release of lysosomal ca + then activates the p and nf-jb pathways, but also the transcription factor eb, resulting in an enhanced anti-tumour t-cell response (chen et al, ) . by stimulating the t-cell-mediated immune response and simultaneously decreasing immune inhibitory cells, including tams and t regs , and cytokines such as tgf-b and il- , cq treatment reduced breast cancer growth and prolonged mice survival in a breast xenograft model . another important aspect of anti-cancer immunity is the activation of immune cells by sensing danger signals (e.g. hmgb ). danger signals are subsequently recognized by receptors, such as tlr on dendritic cells (apetoh et al, ) . one function of tlr is to preserve engulfed tumour antigens from enhanced degradation, and thereby favour antigen presentation. the loss of in rads, hcq treatment predominantly alleviates the symptoms (purple boxes) by inhibiting the production and release of pro-inflammatory cytokines. as a consequence, hcq diminishes skin conditions. there are also indications that hcq both decreases cartilage degradation and consequently reduces joint and muscle pain, and helps to restore saliva production. usage of hcq can cause side effects (orange boxes); the most common are gastrointestinal disturbances, skin discoloration, cutaneous eruptions and elevated muscle enzymes, whereas retinopathy, cardiac myopathy and myotoxicity are rare, but severe. of embo molecular medicine : e | ª the authors antigen presentation capacity in tlr -deficient dendritic cells can be restored by cq, possibly by raising lysosomal ph, which contributed to tumour size reduction in a tlr À/À thymoma mouse model (apetoh et al, ) . along these lines, cq reduced breast cancer growth in mice after irradiation by enhancing apoptotic and immunogenic tumour cell death (ratikan et al, ) . the enhanced immune response was attributed to a decreased degradation of tumour antigens in dendritic cells, resulting in an increased antigen presentation (ratikan et al, ) . hcq and cq can also inhibit cxcl /cxcr signalling, which is involved in chemotaxis and adhesion of tumour cells and of growth factors secretion that are key for cancer progression (sun et al, ; kim et al, ; verbaanderd et al, ) . moreover, hcq and cq interfere with the activation of growth-promoting pathways in cancer stem cells, thereby suppressing the regrowth of tumours (li et al, ; balic et al, ; choi et al, ) . multiple reports further describe the mechanisms by which cq triggers cell death in tumour cells. cq induces apoptosis of cancer cells by either stimulating the mitochondrial apoptotic pathway (du jiang et al, ) or activating the p -dependent transcription of pro-apoptotic genes (zhou et al, ; loehberg et al, loehberg et al, , maclean et al, ; kim et al, ; bieging et al, ) . additionally, several studies have suggested that cq intercalates into dna and disturbs chromatin topology (o'brien et al, ; sternglanz et al, ; field et al, ; yin et al, ) , which could lead to an impairment in dna repair mechanisms, and in turn cause dna damage and enhance cell death (michael & williams, ; liang et al, ; weyerhäuser et al, ) . besides directly targeting tumour cells, cq also affects tumour angiogenesis by altering endothelial cell functionality. cq administration leads to notch accumulation in endothelial cell endosomes, stimulating the downstream signalling that leads to tumour vessel normalization, and resulting in reduced tumour invasion and metastasis (maes et al, ) . therefore, cq also improves the delivery and efficacy of other chemotherapeutics (maes et al, ) . hcq and cq thus show potential in inhibiting tumour growth and modulating tumour immune response through various mechanisms. it is, however, important to reiterate that the doses used to achieve relevant effects in cancer therapies are often substantially higher than the doses used to treat rads. moreover, when treating cancer or viral infections, one has to keep in mind that hcq and cq also have immune suppressive functions that could negatively influence its beneficial effect for the patients. side effects of hcq treatment are rare, but nonetheless exist, and can be very serious, especially during prolonged administration (haładyj et al, ) . in table ev , we provide a comprehensive overview of the known side effects caused by hcq in rads and their prevalence. overall, the most common side effects in rad patients taking hcq or cq are gastrointestinal disturbances, skin discolorations, cutaneous eruptions and elevated muscle enzymes. although rare, retinopathy, neuromuscular and cardiac toxicities (fig ) are the most serious and life-threatening side effects potentially triggered by hcq (plantone & koudriavtseva, ) . prolonged administration of hcq or cq can cause retinopathy and loss of retinal function that, when ignored, can result in permanent vision loss (jorge et al, ) . the primary site of toxicity in the retina is the photoreceptor layer, with secondary degeneration occurring later in retinal pigment epithelium (rpe) cells (de sisternes et al, ; yusuf et al, ) . some studies offer a potential explanation for this severe side effect. by inhibiting the lysosomal degradation capacity and possibly endocytosis in rpe cells, hcq and cq are preventing the degradation of old and spent outer segments of photoreceptors in the rpe, a process that is required to maintain its function and preserve vision (kevany & palczewski, ; yusuf et al, ) . furthermore, hcq entrapment in the rpe might lead to an accumulation of lipofuscin, which is associated with photoreceptor function impairment and consequent vision loss (kevany & palczewski, ; yusuf et al, ) . it has been speculated that, due to this entrapment, retinopathy still continues in some cases after cessation of hcq treatment (michaelides et al, ) . accumulation of cq in the pigmented ocular tissue, which comprises rpe cells, the iris, the choroid and the ciliary body, and eventually in the retina, was also observed in rhesus monkeys when cq was administered for months (rosenthal et al, ) . this caused an initial damage to the photoreceptors and the ganglion cells, followed by a disruption of both the rpe and choroid, which ultimately led to visual impairments and retinopathy (rosenthal et al, ) . high levels of hcq inhibit the function of the organic anion transporting polypeptide a (oatp a ), a plasma membrane importer expressed in many tissues, including rpe cells (xu et al, ) . in particular, oatp a transports all-trans-retinol (atrol), a retinol precursor essential for the classic visual cycle (chan et al, ) , into rpe cells. by blocking this transporter, hcq causes an extracellular accumulation of atrol and disrupts the classic visual cycle (xu et al, ) . cardiac side effects and myotoxicity hcq can cause acute and chronic cardiac adverse effects (chatre et al, ) . acute adverse effects are linked to a very high dose of hcq, which provokes a block of na + and ca + channels. this inhibition can lead to membrane-stabilization effects in cardiac muscle cells, which in turn causes conduction disturbances with atrioventricular block and qrs interval widening (white, ) . chronic adverse effects are connected to long-term treatment with a high cumulative dose of hcq (chatre et al, ) . as described above, hcq treatment impairs the degradative activity of lysosomes, which leads to an accumulation of material such as glycogen and phospholipids in their interior (chatre et al, ) . in myocytes, this causes a vascularization of the cytoplasm and myofibrillar disorganization, which contributes to the development of cardiac myopathy and myocardial fibrosis (yogasundaram et al, ) . this phenomenon can also be seen in the fabry and danon lysosomal storage diseases, which have similar phenotypes (roos et al, ; d'souza et al, ; chatre et al, ) . moreover, hcq-mediated accumulation of autophagosomes in muscles and peripheral nerves can lead to myotoxicity or myotoxicity combined with peripheral nerve dysfunction (shukla et al, hcq is nowadays widely used for the treatment of rads and has shown great success in improving the quality of life of many patients. over the years, research on the molecular and cellular mode of action of hcq (and cq) revealed that this compound modulates molecular processes and cellular responses in multiple ways. at least four mechanisms of action that, directly or indirectly, influence the immune system by synergistically dampening pro-inflammatory responses, have been described. although lysosomal inhibition and autophagy impairment are the most studied, hcq also influences other important immune regulatory pathways by inhibiting specific steps, such as activation of endosomal tlr-, cgas and nox signalling and ca + mobilization for the er. the beneficial therapeutic effect of hcq in rads probably lies in its multifaceted properties, which also makes it a promising candidate in other medical fields, such as oncology (onorati et al, ) and microbiology (savarino et al, ; cortegiani et al, ; yao et al, ) . generally, hcq is considered a safe drug with low prevalence of side effects. these side effects nevertheless exist and can impact the life of a patient tremendously. among them, the most severe, i.e. retinopathy and cardiomyopathy, is linked to the induced lysosomal activity inhibition. this suggests that the unwanted negative effects of hcq could be due to its lysosomotropic properties. in this context, it has been reported that the effect of hcq on endosomal and lysosomal ph at therapeutic concentrations is negligible (ku znik et al, ) and that the ph changes observed in vitro might not reflect the in vivo reality. therefore, a higher dose of hcq (or a higher cumulative dose) could lead to a ph increase in the compartments of the endolysosomal system and thus cause more side effects (latasiewicz et al, ; jorge et al, ) . the well-documented list of side effects caused by hcq during the treatment of rads should be considered when using hcq to treat other pathologies such as cancer (onorati et al, ) , neurodegenerative disorders (hedya et al, ) , metabolic diseases (pasquier, ) and microbial infections (savarino et al, ) , especially since treatment of some pathologies requires high hcq doses (leung et al, ) . while the search for a unifying mechanism of action for hcq is tempting, current knowledge shows that this small molecule has more than a single target. as a result, future research should aim at identifying potential additional cellular and organismal pathways specifically modulated by hcq. the mechanisms by which hcq causes side effects could also provide important information. increasing our understanding of hcq mode of action would improve patient outcome by promoting therapeutic benefits while reducing side effects. (i) investigate whether all hcq modes of action described with in vitro experiments are relevant in patients, and whether one of these mechanisms is predominantly causing the observed side effects. (ii) determine whether hcq has other molecular effects than the ones described, which could help to better understand hcq treatment outcomes in patients. (iii) chemically improve hcq to make it more effective and less toxic, and thereby render it more suitable for the treatment of other diseases (e.g. specific cancers). 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authors apologize in advance to those authors whose contributions have been omitted due to lack of space and felt that their work was one of the highlights. the authors declare that they have no conflict of interest. ackerman nr, jubb sn, marlowe sl ( ) effects of various antiinflammatory and anti-rheumatic agents on the synthesis, secretion, and key: cord- - es zv authors: aggarwal, gaurav; henry, brandon michael; aggarwal, saurabh; bangalore, sripal title: cardiovascular safety of potential drugs for the treatment of coronavirus disease date: - - journal: am j cardiol doi: . /j.amjcard. . . sha: doc_id: cord_uid: es zv coronavirus disease (covid- ) has become a global pandemic. it is still uncontrolled in most countries and no therapies are currently available. various drugs are under investigation for its treatment. the disease is known to have worse outcomes in patients who have underlying cardiovascular disease. chloroquine/hydroxychloroquine, azithromycin, remdesivir and lopinavir/ritonavir are currently being studied in trials and show some promise. conduction disorders, heart failure and mortality have been reported with the use of these drugs. it is important to have a knowledge of potential cardiotoxic effects of these drugs before using them for covid- patients for better allocation of healthcare resources and improvement in clinical outcomes. the current outbreak of coronavirus disease (covid - ) has been declared as a global pandemic. it is caused by the severe acute respiratory distress syndrome coronavirus (sars-cov- ) and has thus far infected > . million people with > , cases in the unites states alone . physicians and scientists are working tirelessly to find a potential drug or vaccine for its treatment. increased disease severity and mortality have been noted in those with cardiovascular disease who develop covid- . moreover, a decreased potassium level has also been reported in patients with covid- , which can cause electrocardiographic changes like prolonged qt interval and may increase the risk of adverse reactions with pharmacotherapies. hence, understanding the cardiovascular risks of potential pharmacotherapies being investigated for covid- is of utmost importance. numerous drugs are currently under investigation, some in early phase clinical trials. the drugs of highest interest to-date include chloroquine/hydroxychloroquine (cq/hcq) alone or in combination with azithromycin, remdesivir, lopinavir/ritonavir and interferon alpha- b . this article reviews the potential cardiovascular risks associated with these drugs. cq/hcq are quinoline medications widely used in treatment of malaria, rheumatoid arthritis (ra) and discoid or systemic lupus erythematosus (sle). however, they have been shown to be cardiotoxic due to lysosomal dysfunction and accumulation of glycogen and phospholipids . the cardiotoxic effects of cq/hcq appear to be related to the cumulative dose. high cumulative doses of cq/hc have been shown to be associated with atrioventricular blocks and cardiac arrest . sick sinus syndrome and qt prolongation have also been reported with high doses , . in some of these cases, baseline qt interval was found to be mildly prolonged and hence qt interval is such patients should be closely monitored to prevent risk of ventricular arrhythmias. given the fact that hypokalemia causes prolongation of qtc interval, low potassium levels in patients with severe covid- may further exacerbate the arrhythmogenic potential of cq/hcq. cq has been found to be more associated with conduction defects compared to hcq. in a study of patients treated with hcq for a minimum of year and who had no underlying cardiac disease, hcq was found to be safe with only two patients developing right bundle branch block and one patient developing left bundle branch block . there were no instances of atrioventricular blocks or qt prolongation. echocardiographic abnormalities have also been reported in patients exposed to high cumulative doses of cq/hcq. in a robust systematic review, chatre et al found that patients with cardiac complications attributed to cq/hcq were mainly female ( %) and had a median age of years . conduction disorders accounted for almost % of the reported cardiac complications. other reported toxicities included left ventricular hypertrophy ( %), heart failure ( %), valvular dysfunction ( %) and pulmonary hypertension ( %). cardiac magnetic resonance imaging (cmri) in such patients has shown patchy delayed contrast enhancement , . endomyocardial biopsy in such patients shows no evidence of inflammation or vasculitis . instead, the important findings are swollen myocytes with vacuolated cytoplasm filled with numerous curvilinear bodies, myeloid bodies and large secondary lysosomes. the curvilinear bodies are membrane bound and closely associated with lysosomes and contain partially digested lipids. after discontinuation of the drug, complete recovery of cardiac function has been reported in < half of the patients . irreversible damage including death and need for pacemaker and heart transplantation has been described in literature . a recent small randomized study has shown beneficial effects of hcq treatment on time to clinical recovery and pneumonia resolution . for patients infected with covid- , cq/hcq are currently recommended for a - -day course. the cumulative dose for this duration may not be high, but the prolonged recovery time and uncertainty about the best duration of treatment may potentially lead to cardiotoxicity. moreover, as noted, the cardiotoxic effects may still occur even with low cumulative doses. azithromycin is a semisynthetic macrolide antibiotic and is the most common prescribed antibiotic in the united states. it works against gram positive, gram negative and atypical pathogens. it has been postulated as a possible cure for covid- in combination with cq/hcq . initially thought to be free of cardiotoxic effects, it was later found to cause qt prolongation and higher risk of cardiovascular morbidity and mortality. multiple studies have shown the risk of qt prolongation and ventricular tachycardia with azithromycin. its use has also been linked to risk of atrial fibrillation and cardiac arrest. in a large multinational casecontrol study, azithromycin use was found to be associated with an increased risk of ventricular . the mechanisms by which azithromycin causes arrhythmias in still under investigation. qt prolongation and ventricular arrhythmias have been postulated to be due to increased na + current and inhibition of outward flow of k + ions from ventricular myocytes . qt interval usually returns to baseline once the drug is stopped. however, this could be however, no difference was found between those on azithromycin versus those on penicillin v (rate ratio . , % ci . to . ). the authors concluded that excess mortality in patients on azithromycin when compared to those not on antibiotics was most likely due to the mortality risk of the underlying infection itself. an interesting finding among some studies is a trend towards higher mortality in first days of azithromycin use compared to other antibiotics, but no difference from day . a large meta-analysis of randomized and observational studies found azithromycin use to be associated with higher risk of cardiovascular death but not with all-cause death . in this meta-analysis, authors also found a higher risk of ventricular arrhythmias and sudden death (rr . , ci . to . ) with the use of azithromycin. as evident from the above, the cardiovascular risks associated with azithromycin has yet to be fully elucidated, and further prospective studies are needed. we suggest clinicians be careful in patients who are elderly, have underlying cardiovascular disease, those who are on drugs known to prolong qt interval and those with renal failure. we recommend electrocardiography (ekg) before starting hcq or azithromycin for all patients, and then serially monitoring qt interval in patients at risk for torsade de pointes. remdesivir is ˊ-cyano-substituted adenosine nucleotide prodrug which inhibits viral rna synthesis which was first studied in treatment for ebolavirus. the data are scant on potential efficacy and risks with remdesivir. the only study evaluating effects of remdesivir in humans randomized patients infected with ebolavirus to different treatment strategies, out of which patients received remdesivir. only patient who received remdesivir had hypotension and subsequently died due to cardiac arrest . however, the authors could not exclude the death in this patient was related to underlying ebolavirus itself. if this drug does show therapeutic efficacy in treatment of covid- , then ongoing surveillance would be needed to study its potential cardiovascular adverse effects. lopinavir/ritonavir are protease-inhibitors frequently used in the treatment of human immunodeficiency virus (hiv) infection. this combination has been studied for treatment of sars and mers. however, a randomized comparison between this combination and standard care showed no difference in mortality in patients with severe covid- illness, though there was a trend towards shortened median time to clinical improvement . in this study, only one patient in the lopinavir/ritonavir group was found to have prolongation of qt interval. more studies are ongoing evaluating its efficacy in patients with covid- . the main cardiac risk associated with lopinavir/ritonavir is progression of atherosclerosis. elevation in plasma levels of total cholesterol, low-density lipoprotein (ldl) and total cholesterol to high-density lipoprotein (hdl) ratio, and decrease in hdl levels has been reported with the use of lopinavir/ritonavir therapy . cardiac conduction defects have also been reported with the use of lopinavir/ritonavir. sinus arrest, first and second degree atrioventricular blocks have been documented with its use . pegylated interferon-alpha (α) has been also studied for treatment of sars and mers. however, it has been linked to cardiovascular adverse effects in prior studies. in a study of patients who were treated with interferon-α for hepatitis c, cardiac complications were noted in patients during treatment and more within one year after end of treatment . a total of patients had arrhythmias, patients had ischemic heart disease and patients developed cardiomyopathy in this study. the increase in tumor necrosis factor-alpha levels during interferon-α therapy may be the underlying mechanism mediating its cardiotoxic effects, though this same mechanism may be beneficial in inhibiting viral replication in patients with covid- . pericardial effusion has been reported to occur with interferon-α therapy . some other therapies being studied for treatment of covid- are favipiravir and high dose vitamin c. data are scant on potential cardiovascular risks with these drugs. favipiravir was reported to be associated with mild prolongation of qt interval in a young patient treated for ebolavirus . high dose of vitamin c was found to be associated with higher cardiovascular mortality in patients with diabetes . human monoclonal antibodies that inhibit interleukin- (il- ) pathway by binding and blocking il- receptor are also bring currently studied for treatment of covid- . they have been shown to cause increases in total cholesterol and ldl levels . a phase trial evaluating colchicine is ongoing for treatment of patients with covid- that plans to enroll participants[clinicaltrials.gov nct ]; colchicine has not been linked to cardiovascular side effects but may worsen bleeding. finally, ace inhibitors and arbs are also under investigation for the treatment of covid. sar-cov- may directly interact with the renin-angiotensin-aldosterone system, with the virus using the angiotensin converting enzyme (ace ) as its host receptor on type ii pneumocytes. as such, a link between angiotensin converting enzyme (ace) inhibitors or angiotensin receptor blockers (arbs) and covid has been proposed. however, there is little, if any, convincing evidence to suggest discontinuation of such drugs and arbs are currently undergoing a clinical trial as a treatment for severe covid- . covid- is a pandemic with high morbidity and mortality burden. the patients who have underlying cardiovascular disease or those who develop cardiac dysfunction during infection with covid- are at higher risk of mortality. various drugs currently under investigation for treatment of the novel coronavirus have been associated with cardiotoxic effects (table ) . though cumulative dose effects impact toxicity, conduction defects, prolongation of qtc interval, cardiomyopathy and ischemic heart disease have been shown to occur with use of hydroxychloroquine, chloroquine, azithromycin, remdesivir, interferon-alpha and lopinavir/ritonavir therapies. caution and careful monitoring should be exercised when prescribing these therapies in patients at risk for cardiac disease. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. who. coronavirus disease (covid- ) pandemic clinical predictors of mortality due to covid- based on an analysis of data of patients from wuhan, china hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial chloroquine: modes of action of an undervalued drug complete heart block in chronic chloroquine poisoning a case of chloroquine-induced cardiomyopathy that presented as sick sinus syndrome conduction disorder and qt prolongation secondary to long-term treatment with chloroquine heart conduction disorders related to antimalarials toxicity: an analysis of electrocardiograms in patients treated with hydroxychloroquine for connective tissue diseases cardiac complications attributed to 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doc_id: cord_uid: tkqmu va chloroquine and closely related structural analogs, employed initially for the treatment of malaria, are now gaining worldwide attention due to the rapidly spreading pandemic caused by severe acute respiratory syndrome-coronavirus- , named coronavirus disease (covid) of (covid- ). although much of this attention has a mechanistic basis, the hard efficacy data for chloroquine/hydroxychloroquine in the management of the clinical syndrome of covid- have been limited thus far. this review aims to present the available in vitro and clinical data for the role of chloroquine/hydroxychloroquine in covid- and attempts to put them into perspective, especially in relation to the different risks/benefits particular to each patient who may require treatment. chloroquine and related drugs were initially developed as antimalarial agents. during the second world war, many clinicians made serendipitous observations that these drugs could be beneficial for treating rheumatological and dermatological conditions. [ ] [ ] [ ] since then, several well-designed studies have established their efficacy in the chronic management of connective tissue disorders, including systemic lupus erythematosus and rheumatoid arthritis, and there has been a growing list of indications to support their therapeutic potential in varied diseases of oncology, cardiology, and nephrology. these agents have also shown a promising role in viral infections, and with the recent declaration on march th, , by the world health organization that coronavirus disease (covid) of (covid- ) is a pandemic, these compounds have rapidly gained worldwide attention for their ability to control the causative virus, severe acute respiratory syndromecoronavirus- (sars-cov- ). in this narrative review, the authors discuss these medications from a mechanistic perspective with specific insights into their potential role in controlling viral infections. we discuss in vitro and in vivo evidence for their emerging therapeutic role in covid- with due concerns to safety when applying the known facts to large populations increasingly affected with covid- . both chloroquine and hydroxychloroquine (hcq) are weak bases that exist in the extracellular environment mostly in a protonated form with a positive charge. this positive charge makes them incapable of crossing the plasma membrane. the non-protonated portion that enters a cell is quickly protonated and concentrated in the acidic, low-ph organelles such as endosomes, golgi vesicles, and lysosomes. the antimalarial actions of these compounds are related to a heavy accumulation of these drugs in the acidic lysosomes of the parasites, which leads to a neutralizing, 'lysosomotropic' effect that prevents the detoxification of ingested 'heme' moiety, resulting in lysis of the malarial parasite. additionally, investigators have identified a series of immunomodulatory and anti-inflammatory effects for these agents. these include inhibition of ligand-based toll-like receptor stimulation, inhibition of nuclear factor kappalight-chain-enhancer of activated b cells (nfkb) pathways in macrophages with resultant reduction in the generation of pro-inflammatory cytokines, reduced processing of the endogenous and exogenous ligands through lysosomes and endosomes with resultant reduction in the availability of processed antigens for presentation to the major issn: - review -chloroquine, hydroxychloroquine, and covid- drugsincontext.com histocompatibility complex-t cell receptor interactions, and downstream activation of cellular immunity. [ ] [ ] [ ] [ ] together, these properties lay the foundation of their use in several rheumatological, cardiovascular, and dermatological diseases. these pleiotropic actions, seen in a variety of chronic diseases, have also guided their exploration for the control of viral infections. to date, these agents have been explored in ebola virus disease, human immunodeficiency virus (hiv) infection, , middle east respiratory syndrome (mers), and sars-cov- infection, , and their promising action against sars-cov- has provided the basis for their putative benefits in treating sars-cov- infection. however, the exact mechanism by which chloroquine/hcq may be of benefit in covid- is largely speculative and appears to be related to a series of actions demonstrated in alternate, yet similar, disease models ( figure ). hcq is known to have significant effects on many mechanisms that drive the viral entry into the host cells. most prominent among them are its actions on the angiotensin converting enzyme (ace) ii receptors. the original experiments during the sars epidemic suggested that sars-cov- binds to ace ii receptors, primarily present in the lung, heart, kidney, and intestine for its entry into the host system. more specifically, sars-cov- binds to the sialic acid moiety of the ace ii receptors. , chloroquine inhibits the intracellular glycosylation of the ace ii, and thus inhibits the addition of sialic acid moiety, which then leads to reduced ligand recognition and internalization of the virus. phylogenetic analysis of sars-cov- has shown about % nucleotide homology with sars-cov- , prompting the evaluation of these drug compounds for covid- . once the virus is bound to the cell membrane, endosomes play an important role in the fusion of viral particles and their internalization. thus, neutralization of the acidic ph of the endosome by chloroquine or hcq may prevent the fusion of sars-cov- with the host cell inhibiting the primary entry. an alternate mechanism hypothesized to inhibit the uptake of the virus into the host cell is based on the ability of chloroquine to be a broad inhibitor of nanoparticle endocytosis by resident macrophages. at the concentrations achieved in routine clinical dosing, chloroquine reduces in vitro and in vivo accumulation of synthetic nanoparticles. , it also reduces the expression of phosphatidylinositol-binding clathrin assembly protein, required for clathrin-mediated endocytosis of nano-sized structures. the ultrastructural studies of sars-cov- show that these virions fall within the same size ( - nm) and shape (spherical) range as the commonly studied synthetic nanoparticles. , thus, these actions against sars-cov- may be applicable at early stages before viral replication, which requires further experimental confirmation. alternatively, these compounds may act at later stages by inhibiting specific enzymes needed for assembly of virion and budding of the virions from the cell membrane. these drugs may accomplish these goals without disruption of the viral particle and liberation of viral nucleic acid and enzymes that are necessary for viral replication from a lysosome. , finally, these compounds have been shown to have a profound effect on the inflammatory cascade. sars-cov, through its ace ii receptor attachment, infects the type pneumocytes in the alveolar epithelium. this results in a local inflammatory reaction with resident neutrophils and macrophage activation as well as activation of the cellular immunity arm with t helper (th )type response. the resultant cytokine storm and alteration in epithelial permeability lead to the development of acute respiratory distress syndrome and associated morbidity and mortality related to covid- . chloroquine/hcq reduces the secretion of the proinflammatory cytokines, in particular the th cytokines, namely interleukin (il)- , il- tumor necrosis factor-α (tnfα), and interferon-gamma (ifnγ), by the alveolar macrophages, and thus may have a role in reducing the peak inflammatory response in covid- . these antiviral effects proposed mechanisms for chloroquine and hydroxychloroquine inhibition of nanoparticle uptake tnf alpha ifn gamma combined with immunomodulatory properties are promising, and thus over the last months, multiple studies have been launched to leverage these benefits in the clinical setting. a majority of the putative antiviral effects of chloroquine/hcq molecules on the sars-cov- are the result of the indirect inferences drawn from the data available on sars-cov- or from an alternate viral model, that is, epstein-barr virus (ebv), hiv, and so forth. led by its putative effects on the endosomal function disruption in one of the earliest in vitro studies, the centers for disease control (cdc) studied the effects of chloroquine in primate vero e cells (african green monkey kidney cells). the investigators found that chloroquine was effective against sars-cov- , and the inhibitory effects were equally potent whether the primate cells were treated before or after exposure to the virus, suggesting both prophylactic and therapeutic applications. they further showed that addition of ammonium chloride to raise the endosomal ph also had a similar inhibitory effect on the viral replication, suggesting that these effects, that is, inhibition of cellular entry as well as postentry viral replication and assembly may be affected by the alterations in the ph of the intracellular organelles. in line with the findings from the cdc study, an investigative team in wuhan, china, performed a similar in vitro time-ofaddition assay involving vero e cells for sars-cov- . in a physiology-based pharmacokinetic model, chloroquine/ hcq concentrations in lung fluid were simulated under five different dosing regimens to explore the most potent and safe regimen. for 'entry' treatment, the drugs were added to the cells for hour before viral attachment. for the 'post-entry' experiment, drugs were added at hours post infection and maintained until the end of the experiment. virus yield in the infected cell supernatants was quantified by quantitative reverse transcription-polymerase chain reaction (qrt-pcr). similar to the cdc experiments, the investigators found that these molecules affected the control of virus at both entry and at post-entry stages. the investigators further found that among the two, hcq was more potent than chloroquine as the effective concentration for a half-maximal response (ec ) was much lower ( . μm) for hcq than for chloroquine ( . μm). based on these experiments, the investigators suggested to treat sars-cov- infection with a loading dose of mg twice daily of hcq sulfate to be given orally on day , followed by mg given twice daily for more days. in another study from china, the investigators found the ec value of chloroquine against the sars-cov- in vero e cells was . μm. the investigators recommended that these concentrations, as evidenced through the pharmacokinetic studies for these molecules performed in the plasma of rheumatoid arthritis patients who received chloroquine at mg per day dose, were clinically achievable. while the in vitro experiments provide a rationale for their use in covid- , there is limited evidence on the clinical efficacy of these compounds for treating covid- . at the time of writing this manuscript, we found there were over randomized studies registered with clinicaltrials.gov for the use of hcq in covid- . the vast majority were still in the pre-recruitment phase, and a few were in the early stages of recruitment, but none of the larger studies had published their results. the in vivo or clinical evidence presented in this review is derived mainly from the small-cohort, observational, and randomized studies (available for review at the time of manuscript preparation) on the clinical outcomes (table ). in one of the first published pieces of evidence (published as a letter), gao and colleagues from china drew attention to the potential role of chloroquine for treating covid- . based on the clinical data collected from ten hospitals in china (wuhan, jingzhou, guangzhou, beijing, shanghai, chongqing, and ningbo) involving > patients, the authors proposed that chloroquine was a promising therapeutic drug. they reported that the patients who received chloroquine phosphate had lower rates of pneumonia exacerbation, greater improvement in lung imaging findings, higher rates of conversion to the virus-negative state, and shorter disease course compared to those who did not receive the drug. unfortunately, the report did not publish specific details about the patient population, response pattern, or analysis of outcomes. were either treated at a nearby facility without hcq or who refused participation. twenty-six patients received hcq ( mg three times daily for days) of which six received it in conjunction with azithromycin ( mg on day followed by mg daily for days). the inclusion of azithromycin was rationalized on the basis of its in vitro activity against zika and ebola viruses , and on the clinical evidence for its ability to prevent severe respiratory tract infections in patients suffering from viral infection. the primary outcome for the study was the virological clearance from nasopharyngeal secretions at day after receiving the drug. the investigators found that patients treated with hcq alone or in combination with azithromycin had significantly greater virological clearance (p= . ) at day ( . and %, respectively), compared to those in the control group ( . %). although encouraging, this ongoing study, as published, had several issn: - review -chloroquine, hydroxychloroquine, and covid- drugsincontext.com concerns to allow for strong recommendations in favor of or against hcq. six of the patients in the treatment group were lost to follow-up and were not included in the analysis, with three transferred to intensive care unit (icu), one death, and two withdrawals, limiting the interpretation of the data. furthermore, the criteria for the selection of patients receiving additional azithromycin were not specified. finally, the study had a very low proportion of patients with lower respiratory tract pathology, and outcomes of the clinical parameters including effects on hemodynamic stability, ventilator parameters, length of icu and hospital stay, and mortality have not been published. nonetheless, early strong indications for the efficacy of this paper prompted another group in france to prospectively study consecutive covid- patients with high comorbidity burden (in out of ) admitted under their care to be treated with the combination regimen of hcq and azithromycin, in doses similar to those used in the marseille study. serious adverse events (one death, two transfers to the icu, and one drug discontinuation due to prolongation of qtc interval) were reported in out of patients, and out of the remaining had persistently positive nasopharyngeal swab pcr after - days of therapy, prompting investigators to conclude that there is no evidence of rapid clearance with this therapy. findings from two additional randomized control studies recently became available for review. in the first one, investigators from wuhan, china (renmin hospital of wuhan university), evaluated the effects of a -day course of hcq ( mg twice-a-day regimen) in addition to a 'standard treatment' comprising oxygen therapy, antibiotics, and immunoglobulin, with or without corticosteroids compared with 'standard treatment' alone. the study utilized clinical measures, such as the return of body temperature and did not find similar benefits with hcq. in this study, patients with confirmed covid- were randomized to either receive conventional treatment only or conventional treatment with the addition of hcq ( mg daily for days). the investigators found that the viral clearance, as judged by the detection of the viral nucleic acid in the pharyngeal swab on day after treatment initiation, was not different for the two groups. they also found that the difference in the median time to achieve afebrile status and in the radiological findings of pneumonia was not different for the two groups. details of the pharmacokinetics and safety profiles for chloroquine/hcq were recently reviewed in this journal and are available. we have more than seven decades of clinical experience with these agents, and overall, the safety of these agents is well established. hcq is the structural analog of the chloroquine molecule with the addition of a β-hydroxyl moiety at one end. although this imparts hcq a comparable clinical efficacy, it has been noticed to do so by providing a better safety profile. in modern medicine, hcq is a more commonly used formulation for most non-malarial indications. both these agents are cheap, safe, and well tolerated by most patient populations, including pregnant women and those with chronic diseases or immunocompromised status. they are administered orally and have a near-complete absorption from the gastrointestinal tract with about % bioavailability. peak drug concentrations are achieved in about - hours after oral administration and are excreted principally through the kidneys, the process facilitated by acidification of the urine. small quantities are also excreted through the bile, sweat, and saliva. they are widely distributed throughout the body, including the lung, and have a large volume of distribution with significant intracellular sequestration, allowing them to have a long functional half-life ( - days) and achieve stable plasma levels usually after - weeks of regular daily dosing. unfortunately, these kinetics have been studied with chronic use of chloroquine/hcq, and its applicability, especially in short courses, concerning alveolar concentration is not known. the most frequent adverse effects related to acute use comprise gastrointestinal intolerance, concerns for acute anemia -especially in patients with pre-existing glucose- phosphate dehydrogenase (g pd) deficiency, and flashing lights as an acute manifestation of retinopathy. while the risk of retinopathy is a major limiting factor for chronic use at higher doses, this is likely to be less of a concern with acute short-term doses recommended for covid- , especially for hcq that has a faster clearance from retinal pigment cells, compared to chloroquine. another concern particularly relevant to the current pandemic is the possibility of myocardial toxicity, qtc interval prolongation, and the possibility of cardiac arrhythmias. several anecdotal reports and cohorts have raised concerns for chloroquine/hcq -induced cardiomyopathy. however, a detailed review of the data suggests that these concerns are significant largely in patients prescribed high doses of these agents. recent reports have also suggested that covid- itself may cause myocardial injury, which in and of itself is associated with the higher incidence of adverse outcomes. it is noteworthy that the risk of adverse outcomes, and hence the need for therapy, is higher in the elderly covid- population with multiple chronic diseases, the exact population at higher chances of having comorbidities or medications causing qtc prolongation. in this regard though, retrospective analyses of the rheumatological cohorts have shown that the incidence of cardiac arrhythmias is lower in patients on therapy with these agents compared to those not on treatment. although reassuring, these mutually contradictory data demand additional caution, as the doses of hcq recommended for the treatment of covid- are higher than those used conventionally for chronic low-dose therapy, and their interplay with direct toxicity of sars-cov- is not known. thus, especially for the high-risk population with significant comorbidity burden, it may be prudent to obtain a routine electrocardiogram prior to initiating hcq therapy. as azithromycin is also known to prolong the qtc interval, patients with prolonged qtc (i.e. ≥ - msec) may be better served by avoiding a combination regimen or with ongoing telemetry monitoring for the occurrence of arrhythmias. the available data taken together show that chloroquine/ hcq appears to have a potential role in the management of the clinical syndrome of the covid- . however, the level of preclinical and clinical evidence is not robust and must be backed by a higher level of data. unfortunately, lack of alternative therapy, high rate of infectiousness and mortality, and the rapidity of spread elevate the nature of public health hazards related to covid- . the pandemic has many health and non-health ramifications, including the global recession. thus, a rising number of regulatory healthcare agencies from across the globe, including china, italy, france, europe, canada, and the usa -list not exhaustive, have included these compounds in their guidelines for treating covid- . at the same time, there are also over studies of varying enrollment targets, and a variety of issn: - review -chloroquine, hydroxychloroquine, and covid- drugsincontext.com clinical, biological, and mortality-related outcome measures registered with the clinicaltrials.gov. prominent among these are two major studies that aim to evaluate the effect of these agents on therapy (who-sponsored pragmatic randomized study; solidarity trial) pending the availability of such confirmatory studies, the use of these agents in covid- should be viewed as experimental at this stage, and it should adhere to local, regional, or national ethics and research guidelines. contributions: ashutosh shukla was involved in the conception, execution, review, and critique of the manuscript; lennox k archibald was involved in the review and critique of the manuscript; aparna wagle shukla was involved in the organization, review, and critique of the manuscript; hiren mehta was involved in the review and critique of the manuscript; kartikeya cherabuddi was 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hydroxychloroquine for connective tissue diseases association of coronavirus disease (covid- ) with myocardial injury and mortality evidence for cardiac safety and antiarrhythmic potential of chloroquine in systemic lupus erythematosus chloroquine in covid- : the evidence who launches global megatrial of the four most promising coronavirus treatments pcori funds registry and large-scale study of effectiveness of hydroxychloroquine to prevent covid- infection in acknowledgements: ashutosh shukla reports the following va merit grant supports from the department of veterans affairs (i cx : key: cord- -m xdzk authors: di castelnuovo, a.; costanzo, s.; cassone, a.; cauda, r.; de gaetano, g.; iacoviello, l. title: low dose hydroxychloroquine is associated with lower mortality in covid- : a meta-analysis of studies and , patients date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: m xdzk background: hydroxychloroquine (hcq) was proposed as potential treatment for covid- , but its association with mortality is not well characterized. we conducted two meta-analyses to evaluate the association between hcq (with or without azithromycin (azm)) and total mortality in covid- patients. methods: articles were retrieved until october th, by searching in seven databases. data were combined using the general variance-based method on relative risk estimates. results: a total of articles were found (n= , covid- patients, including n= , from randomized clinical trials (rcts)); studies were valuable for analysing the association of hcq+azm. overall, the use of hcq was associated with % lower mortality risk (pooled risk ratio: . , %ci: . to . ; high level of heterogeneity: i = %, random effects). this association vanished ( . , %ci: . to . and . , %ci: . to . ) when daily dose > mg or total dose > , mg were used, respectively). hcq+azm was also associated with % lower mortality risk, but uncertainty was large ( %ci: . to . ; p= . ). no association was apparent when only pooling the rcts ( . % of the overall weight; pooled risk ratio: . , %ci: . to . ). conclusions: hcq use was not associated with either increased or decreased mortality in covid- patients when rcts only were evaluated, while a % to % reduced mortality was observed when observational studies were also included. the association was mainly apparent when pooling studies using lower doses of hcq. these findings can help disentangling the debate on hcq use in covid- . the aminoquinoline hydroxychloroquine (hcq) is an anti-malaria drug, with immunomodulatory and anti-thrombotic properties, currently used in the treatment of autoimmune diseases like rheumatoid arthritis, systemic lupus erythematosus and anti-phospholipid syndrome [ , ] . at the beginning of the pandemic, it was proposed as a possible therapy in covid- patients since it could directly inhibit viral entry and spread in several in vitro and in vivo models [ ] . indeed, hcq has been used in ebola virus disease [ ] , human immunodeficiency virus (hiv) infection [ ] , sars-cov- infection and the middle east respiratory syndrome [ ] . despite the lack of evidence of efficacy from few randomized clinical trials, hcq became very popular and widely used by many clinicians. in italy over % of covid- hospitalised patients were treated with hcq [ ] . on the other hand, this drug was given an inexplicable political connotation that shifted the focus more to a political battle than to a discussion based on scientific evidence. the publication of a very questionable study [ ] by one of the most reliable scientific journals showing that the use of hcq was associated to an increased risk of death, lead to the stop of several clinical trials in their tracks and of the hcq arm in the recovery trial [ ] . this lancet study was retracted days after publication [ ] , because its data turned out to be fabricated. anyway, the principal drug agencies decided to suspend the authorization to use hcq for covid- . consequently, the review process of papers on hcq became increasingly difficult, creating a publication bias that affected all meta-analyses published until now. however, a number of questions remain open on the relationship between hcq treatments in covid- patients: is there a dose issue? does mortality rate of a population or the severity of the disease affect hcq efficacy? is there any interaction with other anti-covid drugs? in the last few months, at least three large, well-conducted observational studies have been published showing a protective effect of hcq on mortality risk in hospitalized cobid- patients [ , , ] . all three studies have not been included in previous meta-analysis [ ] , and used hcq doses lower than those administered in randomized clinical trial (rct), as the recovery or the solidarity trials [ , ] . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint therefore, we decided to conduct an updated meta-analysis on observational and rct studies on hcq use and the mortality outcome in patients hospitalized for covid- . we also performed subgroup analyses to dissect whether treatment effects differ according to methodological or clinical characteristics of the primary studies. this study was conducted according to the recommendations outlined in the cochrane handbook for systematic reviews of interventions, version . . , and reported in line with the prisma statement. institutional review board approval was not required as the study did not directly involve human participants. flow diagram for study selection is reported in figure . articles published in english were retrieved from inception to october th , by searching in medline, embase, pubmed, web of science, cochrane central database, medrvix and preprints.org, with the search terms: "(covid- or cov-sars- ) and (hydroxychloroquine or chloroquine)". in addition, the reference lists of relevant articles for potential studies were also manually reviewed. after initial search, the duplicate results were removed. the remaining articles were screened for relevance by their titles and abstracts by two of us independently (sc and adc). all selected potential articles were then reviewed by the remaining investigators to ensure their eligibility for inclusion. disagreements about eligibility of the literature were resolved by consensus based on the agreements of all investigators. to be included in this meta-analysis, the study had to meet the following criteria: ( ) clinical trials or cross-sectional studies or cohort studies; ( ) quantitatively investigating the difference in mortality risk in unselected covid- patients according to use or not of hcq. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint twenty-seven articles were identified [ , , , , . for ten of them [ , , , , , , , [ ] [ ] [ ] it was possible to extract data necessary for comparing hcq+azm versus no hcq+azm. for the other studies, it was not possible to systematically distinguish if hcq therapy was complemented or not with azm. the rct by mitjà et al. [ ] was not included since the authors found zero deaths both in hcq and control group. the study of mehra et al. [ ] was excluded since the authors retracted it owing to several concerns on the veracity of the data [ ] . for each study, odds ratio (or) or hazard ratio (hr) and/or number of events (number of deaths and number of total covid- patients) in both the hcq (or hcq+azm) and respective control groups were extracted. if available, measure of association adjusted for covariates were retrieved. number of events were used to calculate relative risk and % confidence intervals (cis) when other measures of association were not available from the primary study. the following information was also extracted: study design, if the article was not peer-reviewed, region, level of adjustment, sample size, mortality rate in the entire cohort, percentage of patients treated with hcq, mean duration of the treatment, mean daily dose after the first day and mean total dose of hcq used. the total dose of hcq was calculated as the sum of the amount of drug used in the first day plus daily dose multiplied by number of days of treatment after the first. pre-specified subgroup analyses have been conducted for all the additional characteristics retrieved. all analyses were performed using standard statistical procedures provided in revman . (the cochrane collaboration, oxford, united kingdom). data were combined using the general variance-based method, that requires information on the relative risk (or or or hr) estimate and their % ci for each study. % ci were used to assess the variance and the relative weight of each study. heterogeneity was assessed using the higgin's i metric. fixed and random effects were considered, but due to the large heterogeneity observed, findings from random effects were is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint considered as primary analysis. the hypothesis that publication bias might have affected the validity of the estimates was visually tested by a funnel plot-based approach. the workflow of the process of study selection is reported in figure . a total of articles were found in the search. twenty-six of these eligible studies were enrolled for analysing the association with mortality of hcq use in patients with covid- , and of them were valuable for analysing the association of hcq+azm. the main characteristics of the studies included in the meta-analysis are shown in table . we found rct studies [ , , , ] and observational studies; articles were not published in peer reviewed journals; studies reported not adjusted measure of association between hcq and mortality; studies have been conducted in europe, in north america and in other countries. the outcome considered was the mortality for any cause; for the large majority of the studies, the mortality was intra-hospital. in all studies the control group was formed by patients without hcq exposure (hcq or hcq+azm). all studies included adult men and women covid- patients, with the exception of two rct [ , ] , that included a portion of individuals with uncertain positivity to sars-cov- . a total of n= , covid- patients (including n= , from the rcts) were counted in the meta-analysis of hcq, and n= , in the meta-analysis of hcq+azm. forest plot on the association between hcq and mortality is reported in figure . pooling of data from observational studies, which accounted for . % of the total weight, the use of hcq has been associated with % lower mortality risk (pooled risk ratio: . , %ci: . to . ; high level of heterogeneity: i = %, random effects). conversely, the association was not evident in the is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint rcts ( . % of the weight; pooled risk ratio: . , %ci: . to . ; i = %). grand total by pooling of both rcts and observational studies (weight %) pointed to a relative reduction of mortality in support of hcq by % to % (figure ). subgroup analyses according to main features of primary studies are presented in table . the association of hcq with lower mortality was observed with very low differences in all subgroups, with the exception of dose grouping. the reduced mortality was in fact confined to studies that used a daily dose ≤ mg (as estimated in days of treatment after the first, in which a higher (double for most) dose of drug was administered); pooling n= studies which used more than mg of hcq daily resulted in an overall measure of association equal to . ( %ci: . to . ; table and supplementary figure ). also, the magnitude of the association was higher in studies which used hcq for or less days (table and is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint pooled analyses of rcts on the relationship between hcq and incidence of adverse effects are reported in supplementary figure . hcq use was associated with an increased risk of adverse effects of any type (panel a). on the contrary, patients treated with hcq in rcts showed a similar rate of serious adverse events (panel b), as that non-treated with hcq (pooled risk ratio: . , %ci: . to . ; p for testing of overall effect= . ). in a meta-analysis of studies ( rcts) involving , covid- patients, the use of hcq was associated with a % lower risk of total mortality. the association was apparent by pooling observational studies and was more evident in studies which used lower hcq doses. no association was found pooling rcts. use of hcq was not associated with severe adverse events. the potential for selection bias in observational studies is not negligible. the decision from the clinician to utilize or not a drug may depend on comorbidities and baseline risk of the patient. however, in the pandemic, and in absence of guidelines and specific anti covid- drugs, allocation of hcq in observational studies was not associated systematically with a lower or higher baseline risk profile. for example, in the corist study [ ] patients receiving hcq were more likely younger and less likely had ischemic heart disease, cancer or chronic kidney disease, but, on the contrary, they were more likely men and had higher levels of c-reactive protein. as a consequence, it is not clear if in that particular study hcq patients were potentially at higher or lower risk of a negative prognosis. in attempting to account for baseline differences between patients who received hcq and those who did not, we used the results for adjusted measure of association for each study, and this was possible for out of studies included in the metaanalysis. after the exclusion of unadjusted studies [ , , ] , the strength of the overall association of hcq with mortality was merely reduced from . to . . although we attempted . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint to control for potential confounding factors inherent to patient and clinical characteristics, it is possible that unmeasurable confounding still remains, and this may explain the different finding between observational and rct studies. however, it is hard to determine which are, if any, the unmeasured characteristics that have confused so strongly the association between hcq and mortality in covid- that was observed in observational studies. in fact, these features must be a) unmeasured in observational studies; b) associated with mortality in covid- and c) associated with hcq use, in a way that when the risky conditions are present the clinicians tend systematically to avoid using hcq. for example, hcq is contraindicated in patients with cardiomyopathy but this condition has been mostly measured in observational studies and was not recognised as a risk factor for mortality in covid- patients. the dissimilar findings between observational and rcts we found might also be explained by differences in hcq dosage [ ] . interestingly, we observed that the reduced mortality associated with hcq treatment is actually confined to studies that used a daily dose ≤ mg, or a total dose ≤ , mg or which used hcq for or less days. obviously these three conditions largely overlapped in studies, that we can now designate as "at low hcq dose". remarkably, none of the rcts are in this category. in detail, the recovery [ ] and the solidarity study [ ] used mg/day for or days (after the first), respectively and a total dose of or mg of hcq (including the dose at first day) respectively, a very high dose regimen as confronted to the rest of studies, particularly of the observational ones. the possibility that hcq reduced the risk of negative prognosis in covid- patients when only administered at "low dose" cannot be here undoubtedly proven starting from our findings, but it is a plausible hypothesis that may explain the different result between observational and rct studies and, more importantly, might be useful in disentangling the debate on hcq use in covid- . high levels of hcq administration were used in rcts to maximise the antiviral activity of the drug that was considered to be the main mechanism of action of hcq in this context. in some studies, the is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint inverse association of hcq with inpatient mortality was more evident in elderly, in patients who experienced a higher degree of covid- severity or having elevated c-reactive protein levels [ ] , suggesting that the anti-inflammatory potential of hcq may have had a more important role than its antiviral properties. hcq, indeed, beside an antiviral activity, may have both anti-inflammatory and anti-thrombotic effects [ ] . this can justify its effect in reducing mortality risk, since sars-cov- can induce pulmonary microthrombi and coagulopathy, that are a possible cause of its severity [ , ] and the lack in preventing sars-cov- infection after exposure [ ] . on the other end, national guidelines suggested to use hcq mg twice daily for - days probably to maintain a better risk benefit profile hypothesizing that low doses could be more effective and safer. indeed, non-sigmoidal, bell-shaped dose-response curves are possible with drugs having complex biological effects, multiple-binding sites or cellular and organ targets. on the other hand, anti sars- -cov- activity of hcq has been confirmed in vero cells [ ] . hcq is also reported to reduce secretion of ifn-γ and il- in activated th and th cells, respectively [ ] . the concomitant use of azithromycin seems to not neither increase nor decrease the effect, if any, of the hcq since the combination of the two drugs was associated with a lower mortality risk at very similar extent to that observed for hcq alone, but the assumption is inconclusive because of the very large uncertainty in the findings. a main concern with hcq treatment have been its side effects, in particular a severe cardiovascular toxicity. indeed, hcq can cause prolongation of the qt interval on electrocardiogram [ ] , which could be exacerbated by coadministration with azithromycin, widely prescribed as co-treatment in covid- treatment. our meta-analysis of data from rcts, that allowed a proper evaluation of side effects, shows that use of hcq was associated with an increase in side effects of any type, but not of major type, including cardiovascular events. this despite the high prevalence of cardiovascular disease in patients with covid- or the high dose used in rtcs. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint this meta-analysis has the strength of including all available data recently published and that have not been included in previous meta-analyses [ , ] , and of considering modification of effect by dosing of hcq. as major limitations, we recognised that majority of the primary studies were observational, the pooled findings suffer of a high degree of heterogeneity and that results in observational and rct studies were different. in conclusion, hcq was not associated with increased or decreased mortality in covid- patients when only rcts were pooled, but it was associated with % to % reduced mortality when observational studies were also included. the association was mainly apparent by pooling studies using lower doses of hcq. use of hcq was not associated with severe adverse events. these results should be considered with caution, because the majority of the studies included were observational and retrospective and the possibility of confounding could not be fully excluded. however, at present, this is the largest comprehensive quantitative overview on the association of hcq with mortality in covid- patients, and our findings underscoring hcq dosage effects can help disentangling the debate on hcq use and encourage the planning of rcts using low doses of hcq in hospitalised covid- patients. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint adc and li contributed to the conception and design of the work and interpretation of data; sc and adc managed study selection and data extraction and critically reviewed the results; sc analysed the data; adc and li wrote the paper; li, ac, rc and gdg originally inspired the research and critically reviewed the manuscript. all authors approved the final version of the manuscript. the authors report no conflict of interest related to the current work. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint forest plot for association of hydroxychloroquine use with covid- mortality (random effects), according to daily dose (as estimated in the days of treatment following the first) . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint forest plot for association of hydroxychloroquine use with covid- mortality (random effects), according to duration of treatment . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint forest plot for association of hydroxychloroquine use with covid- mortality (random effects), according to total dose (calculated as the sum of the amount of drug used in the first day plus daily dose multiplied by number of days of treatment following the first) . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint forest plot for association between hcq treatment (panel a) or hcq+azm (panel b) and mortality in covid- patients rct mean randomized controlled trial; rr means relative risk; se means standard error; hcq means hydroxychloroquine; azm means azithromycin supplementary figure forest plot for association of hydroxychloroquine use with any (panel a) or major (panel b) adverse effects. data are from randomised clinical trials. the study by mitja et al. (ref=meta esclusi ) was not included in the meta-analysis on mortality since the authors found no deaths both in hcq and control group. . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint expanding horizons for clinical applications of chloroquine, hydroxychloroquine, and related structural analogues effects of chloroquine on viral infections: an old drug against today's diseases? the possible mechanisms of action of -aminoquinolines (chloroquine/hydroxychloroquine) against sars-cov- infection (covid- ): a role for iron homeostasis? chloroquine inhibited ebola virus replication in vitro but failed to protect against infection and disease in the in vivo guinea pig model chloroquine and beyond: exploring anti-rheumatic drugs to reduce immune hyperactivation in hiv/aids hydroxychloroquine or chloroquine with or without a macrolide for treatment of covid- : a multinational registry analysis repurposed antiviral drugs for covid- ; interim who solidarity trial results retraction-hydroxychloroquine or chloroquine with or without a macrolide for treatment of covid- : a multinational registry analysis treatment with hydroxychloroquine, azithromycin, and combination in patients hospitalized with covid- low-dose hydroxychloroquine therapy and mortality in hospitalised patients with covid- : a nationwide observational study of participants effect of hydroxychloroquine with or without azithromycin on the mortality of coronavirus disease (covid- ) patients: a systematic review and meta-analysis epub ahead of print effect of hydroxychloroquine in hospitalized patients with covid- impact of azithromycin and/or hydroxychloroquine on hospital mortality in covid- the association of treatment with hydroxychloroquine and hospital mortality in covid- patients hydroxychloroquine with or without azithromycin in mild-to-moderate covid- observational study of hydroxychloroquine in hospitalized patients with covid- hydroxychloroquine and tocilizumab therapy in covid- patients-an observational study hydroxychloroquine use in hospitalised patients with covid- : an observational matched cohort study hydroxychloroquine for early treatment of adults with mild covid- : a randomized-controlled trial compassionate use of hydroxychloroquine in clinical practice for patients with mild to severe covid- in a french university hospital epub ahead of print association of treatment with hydroxychloroquine or azithromycin with in-hospital mortality in patients with covid- in new york state hydroxychloroquine with or without azithromycin and in-hospital mortality or discharge in patients hospitalized for covid- infection: a cohort study of , in-patients in france. medrxiv . . outcomes of hydroxychloroquine treatment among hospitalized covid- patients in the united states-real-world evidence from a federated electronic medical record network hydroxychloroquine in nonhospitalized adults with early covid- : a randomized trial low dose of hydroxychloroquine reduces fatality of critically ill patients with covid- . sci china life sci dose optimization of hydroxychloroquine for coronavirus infection : do blood concentrations matter? the role of cytokines including interleukin- in covid- induced pneumonia and macrophage activation syndrome-like disease the versatile heparin in covid- a randomized trial of hydroxychloroquine as postexposure prophylaxis for covid- hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro chloroquine inhibits human cd (+) t-cell activation by ap- signaling modulation acknowledgments: sc was the recipient of a fondazione umberto veronesi travel grant. key: cord- - qpk x authors: elsawah, hozaifa khalil; elsokary, mohamed ahmed; elrazzaz, mahmoud gamal; elshafey, ahmed hane title: hydroxychloroquine for treatment of non‐severe covid‐ patients; systematic review and meta‐analysis of controlled clinical trials date: - - journal: j med virol doi: . /jmv. sha: doc_id: cord_uid: qpk x background: being a pandemic and having a high global case fatality rate directed us to assess the evidence strength of hydroxychloroquine efficacy in treating covid‐ arising from clinical trials and to update the practice with the most reliable clinical evidence. methods: a comprehensive search was started in june up to july‐ , in many databases, including pubmed, embase and others. of studies found, only six studies fulfilled the inclusion criteria which includes: clinical trials, age> years with non‐severe covid‐ , pcr‐confirmed covid‐ , hydroxychloroquine is the intervention beyond the usual care. data extraction and bias risk assessment were done by two independent authors. both fixed‐effect and random‐effect models were utilized for pooling data using risk difference as a summary measure. the primary outcomes are clinical and radiological covid‐ progression, sars‐cov‐ clearance in the pharyngeal swab, and mortality. the secondary outcomes are the adverse effects of hydroxychloroquine. results: among covid‐ confirmed patients obtained from pooling studies, patients received hydroxychloroquine and patients served as a control. hydroxychloroquine significantly prevent early radiological progression relative to control with risk difference and % confidence interval of ‐ . (‐ . to ‐ . ). on the other hand, hydroxychloroquine did not prevent clinical covid‐ progression, reduce ‐days mortality, or enhance viral clearance on days , , . moreover, many adverse effects were reported with hydroxychloroquine therapy. conclusions: failure of hydroxychloroquine to show viral clearance or clinical benefits with additional adverse effects outweigh its protective effect from radiological progression in non‐severe covid‐ patients. benefit‐risk balance should guide hydroxychloroquine use in covid‐ . this article is protected by copyright. all rights reserved. authorization was also issued for hcq in march and revoked in june due to safety and efficacy concerns . hcq is a weak base -aminoquinoline, developed in as an antimalarial agent, which is a safer derivative than chloroquine . the antiviral activity of hcq against viral diseases such as hiv and severe acute respiratory syndrome studied many years ago . it also showed in vitro activity against sars cov- by inhibiting viral entry through targeting early endosomes and endolysosomes . moreover, hcq could modulate the immune response and reduce proinflammatory cytokines which are important inducers of acute respiratory distress syndrome . few retrospective observational studies reported some benefits in treating covid- patients, where hcq decreased mortality and il- level , decreased case fatality rate , and improved patient survival . on the other hand, other observational studies reported no benefits and more frequent side effects while using hcq [ ] [ ] [ ] . the same controversies are found in the randomized clinical trials (rct) that investigated hcq efficacy in covid- . additionally, hcq is one of the most widely used agents for treating covid- infection despite insufficient supporting evidence. a few numbers of meta-analyses investigating this subject were conducted. however, they were criticized for some flaws addressed and discussed afterward. therefore, there is an urgency to conduct a systematic review and meta-analysis including all available clinical trials that meet the prespecified inclusion criteria. the objectives are to summarize efficacy of hcq use in covid- relative to control based on available clinical trials indicated by all possible improvements of the disease this article is protected by copyright. all rights reserved. and to pool all short-term possible side effects related to hcq therapy in covid- patients. the preferred reporting items for systematic reviews and meta-analyses (prisma) statement were followed to improve reporting the present systematic review . the protocol was registered in the international prospective reregister of systematic review (prospero) with registration number crd in june . the inclusion criteria for the eligible studies for systematic review and metaanalysis include: -clinical trials either randomized or not -covid- patients > years -infected with sars cov- and had a pcr confirmation test; the test should be based on nasopharyngeal or oropharyngeal swab. -non-severe infection (mild and moderate) based on clinical assessment by each study. -the treatment arm is hcq ± usual treatment that was given according to each hospital and was not proven to be anti-covid- . this article is protected by copyright. all rights reserved. -control group is only on the usual treatment -outcomes: any clinical outcomes or drug-related side effects during the follow-up period. the following databases were used for studies identification: pubmed, embase, cochrane, google scholar, clinicaltrial.gov, proquest, science direct, chinese clinical trial registry (chictr) and medrxiv. the search started in june and continued through july to track all new studies. the advanced search was used in different databases with limitation to clinical trials and fields of title and abstract without other limitations. the synonyms applied in search terms were sars or covid and hydroxychloroquine or hcq or plaquenil. three researchers independently underwent comprehensive searching and identified certain studies after removing duplicated ones. according to the prisma flow diagram (figure ) , the selection of eligible studies for meta-analysis from identified ones was conducted by two researchers through three steps; abstracts screening for relevant studies, full-text articles assessment for eligibility, and effect measures assessment for quantitative synthesis. disagreements were resolved by discussion among the authors. this article is protected by copyright. all rights reserved. . . data collection process: "data collection form for intervention reviews: rcts and non-rcts" developed by cochrane was used for data extraction . numbers were extracted directly from text and tables and indirectly from graphs using getdata graph digitizer version . . . . data extraction was done by three independent authors. there are three types of variables; ) independent variable is hcq therapy, ) dependent variables include viral clearance in the pharyngeal swab, clinical progression (increase in the baseline severity), radiological progression, adverse effects, and mortality, ) confounders include usual treatment that varied among studies, age, sex, disease onset, and different hcq doses. the cochrane risk of bias tools to assess the bias risk were followed . it includes domains: selection bias, reporting bias, performance bias, detection bias, attrition bias, and other sources of bias. the consolidated standards of reporting trials (consort) were also utilized . the risk of bias was assessed by three authors and a final consensus was done. publication bias couldn't be assessed because of the low number of the included studies. sensitivity analysis was performed after removing the low-quality studies. the principal summary measures were risk difference (rd) for the outcomes, odds ratio (or) for gender, and mean difference (md) for age with % confidence this article is protected by copyright. all rights reserved. interval (ci) to compare between hcq arm and control arm using revman version . . statistical heterogeneity was tested using the q statistic and quantified with i value. each of fixed-effect and random effect models was used to pool the effect sizes according to the heterogeneity of each outcome . mantel hazel method and inverse variance method were used for dichotomous data and continuous data respectively. all-time point meta-analysis was used to summarize the result of viral clearance at each available time point. prisma flow diagram in figure ( ) shows that studies were identified after the removal of duplications, studies were removed after screening titles and abstracts based on their relevance, and studies were removed after assessment of full article for eligibility based on the inclusion criteria. the remaining studies were included in the systematic review and meta-analysis. some studies were excluded because of their retrospective design , did not recruit pcr-confirmed cases , , or recruited less than years-old patients . the population of the included studies had non-severe covid- except severe patients in tang w. et al study . the disease severity definition slightly varied by the studies. it was based on chinese guidelines in three studies [ ] [ ] [ ] [ ] in barbosa j. et al study . the disease onset before hcq treatment varied from and days in three studies [ ] [ ] [ ] to days in another study . hcq regimens varied among the studies; only three studies used loading doses of and mg/d , , . maintenance daily doses of , , , mg were also used according to each study (table ) . usual treatment was given to all patients according to needs and varied widely among the studies. it included: supportive care, symptomatic treatment, steroids, antibiotics, and antivirals - , , (table ). the two groups were comparable in all mentioned baseline factors in each included study. the risk of bias is summarized in figure ( ) . the study design of barbosa j. et al was included as it emulated clinical trial design and the recall bias was unlikely heterogeneity was not significant (χ = . , p = . ). (figure ) -twenty-eight-days mortality was not statistically significant between the two groups. rd pooled from two studies , using fixed-effect model was . (- . to . ). heterogeneity was not significant (χ = . , p = ). (figure after excluding the two low-quality studies , , the same analyses on the applicable outcomes were performed. no difference was observed between the two analyses on the progression, mortality and viral clearance. hcq therapy in covid is still a matter of debate among healthcare providers . it was introduced early in the pandemic based on early studies this article is protected by copyright. all rights reserved. observational and interventional studies raised concerns about the safety of the drug and even prematurely terminated due to serious cardiac side effects , - . sensitive indicators for a possible efficacy of anti-covid- drugs should rely on the improvement in the disease clinical course and modification in possible causes of the related mortality. radiological abnormalities of the lung could be a good measure of the drug efficacy. lung abnormalities on chest ct in covid- patients changed gradually from ground-glass opacities on the first days to an increase in the crazypaving pattern after one week, then became consolidated on day and started to resolve after weeks of the disease course . ability of a drug to prevent disease progression from mild/moderate to severe has been targeted as a reliable efficacy measure . accordingly, it could inhibit the pathophysiological pathways of the virus. the disease severity was defined by who as spo < % on room air, including those who require any form of supplemental oxygen . viral clearance is of clinical importance as it correlated with the clinical and biochemical outcomes , but may underestimate the immunomodulators effect including hcq , . on the other hand, low rates of mortality were reported among non-severe covid- , large number is required to get enough power to show a significant difference . mortality may not be a sensitive indicator among those with non-severe covid- . the present meta-analysis targeted non-severe covid- patients to assess the efficacy and safety of hcq based on the available evidence. in addition, the minimal age of the inclusion criteria was expanded to years to add more studies. hcq was used in the recovery trial for infants more than months without concerns , but it could not be included in this review as it also had no pcr-based confirmation test. this article is protected by copyright. all rights reserved. the present study offers moderate-quality evidence built on five clinical trials and one quasi-trial. the meta-analysis investigated five measurable objective outcomes, two of them showed statistical significance; chest ct progression and incidence of some adverse drug effects. however, clinical progression, viral clearance at three time points, and -days mortality did not differ between the two groups. all time points meta-analysis to summarize the effect size on , , and -days was performed to get more accurate results . the chest ct-based disease assessment was performed on days and to evaluate the disease progression in chen z. et al study . it depended on pneumonia absorption on ct and weather it was absorbed by more or less %, it also depended on pneumonia absorption on ct in chen j. et al study . the clinical progression definition was consistent in three studies [ ] [ ] [ ] large rct with sufficient power is required with longer follow-up period, it should report more sensitive outcomes stratified by the disease severity and based on the proposed mechanisms of action of hcq to improve the clinical course of covid- . a lot of limitations faced the investigators due to conflicts between the included trials, high level of heterogeneity which is present among some studies methodologies and outcomes such as covid- severity definitions and the background treatment. the this article is protected by copyright. all rights reserved. low number of studies with relatively small sample size and low quality is also another challenge. there are no tangible beneficial effects of adding hcq to the treatment of patients suffering from non-severe pcr confirmed covid- infection. reducing the chest ct progression by hcq was neither sufficient to reduce the early mortality nor promote the early clinical progression more than the usual therapy used. its use was accompanied with a significant incidence of adverse effects without any effect on viral clearance. funding: none the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- positive rate of rt-pcr detection of sars-cov- infection in cases from one hospital in who declares covid- a pandemic coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. microbiology and molecular biology reviews epidemiology, genetic recombination, and pathogenesis of coronaviruses global comparison of influenza type a and b with covid- : a systematic review and meta-analysis on clinical, laboratory, and radiographic findings. laboratory, and radiographic findings case-fatality rate and 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covid- : a systematic review and meta-analysis hydroxychloroquine versus covid- : a periodic systematic review and meta-analysis key: cord- - ydn bvy authors: kumar, neeraj; awasthi, amardeep; kumari, anchala; sood, damini; jain, pallavi; singh, taru; sharma, neera; grover, abhinav; chandra, ramesh title: antitussive noscapine and antiviral drug conjugates as arsenal against covid- : a comprehensive chemoinformatics analysis date: - - journal: journal of biomolecular structure & dynamics doi: . / . . sha: doc_id: cord_uid: ydn bvy coronavirus pandemic has caused a vast number of deaths worldwide. thus creating an urgent need to develop effective counteragents against novel coronavirus disease (covid- ). many antiviral drugs have been repurposed for treatment but implicated minimal recovery, which further advanced the need for clearer insights and innovation to derive effective therapeutics. strategically, noscapine, an approved antitussive drug with positive effects on lung linings may show favorable outcomes synergistically with antiviral drugs in trials. hence, we have theoretically examined the combinatorial drug therapy by culminating the existing experimental results with in silico analyses. we employed the antitussive noscapine in conjugation with antiviral drugs (chloroquine, umifenovir, hydroxychloroquine, favlplravir and galidesivir). we found that noscapine-hydroxychloroquine (nos-hcq) conjugate has strong binding affinity for the main protease (mpro) of sars-cov- , which performs key biological function in virus infection and progression. nos-hcq was analyzed through molecular dynamics simulation. the md simulation for ns affirmed the stable binding of conjugation unprecedentedly through rmsd and radius of gyration plots along with critical reaction coordinate binding free energy profile. also, dynamical residue cross-correlation map with principal component analysis depicted the stable binding of nos-hcq conjugate to mpro domains with optimal secondary structure statistics of complex dynamics. also, we reveal the drugs with stable binding to major domains of mpro can significantly improve the work profile of reaction coordinates, drug accession and inhibitory regulation of mpro. the designed combinatorial therapy paves way for further prioritized in vitro and in vivo investigations for drug with robust binding against mpro of sars-cov- . the recent outbreak of the novel severe acute respiratory syndrome coronavirus- (sars-cov- ) is unprecedented. the world health organization (who) has given the guidelines that the current pandemic might go long, and the virus may become endemic in our society. as per the statistics of who, seven million people (as of th june ) have already been infected with coronavirus disease- and expected to increase exponentially in some of the regions (who, ) . the coronavirus is a single-stranded rna virus that typically affects the vertebrates and causes lethal effects (fung & liu, ) . the transmission of the sars-cov- virus between different host species is generally through respiratory droplets, in aerosol form, and fomites . due to the genetic proximity of this novel betacoronavirus, it is similar to the previous outbreaks of severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) (zhu et al., ) . the diverse spike protein of sars-cov- makes it more lethal than previous sars and mers family viruses. among the various routes for its treatment/ prevention by the genetic code information of coronavirus, reported by the chinese research group in march (ren et al., ) provided an early opportunity for scientists across the globe to develop the vaccine for the covid- disease at earliest. however, it is too early to assume success for the covid- vaccine because the development of vaccination is tricky; it may take time to manufacture an effective and safe vaccine on a large scale. alternatively, to the current situation, drug repurposing seems to be an effective drug discovery strategy by utilizing the existing drugs and natural plant products for the treatment of the sars-cov- outbreak. drug repurposing strategy can potentially shorten the time and as well as reduce the cost compared to develop novel drug discovery and their clinical trials (cheng, ; cheng et al., cheng et al., , , . overall, the world research community is repurposing known fda approved drugs and improving them by conjugation or synthesizing potential analogs. remdesivir (beigel et al., ) , hydroxychloroquine (colson et al., ) , chloroquine , favipiravir (t- ) (coomes & haghbayan, ) , and galidesivir (bcx ) (li & de clercq, ) are some of the most successful repurposed small molecule drugs which has shown broad-spectrum activity against coronavirus. these repurposed drugs had initially shown good results; however, there were some major limitations as well, likewise potency, effectiveness, specificity to viral targets, and in some cases, side effects due to non-specificity, etc. interestingly, combination therapy of approved drugs suggested for being an effective strategy to treat corona infection. recently, hydroxychloroquine (hcq), a well-known drug for malaria, has been repurposed, which brought attention to potentially helping patients to recover from covid- . also, it has been used in the combination therapy and reaches to the trials such as; hydroxychloroquine with mivermectin (patr ı & fabbrocini, ), hydroxychloroquine with azithromycin (magagnoli et al., ) , hydroxychloroquine with nitazoxanide (amawi et al., ) , etc. however, these have not shown expected outcomes and associated with issues like high mortality rates and increased frequency of irregular heartbeat. as per who reports, these hcq based combination therapies took major setbacks after not performing significantly in clinical trials. hence in present work, we propose a drug conjugate of noscapine along with major antiviral drugs in repurposing trials (chloroquine, hydroxychloroquine, umifenovir, favlplravir, and galidesivir) . noscapine is an approved antitussive drug for the treatment of cough through simulating the lung linings. it is known to inhibit bradykinin enhanced cough response (ebrahimi et al., ) . we are aware of the fact that the primary target for sars-cov is the lungs, and noscapine has been widely used to inhibit bradykinin, eventually inhibiting inflammation and lung damage (ebrahimi, ) . hence it makes it sense to conjugate noscapine with antiviral drugs, which can decrease the severe symptoms due to the lung damage; this can overcome the setbacks of previous combinations therapies, including hcq conjugations, by attenuating the high mortality rates due to covid- . noscapine is known to obtain from the naturally occurring opium poppy (papaver sominiferum) medicinal plant, and it is characteristically a non-toxic phthalide-isoquinoline alkaloid (aneja et al., ) . we aimed to design the combinatorial therapy against the main protease (mpro or clpro) enzyme, a key regulator of the life cycle of the coronavirus. mpro is essential for the replication of the virus as it processes the polyproteins that are translated from the viral rna to yield functional viral proteins (chen et al., ) . this makes the mpro enzyme a potential drug target to combat coronaviruses. this fact has led to some of the research groups to develop a potential inhibitor to the protease (mengist et al., ; zhang et al., ) , . these inhibitors prevent the replication of the virus after entering into the host cells. mpro protease inhibitors are reported to modify the polypeptides essential for sars-cov- infection and progression in the host. also, our group has recently reported the potent interaction of noscapine analogs and subsequent inhibition of the mpro protease of the sars-cov- (kumar, kumari, et al., ) . with detailed literature, we designed and analyzed the noscapine conjugates with antiviral drugs (chloroquine, hydroxychloroquine, favipiravir, and galidesivir that are currently being clinically tested (table ). the molecular binding affinity of conjugates to inhibit the mpro has been evaluated using high throughput computational assays. the molecular dynamics studies have been widely used in modern drug discovery and development. computer-aided drug design has been employed to annotate the molecular binding mechanism of drugs to their specific targets (singh et al., ; , . in many studies, advanced molecular docking approach has been harnessed to screen an extensive library, define the binding affinity of drugs and to elucidate the involved molecular interactions in the complexation of the ligand with receptor protein chaudhary et al., ) , . molecular dynamics (md) analysis helps to insight into the binding mechanism of the drugs through the root-mean-square deviation (rmsd), radius of gyration binding energy calculations, and relevant calculations (kumar, sood, sharma, et al., ; kumar, sood, tomar et al., ; singh et al., ; ., . with employing these advanced computational approaches, deigned noscapine conjugations with antiviral drugs were examined to derive the highly efficient combination against the mpro of sars-cov- for further validation with experimental lab assays. . . drug target main protease (mpro) of sars-cov- retrieval and structural assessments the main protease regulatory enzyme of sars-cov- has been reported to play an essential role in coronavirus infection and progression in the host (xue et al., ) . to design the drug therapy against mpro, a three-dimensional crystal structure of mpro was retrieved from the protein data bank (pdb id lu we have designed the noscapine based antiviral cognates with antiviral drugs in trials (chloroquine, umifenovir, hydroxychloroquine, favlplravir, and galidesivir) . noscapine is reported possess the appeased effects on lung and respiratory infection with its antitussive properties (kim et al., ) . noscapine conjugates were drawn using the chemdraw software and assessed for their compatibility and deformation. the molecular interaction analyses of all conjugates were performed with the mpro to illustrate their binding affinity. to perform the docking calculations, the target mpro was prepared and optimized by the whatif server (vriend, ) . mpro was confined to add missing hydrogen atom, amino acid chain, any other unwanted chain and removing the water molecules. similarly, ligand files of noscapine-antiviral conjugates were prepared and optimized for any missing atom and chain. the molecular docking was performed using the hex . fast fourier algorithm-based module (macindoe et al., ) . by incorporating the binding sites, grid box in all axes to mpro was formed and ligand files were inserted to the server. the receptor grid was set to . Å for x, y, and z coordinates of mpro structure. the translational step, twist range of and protein flip ranges were set as per the molecular docking manual. moreover, to strengthen our results, we redocked the high-rank noscapine conjugate with another molecular docking tool with the swissdock server. swissdock works on the basis of the eadock dss algorithm with curated scripts of receptors and ligands (grosdidier et al., ) . from the obtained various docked conformation of mpro-conjugates, the model with the lowest energy was analyzed for the binding mechanism by protein-ligand profiles (laskowski & swindells, ; salentin et al., ) , . the model with the lowest docked energy score in all drug conjugates was selected for molecular dynamics simulation analysis. to mechanistic insights into the binding of the conjugate (nos-hcq), the molecular dynamics simulation analysis was performed. md simulations were implemented through gromacs v . under the force field gromos a having water model spc along with the time step of fs for ns . we investigated the atomic motions, conformational changes, structural stability, and binding energy contributing to the binding of the ligand to the target molecule. in an explicit water solvent, internal atomic motion simulation was initiated. the binding complex of nos-hcq conjugate was solvated in the octahedron system and stretched to Å in all directions. the complex operation was minimized through four steps, and all atoms were fixed. in the minimization process steepest descent steps were executed, and all atoms were relaxed at k with one atmospheric pressure in boundary states. npt ensembles, along with periodic boundary conditions, were utilized to carry out md simulations. a cut-off of about Å was used in order to manage the vander waals forces. the particle mesh ewald model having a cut-off of Å was further utilized to calculate the electrostatic interactions. the obtained trajectory for ns md simulation run was analyzed for root mean square deviation (rmsd), root mean square fluctuations (rmsf), hydrogen bond analyses, solvent accessible surface area analysis (sasa) and radius of gyration (rg) plot analysis (grant et al., ) . also, the native contacts, along with the principal component analysis (pca), cross-correlation map analyses were performed throughout the simulation run (mcgibbon et al., ) . moreover, the binding energy calculation lead conjugate with mpro was investigated through the reaction coordinates analysis. it determined the ligand compatibility with threshold energy and the progression of ligand for stabilized binding with the target receptor. a total of frames of interactions complex trajectory was analyzed for binding energy computation. the equation employed for the calculation is dgcomplex, dgreceptor, dgligand represents the binding energy of complex, binding energy receptor, binding energy of ligand. . . sars cov- mpro enzyme as a potential target sars coronavirus is an enveloped positive-stranded rna virus, which majorly affects the respiratory system and enteric system (graham et al., ) . sars-cov- infection and progression in the host are regulated by multiple structural proteins. among various structural proteins, the main protease enzyme of coronavirus is reported to play significant roles in viral replication through proteolytic machinery and involved in transcription, translation, and amplification of viral proteins (paules et al., ) . mpro enzyme is of size amino acids and possesses high similarity with protease enzyme from different human and animal by sequence analysis. the protein annotation results depicted mpro has various roles in coronavirus viz; a nucleic acid-binding domain of sars coronavirus, polyprotein cleavage domain, coronavirus endopeptidase, corona nsp , nsp and nsp replication implication regulatory roles, reported through pfam annotation database. moreover, d structural analysis of mpro by the cath server showed it consisted of beta-barrel and alpha domain (figure ). the secondary structural analysis by the dssp server showed it comprised of % alpha-helices ( helices, residues) and % beta-barrel sheets ( strands). these significant analyses along with the literature survey, suggested the mpro enzyme as a potential target to design the covid- therapeutic drugs. moreover, prior to molecular binding analyses the d structure of the mpro enzyme was assessed for structural conformations and physicochemical by ramachandran plot and saves server. the ramachandran plot analyses depicted . % residues are in the favorable region, and . % residues and . % residues are in the outlier region of the plot. the local quality assessment showed good structural quality factors with high homology ( . to . Å) with the native structures in database of , known protein structures. with noscapine significant roles in lung linings and dilation and coronavirus movements into the lung cells evokes the cytokine storm and leads to neutrophilic infiltration and other implication (martin & ernst, ) . hence, it was essential to investigate the effectiveness of antitussive noscapine in conjugation with antiviral drugs against mpro. we chose and designed the noscapine conjugation with potential antiviral drugs (chloroquine, umifenovir, hydroxychloroquine, favlplravir and galidesivir) using the chemdraw and simultaneously assessed their applicability and conformations. for molecular docking, both receptor mpro and ligand files of noscapine conjugates were prepared for molecular docking using the whatif prepdock module and ligprep server. for performing the molecular docking, binding site of mpro enzyme-n inhibitor crystal structure, recently submitted to the protein data bank, was analyzed for binding sites and insights into the molecular mechanism of inhibition by drugs . similar binding sites were employed for performing the molecular docking of the noscapine conjugations with the target mpro of coronavirus using the hex . and swissdock servers. the molecular docking was performed using hex . , which works based on a fast fourier transform by spf shape-density correlations, and results were recalculated with electrostatic association additionally. first, we have performed the molecular docking analysis of drug alones which depicted the high binding score for hydroxychloroquine with À . kj/mol, among all drugs. further we assessed their drug likeliness properties of all drugs, none of them violated lipinski rule of five except galidesivir which has six hydrogen donor groups (table ) . taking these results altogether, we performed the docking for all conjugates. interestingly, obtained results showed the strong binding of noscapine conjugates with mpro of coronavirus. the nos-hcq conjugate depicted the most substantial binding with an energy score of À . kj/mol, among all conjugates. besides, to assess the effectivity of noscapine (singlet state) against the mpro, it was also docked using the same grid, which showed the binding score of À . kj/mol, larger than the known molecule n molecule bound to mpro with binding score of À . kj/mol. it can be seen from the molecular binding analyses that hydroxychloroquine depicted the high binding score in drug alone analyses, whose binding efficiency was enhanced by conjugation of noscapine by about double with a docking score of À . kj/mol to mpro (table ) . moreover, lead conjugation of nos-hcq does not found to follow the drug likeliness lipinski rule of five for high molecular weight and hydrogen bond acceptor groups with combinations of two drugs. in addition, to strengthen our results, we redocked the all conjugates with mpro using the swissdock module. the outcomes affirmed the highest efficacy of nos-hcq conjugate with binding dg energy score of À . kcal/mol and combined full fitness score of À . kcal/mol, among all noscapine conjugates (figure ). with these significant results, it can be attributed that nos-hcq conjugate has a high potential to bind the target mpro enzyme and further can be used as effective therapeutics for sars-cov- . the detailed characterization of contacts of noscapine conjugates is vital to recognize the interaction pattern of ligands for stringent binding to target mpro. the protein-ligand profiler was employed to determine the non-covalent contacts of complex systems. the mpro structural domain analysis by the cath server showed it forms majorly three ligand-binding domains and one additional domain. the globular structure analysis by chimera modeling application suggested the plausibly first domain from ser -pro , second binding remained confined to domain- and domain- ( figure ). from these contact analyses of all conjugates, it can be attributed that nos-hcq conjugate has high affinity and wide molecular contacts for major domains of mpro, among all conjugates. moreover, it was further studied through molecular dynamics to get insight into inhibitory patterns and efficacy to combat the mpro enzyme of coronavirus. the binding affinity of the nos-hcq conjugate was investigated by the molecular dynamics simulation analyses for ns. the stable binding of nos-hcq along with protein dynamics in the target mpro was assessed through the gromos a force field with water model spc consisting time step of fs. the obtained trajectory was solvated in the octahedron box in an explicit water state to provide sustainable conformations. the system minimization was done for steps by the steepest descent program and further steps by conjugated gradient method through inserted force fields. the root means square deviation (rmsd), root means square fluctuation (rmsf), and radius of gyration (rg) were computed for the resulting trajectory to assess the stable binding of lead combination (nos-hcq) with mpro of coronavirus. moreover, to compare and determine the boosting power and synergetic contribution, md simulation was also performed for the singlet state of mpro. the steep rmsd variation can be considered an implication of a malleable and instinctive protein. the rmsd (average backbone range of complex system) for the frames of native contacts with target mpro depicted the stable binding of lead nos-hcq conjugate to mpro with fluctuation in the range of . - . nm and for mpro singlet state fluctuation in the range of . - . nm ( figure ) . interestingly, rmsd of the complex system was more stabilized than the native form of target receptor mpro. moreover, the secondary structure analysis showed the compelling flexibility in terminal residues and other loops of beta linker segments of the mpro (residues - ) to about . nm and mpro-conjugate (residues - and - ) to about . - . nm. also, we found the residues to have comparatively lesser discrepancies in rmsf statistics, which are corresponding to the interacting segment of the mpro-conjugate complex after md simulation. interestingly, the binding region for lead conjugation before md simulation was also within the residues to , which remains the same after simulation, suggesting a strong binding of nos-hcq towards the mpro and provides stability to the mpro-conjugate. after that, the obtained trajectory was computed to evaluate the radius of gyration of the mpro and compared with the noscapine singly. the lead combination with the mpro protein showed the rg fluctuation in the range of . - . nm and for mpro in range of . - . nm and demonstrated the stable and robust binding of nos-hcq with mpro ( figure ) . furthermore, similar outcomes were obtained by sasa computation, which represented the solvent-accessible protein surface, and various orientations through the folding and proceeding to alterations in buried and exposed regions of the surface area of the protein complex. similar observations were determined through sasa analysis representing the solvent defined protein surface and its orientation through folding, making the alterations in the exposed and remains same as before till ns ( figure ). here, mpro-noshcq solvation profile shows a convincing sasa value suggesting a stable structure and sturdy binding. also, the secondary structure analysis of complex trajectory and native mpro trajectory was performed by employing the dssp tool of gromacs. the cluster analyses of both depicted the conformational changes before and after the simulation run. mpro-noshcq and mpro were consisted of mainly conserved b-sheets along with loops of b linker segments and other random coil regions infused with the various bend, a-helices, turn and b bridges. the observations of both plot analyses showed the mpro rationale unfolded and implemented surface with b-sheets to bind stably with lead conjugation nos-hcq. binding energy landscape mm/pb (gb) surface area (sa) calculations were performed to estimate the lead conjugation binding affinities to mpro protein. the system was found to be the most favorable in terms of the sum of various binding energy computations. mm/gbsa free binding energy calculations were executed with two fifty frames dynamically equilibrated for the stabilized trajectory of the mpro-noshcq complex system between ns to ns. the electrostatic energy distribution in molecular mechanics was obtained through the high vander waal energy (vdw) À . ± . kj/mol, electrostatic energy À . ± . kj/mol and total binding energy of À . ± . kj/mol, which are considered as most significant contributor in the binding of ligand (nos-hcq conjugation) to the target protein. the polar solvation energy (pbsol) was calculated to be À . ± . kj/ mol, which added a vital contribution to the stable binding of the ligand to target protein. further, the stability and flexibility of the binding of nos-hcq with mpro were strengthening with the stabilized run of trajectory with set nvt parameters. the average temperature for both native mpro and in conjugation was close to kelvin; average pressure was found to be close to . bars and an average density of . kg/m ( figure ). these outcomes through ns md simulation suggested the stable binding of lead combination with significant contribution of binding energy to the target mpro of the coronavirus. also, the energy work profile of ligand binding and mpro protein dynamics was investigated by smd trajectory based on jarzynski relationships in molecular dynamics simulation. the reaction coordinate work profile demonstrated the contribution of binding energies in potential interaction. the calculated work profile for ligand binding through the reaction coordinates stated the stabilized coupling with energy contribution reached a free energy profile of kcal/mol at an rc value of Å and sustained throughout binding with the less flexible atomic motions. in the early stages, it reached to the maximum value and stabilized at kcal/mol with hydrogen bonds and reached to work profile of kcal/mol at rc Å in later stages. the atomic energy profiles for mpro-nos-hcq reaction coordinates showed an average radius of . Å, with overall priority of conjugated reaction progression about . %. along with the binding energy calculations, hydrogen bond consistency and dynamic contribution of bound nos-hcq revealed the involvement of a high number of hydrogen bonds throughout the simulation run for both native mpro and complex system ( figure ). mpro formed an average of hydrogen bonds with the lead combination during the simulation and played an essential role in the selectivity of intermolecular interactions. these outcomes through the md simulation signified for the stable binding of nos-hcq conjugation in correlation with molecular docking results. the principal component analysis (pca) was performed to insight into the binding clusters variances analyses of the complex system. the binding cluster frames were categorized into two coordinate clusters in black and red color. pca calculations of the atomic backbone of the complex system were computed by three conformations pc , pc , and pc by normal mode molecular dynamics. obtained results showed that the pca cluster possesses the highest variability of . % by binding of lead combination to mpro in terms of internal motions of trajectory. after that, pc statistics depicted the minimal variability with . %, and consequently, pc calculations . % and suggested the stabilized binding with eigen scores and minimal atomic motions variability of nos-hcq in complexation with mpro (figure ) . both the conformations in two groups in different pca analyses were superimposed and aligned, as shown in figure d . the pca trajectory analyses showed a conformational change in clusters from the first blue cluster to white cluster progressively to red color cluster, similarly recovered from first cluster (black color) to second cluster (red color) through simple clustering in pc subspace (figure ) . furthermore, the dynamical residue cross-correlation map (dccm) analysis was performed, which depicted the pairwise correlation of atomic coordinates of nos-hcq to the mpro through the magnitude of pairwise cross-correlation coefficient. the map showed the correlation residues (> . ) of the complex system in blue color, and anti-correlated residues (<- . ) depicted in red color. the highly pairwise correlated residues (light blue color) of complex trajectory in the map showed the stable binding of ligand file nos-hcq. the outcomes were also found in correlation with optimal secondary structures of complex protein dynamics during the md simulation run. the binding ability of nos-hcq towards the mpro and its essence for coronavirus for replication and regulation suggests the potency of nos-hcq and possible role in covid- clinical drug development. with the computational rationalization of lead conjugate nos-hcq against the mpro, we propose the plausible scheme of chemical synthesis. the drug conjugates of noscapine and selected re-purposed small molecules drugs depict the feasible tentative synthetic methodology (scheme ). earlier in , our research group developed a series of microtubule-interfering halogenated noscapine analogs that perturb mitosis in cancer cells followed by cell death. iodo-nos was one of the crucial analogs of halogenated noscapine. -iodo-nos was synthesized by the iodination of noscapine using pyridine-iodine chloride in acetonitrile solvent with a % yield (aneja et al., ) . the target compound can be achieved by the application of a well-known stille metal cross-coupling reaction. compound , organostannanes, which can act as a precursor for the stille cross-coupling reaction, can be achieved via radical pathways in the presence of snme , dipea (base) and mecn (solvent) (chen et al., ) . the presence of the chloro group at hydroxychloroquine facile stille coupling reaction to take place at ease. the pd(pph ) catalytic system with copper(i) iodide and cesium fluoride in dmf is most effective for coupling iodides. copper(i) iodide and cesium fluoride combination facile the synthesis of sterically hindered systems such as (figure ). we report the efficient combinatorial therapy by conjugating the noscapine (antitussive drug) with potential hydroxychloroquine (nos-hcq) against the sars-cov- , through the computational assays with insights into the experimental results. the conjugate nos-hcq showed the most definite binding affinity towards the mpro, among the various noscapine based conjugates with (chloroquine, umifenovir, favlplravir, and galidesivir). noscapine (natural source opium poppy) is deemed to form the conjugation with antiviral drugs in trials against sars-cov- . the molecular docking assays depicted that by conjugation binding affinity score almost doubled with nos-hcq than with noscapine in the singlet state. the binding mechanism analyses of all conjugations illustrated that the lead conjugation (nos-hcq) has strong binding to three major domains of mpro. moreover, md simulation analyses of the trajectory (mpro-nos-hcq) for ns in explicit solvent depicted the stable binding of nos-hcq conjugate to mpro with minimal conformational variations in rmsd and rg plots. we reveal the drug binding with three major domains of mpro has high reaction coordinate completion efficacy (energy work profiles) with a significant contribution of immense binding energies and meticulous atomic level stabilization. the dccp and pca analyses confirmed the pivotal interactions and stabilize the binding of lead conjugation with mpro. besides rationalizing the combinatorial drug therapy, our study provides the fundamental plausible route of chemical synthesis of nos-hcq conjugate. this work paves the way for boosting the current medications into the effective combinational strategy to bind target mpro strongly, which can enhance the inhibitory action of drugs and contributes to fighting against the pandemic covid- with saving time, cost with minimal time screening in the current emergency. further, the warranted lead drug conjugation needs to be validated experimentally in laboratories and clinical trials. the authors declare no competing financial interest. covid- pandemic: an overview of epidemiology, parthenogenesis, diagnostics and potential vaccines and therapeutics synthesis of microtubule-interfering halogenated noscapine analogs that perturb mitosis in cancer cells followed by cell death remdesivir for the treatment of covid- -preliminary report -bromo- '-chloro pyrazoline analog of curcumin augmented anticancer activity against human cervical cancer, hela cells: in silicoguided analysis, synthesis, and in vitro cytotoxicity synthesis of aryl trimethylstannanes from aryl halides: an efficient photochemical method emerging coronaviruses: genome structure, replication, and pathogenesis in silico oncology drug repositioning and polypharmacology individualized networkbased drug repositioning infrastructure for precision oncology in the panomics era drug repurposing: new treatments for zika virus infection? chloroquine and hydroxychloroquine as available weapons to fight covid- favipiravir, an antiviral for covid- noscapine, a possible drug candidate for attenuation of cytokine release associated with sars-cov- . drug development research interaction of noscapine with the bradykinin mediation of the cough response human coronavirus: host-pathogen interaction breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies a decade after sars: strategies for controlling emerging coronaviruses bio d: an r package for the comparative analysis of protein structures swissdock, a proteinsmall molecule docking web service based on eadock dss structure of mpro from covid virus and discovery of its inhibitors natural bis-benzylisoquinoline alkaloids-tetrandrine, fangchinoline, and cepharanthine, inhibit human coronavirus oc infection of mrc- human lung cells understanding the binding affinity of noscapine with protease of covid- using md simulation at different temperature multiepitope subunit vaccine to evoke immune response against acute encephalitis antimicrobial peptide designing and optimization employing large-scale flexibility analysis of protein-peptide fragments preclinical evaluation and molecular docking of , -benzodioxole propargyl ether derivatives as novel inhibitor for combating the histone deacetylase enzyme in cancer ligplot þ : multiple ligand-protein interaction diagrams for drug discovery procheck: a program to check the stereochemical quality of protein structures therapeutic options for the novel coronavirus ( -ncov) research and development on therapeutic agents and vaccines for covid- and related human coronavirus diseases hexserver: an fft-based protein docking server powered by graphics processors outcomes of hydroxychloroquine usage in united states veterans hospitalized with covid- antiviral agents from plants and herbs: a systematic review mdtraj: a modern open library for the analysis of molecular dynamics trajectories designing of improved drugs for covid- : crystal structure of sars-cov- main protease m pro stereochemical quality of protein structure coordinates hydroxychloroquine and ivermectin: a synergistic combination for covid- chemoprophylaxis and treatment? coronavirus infections-more than just the common cold identification of a novel coronavirus causing severe pneumonia in human: a descriptive study plip: fully automated protein-ligand interaction profiler designing of a novel indoline scaffold based antibacterial compound and pharmacological evaluation using chemoinformatics approach a novel peptide thrombopoietin mimetic designing and optimization using computational approach mechanistic interaction study of bromo-noscapine with bovine serum albumin employing spectroscopic and chemoinformatics approaches antibacterial and pharmacological evaluation of fluoroquinolones: a chemoinformatics approach what if: a molecular modeling and drug design program structures of two coronavirus main proteases: implications for substrate binding and antiviral drug design larmd: integration of bioinformatic resources to profile ligand-driven protein dynamics with a case on the activation of estrogen receptor crystal structure of sars-cov- main protease provides a basis for design of improved a-ketoamide inhibitors network-based drug repurposing for novel coronavirus -ncov/ sars-cov- a novel coronavirus from patients with pneumonia in china £-nk and aa contributed equally. n.k, a.a., d.s., r.c., and n.s. designed the studies. n.k., d.s., and a.a, a.k., carried out the in silico experiments. n.k, a.a., a.g., and d.s. wrote the manuscript. no potential conflict of interest was reported by the authors. key: cord- -qwh inx authors: mendoza, jose luis accini; estrada, victor hugo nieto; lópez, nelly beltrán; bolaños, elisabeth ramos; franco, daniel molano; castell, carmelo dueñas; moreno, albert alexander valencia; amaya, iván camilo alarcón; flórez, john serna; valencia, bladimir alejandro gil; camilo pizarro, g; polo, yulieth maría zabaleta; meza, carmen lucia chica title: actualizacion de la declaraciÓn de consenso en medicina critica para la atenciÓn multidisciplinaria del paciente con sospecha o confirmaciÓn diagnÓstica de covid- date: - - journal: nan doi: . /j.acci. . . sha: doc_id: cord_uid: qwh inx antecedentes y objetivos: la enfermedad por coronavirus de (covid- ) es una enfermedad ocasionada por el nuevo coronavirus del síndrome respiratorio agudo grave (sars-cov- ). se identificó por primera vez en diciembre de en la ciudad de wuhan, en los meses siguientes se expandió rápidamente a todos los continentes y la organización mundial de la salud (oms), la reconoció como una pandemia global el de marzo de . la mayoría de los individuos son asintomáticos pero una baja proporción ingresan a cuidados intensivos con una alta morbilidad y mortalidad. este consenso tiene como objetivo actualizar la declaratoria inicial emitida por la asociación colombiana de medicina crítica (amci) para el manejo del paciente críticamente enfermo con covid- dentro de las áreas críticas de las instituciones de salud. métodos: este estudio utilizó dos técnicas de consenso formal para construir las recomendaciones finales: delphi modificada y grupos nominales. se construyeron preguntas por la estrategia pico. grupos nominales desarrollaron recomendaciones para cada unidad temática. el producto del consenso fue evaluado y calificado en una ronda delphi y se discutió de forma virtual por los relatores de cada núcleo y los representantes de sociedades médicas científicas afines al manejo del paciente con coid- . resultados: expertos nacionales participaron en la actualización del consenso amci, especialistas en medicina critica y cuidados intensivos, nefrología, neurología, neumología, bioeticistas, medicina interna, anestesia, cirugía general, cirugía de cabeza y cuello, cuidados paliativos, enfermeras especialistas en medicina crítica, terapeutas respiratorias especialistas en medicina crítica y fisioterapia, con experiencia clínica en la atención del paciente críticamente enfermo. la declaratoria emite recomendaciones en los ámbitos más relevantes para la atención en salud de los casos de covid- al interior de las unidades de cuidados intensivos en el contexto nacional de colombia. conclusiones: un grupo significativo multidisciplinario de profesionales expertos en medicina crítica emiten mediante técnicas de consenso formal recomendaciones sobre la mejor práctica para la atención del paciente críticamente enfermo con covid- . las recomendaciones deben ser adaptadas a las condiciones específicas, administrativas y estructurales de las distintas unidades de cuidados intensivos del país. background and objectives: the coronavirus disease (covid- ) is caused by the new severe acute respiratory syndrome coronavirus (sars-cov- ). it was first identified in december in wuhan, china. in the following months it spread quickly to all continents and was recognised as a global pandemic by the world health organization (who) on march th, . most cases of infection remain asymptomatic, while a low proportion require intensive care, experiencing high morbidity and mortality. this consensus aims to update the initial statement issued by the colombian association of critical medicine (amci) for the management of the critically ill patient with covid- within the critical areas of health institutions. methods: this study used two formal consensus techniques to construct the final recommendations: modified delphi and nominal groups. questions were constructed using the pico strategy. recommendations for each thematic unit were developed by nominal groups. the consensus product was evaluated and qualified in a delphi round, and was discussed virtually by the speaker of each nucleus, as well as the representatives of scientific medical societies related to the management of the patient with covid- . results: a total of national experts participated in the update of the amci consensus, all specialists in critical and intensive care medicine, nephrologists, neurologists, chest physician, bioethicists, internal medicine specialists, anaesthetists, general surgeons, head and neck surgery, palliative care, nurses specialised in critical medicine, respiratory therapists specialised in critical medicine and physiotherapy, with clinical experience in the care of critically ill patients. this update issues recommendations in the most relevant areas for health care of covid- patients within the intensive care units, contextualised for colombia. conclusions: a significant multidisciplinary group of professionals, who are experts in critical medicine, reviewed and issued recommendations on best practice for the care of critically ill patients with covid- through formal consensus techniques. recommendations must be adapted to the specific, administrative, and structural conditions of the different intensive care units in the country. para la actualización de la declaratoria se utilizaron dos técnicas para el desarrollo de consensos de tipo formal, técnica delphi modificada y grupos nominales. un consenso formal permite integrar las opiniones de un colectivo de expertos que están expuestos a un tema específico (experto afectado) con la mejor evidencia científica disponible, utilizando técnicas que permitan reducir los sesgos de subjetividad. la técnica delphi es una metodología que plantea enviar cuestionarios a un grupo de expertos, para que califiquen una serie de recomendaciones en rondas reiteradas con retroalimentación de los resultados y respuestas anónimas, la técnica delphi empleada fue modificada, variante a la versión original propuesta por la corporación rand en , pero se mantuvo las ventajas de la técnica, la iteración y retroalimentación para reflexión de las propias opiniones. los grupos nominales es una técnica que reúne a un grupo de expertos bajo la coordinación de un facilitador para evaluar y calificar información o preguntas ( , ) . para la actualización se convocó grupos nominales con expertos multidisciplinarios cada uno con un líder o jefe de núcleo. los grupos construyeron las preguntas por metodología pico y desarrollaron progresivamente las recomendaciones hasta las versiones finales. el proyecto se desarrolló en fases, fase : formulación del problema y socialización; fase : elaboración de las preguntas, fase : formulación de las recomendaciones y ronda de calificación. las estrategias de búsqueda se desarrollaron en bases de datos especializadas (medline, embase, lilacs, central), en las circunstancias donde no se encontró evidencia directa, se utilizó y se adaptó evidencia indirecta del tópico relevante en el paciente críticamente enfermo general. expertos con un promedio de años de experiencia en la atención del paciente crítico evaluaron y calificación las recomendaciones en la metodología delphi mediante un cuestionario distribuido por medio de correo electrónico, respetando la política de privacidad de datos vigente. recomendaciÓn se recomienda que los prestadores de servicios de acuerdo con su infraestructura física y la disponibilidad de recursos (tecnológico, humano, de interdependencia y apoyo) definan su modelo de atención para pacientes con covid- en estado crítico basado en principios de factibilidad, efectividad, seguridad y la relación entre la demanda (momento epidemiológico) y capacidad/capacidades de respuesta:  modelo . atención de pacientes con sospecha o confirmación diagnóstica covid- . este enfoque permite concentrar, optimizar y racionalizar recursos y reducir el riesgo potencial de contagio al equipo de atención, de apoyo y de pacientes.  modelo . atención mixta, de pacientes con y sin diagnóstico de covid- , en escenarios que cuentan con unidades de aislamiento normatizados (presión negativa y > -renovaciones completas de aire por hora) soportado en el documento institucional de gestión organizacional y operativo del servicio de cuidados intensivos, descrito en los procesos prioritarios. amci ® menos accidentalidad y violencia). la reducción de procedimientos quirúrgicos complejos electivos es una opción razonable condicionado a las posibilidades del paciente. sin embargo, situaciones como la progresión y descompensación de las patologías crónicas asociadas a las medidas de restricción social puede plantear un efecto bumerang con mayor demanda de camas de uci. con base en las predicciones simuladas de las tasas esperadas de ingreso a uci de pacientes con covid- contrastado con el déficit de servicios y camas de cuidados críticos en el país a partir de la capacidad instalada se han planteado fases de desarrollo cuyas características en términos de servicios, recursos y cronología se aprecian en la ilustración y .  fase (para el gobierno nacional: ampliación de la capacidad instalada*). parte de la liberación de camas de cuidados intensivos destinadas para atención covid- bajo el modelo y/o . la liberación de camas y servicios con mínima adaptación es la fase más inmediata y resolutiva que debe acogerse a la exigencia normativa (resolución de ), en la que se espera menos mortalidad, morbilidad y tasa de complicaciones asociados con la atención de pacientes con covid- en estado crítico.  fase (optimización para el gobierno nacional*) representan el reordenamiento de las camas de cuidados intermedios adultos en camas de cuidados intensivos y de hospitalización en intermedio. de adultos. el gobierno nacional toma en cuenta la ampliación de la capacidad instalada hospitalaria descrita en el plan territorial. para esta fase se necesitan equipos de ventilación mecánica (excluidos por la norma de estos servicios), monitoreo básico y avanzado y un número mayor de talento humano multidisciplinario competente. amci ® acuerdo con las etapas ( - ) y los perfiles requeridos priorizados como primera, segunda y tercera línea de respuesta*. en la ilustración podemos observar que avanzar de las fases a se va a necesitar mayor intervención en términos de organización, planeación operativa, formación por competencias y apoyo por telesalud (teleapoyo o teleexperticia). el personal no especializado o especializado de servicios hospitalarios diferente a urgencia, quirófano o uci pueden constituir grupos de apoyo para la gestión administrativa y de índole humanitaria (líneas de respuesta) la integración de estos requerimientos adaptativos se ha puesto de manifiesto en la experiencia del centro médico new york -presbyterian weill en la ciudad de nueva york, en donde la demanda de camas de unidades de cuidados intensivos (uci) y ventilación mecánica excedió su capacidad.( ) se recurrió a los quirófanos y de recuperación las cuales no estaban en uso porque los procedimientos electivos habían sido pospuestos. se hicieron adecuaciones físicas para garantizar la vigilancia continua de los pacientes y la seguridad del equipo de atención. se capacito a todo el personal de cuidado perioperatorio disponible y fueron distribuidos en las áreas recién configuradas. las enfermeras familiarizadas con las máquinas de anestesia asumieron como terapeutas respiratorios y los intensivistas de anestesia supervisaban estas unidades. recomendaciÓn se recomienda la adopción de un modelo simulado de predicción (basado en el cociente de fatalidad, tasa de ingreso a uci y el número de reproducción ro) para proyectar, de manera anticipada las necesidades de recurso físico, tecnológico y humano de cuidados críticos en fase de preparación de la pandemia por covid- . las tomas de decisiones relacionadas con el fortalecimiento de la capacidad/capacidades de la oferta de cuidados críticos (habilitadas o adaptadas) pueden deducirse mediante la aplicación de modelos matemáticos que intentan, desde la dinámica epidemiológica, establecer el efecto simulado de las medidas de mitigación o supresión adoptadas sobre la tasa de contagio a través del tiempo. con ello se busca planear el uso de recursos e implementar acciones de prevención y de distanciamiento social más eficientes, así como establecer las necesidades en materia de cama de cuidados críticos (intensivos e intermedios) habilitadas o adaptadas en áreas de expansión y con ello los recursos resolutivos como tecnologías, interdependencias y talento humano capacitado. la capacidad predictiva del modelo resulta de establecer: la información en términos de casos proyectados (población susceptible), el modelo de transmisión, el impacto de las amci ® intervenciones no farmacológicas (mitigación y supresión) para lograr disminuir el número de reproducción r (tasa de contagio), la distribución de la gravedad de la enfermedad y con ello el porcentaje esperado de casos críticos el decreto del . . ( ) , por el cual se declara un estado de emergencia económica, social y ecológica en todo el territorio nacional proyectó para una tasa de contagio de . y cerca de millones de casos, la ocurrencia de mil casos de pacientes con covid- en estado crítico ( . %) y la necesidad de incrementar las camas de cuidados intensivos en un % que con una estancia promedio de días tendría un costo de mil millones de pesos. colombia cuenta con cerca de camas de cuidados intensivos ( / parte son intermedio) de las cuales el % podría ser utilizada para la atención de pacientes covid- ( ). con base en el modelo matemático ha proyectado la necesidad de camas de uci indicando por deducción la necesidad de camas a partir de un plan adaptativo de expansión y extensión. colombia reporta a la fecha actual , casos confirmados y nuevos casos de covid- confirmados y cerca del % ocupan una cama de cuidados intensivos (esta cifra es mayor si se tomaran en cuenta los casos sospechosos). de este modo, los modelos matemáticos permiten predecir el comportamiento epidemiológico de la enfermedad y con esto anticiparse a proyectar el plan de fortalecimiento hospitalario (incluyendo camas, tecnologías, talento humano) y los recursos financieros para respaldar la expansión del cuidado críticos. estos modelos deben ser predictivos y no reactivos al comportamiento epidemiológico de la enfermedad y solo debe des escalarse hasta después de reducirse a menos de . el índice ro. ( - ) recomendaciÓn se sugiere la implementación de una estrategia de telesalud (teleapoyo o teleexperticia) en el marco de la pandemia covid- , cuando no se cuente con un intensivista presencial, que, mediante una tecnología adecuada complemente la atención en las áreas de cuidados críticos realizado por personal capacitado. aún cuando es considerada ventajosa sus implicaciones en términos de resultados clínicos, económicos y de riesgos legales no se ha demostrado. en situaciones donde se declara una pandemia los sistemas de salud pueden tener dificultades para hacer frente a una demanda exponencial y fuera de control. esto puede ser así en el marco pandemia covid- que prevé un % de pacientes en condición crítica y que amerita reorganización y/o adaptación de su capacidad de respuesta. incrementar la disponibilidad de camas y servicios de cuidados críticos mediante una estrategia de expansión supone retos asociados a la insuficiencia que se puede presentar en talento humano especializado específicamente de especialistas en medicina crítica y cuidado amci ® intensivo, escenario que se puede complicar en la medida que intensivistas sean separados o aislados en el curso de la epidemia. en este contexto se hace necesaria la implementación de modelos ágiles de telesalud (ts) para el acompañamiento de las unidades de cuidado crítico en expansión y de instituciones prestadoras de servicios de salud de baja y mediana complejidad para la regulación con los equipos de referencia y contrarreferencia de pacientes que pueden necesitar atención en cuidados intensivos. ( ) ( ) ( ) ( ) ( ) ( ) el decreto ley de ( ) plantea la adopción de medidas en el sector salud para garantizar la prestación de los servicios de salud y para facilitar la implementación de modelos de atención que incluyan la telesalud y la prestación de los servicios en la modalidad de telemedicina se determinan algunas medidas temporales para: i) adecuar temporalmente un lugar no destinado a la prestación de servicios de salud, intra o extra mural. ii) prestar servicios en modalidades o complejidades diferentes a las habilitadas dentro de las cuales puede estar la telemedicina iii) prestar servicios de salud no habilitados. en este decreto también se establecen condiciones temporales para la implementación de plataformas tecnológicas para la telesalud. en complemento, la resolución de ( ) (estándares de habilitación) plantea la modalidad de la telemedicina (prestador remisor-prestador de referencia) para las unidades de cuidado intermedio e intensivo. la telesalud se puede prestar de dos maneras: teleapoyo y teleexperticia. tabla . teleapoyo (ta) soporte solicitado por un profesional de la salud a otro profesional de la salud a través de tic siendo responsable de la conducta quién solicita el apoyo. no requiere habilitación y por tanto no requiere autorización transitoria relación a distancia con comunicación sincrónica o asincrónica utilizando tic entre dos profesionales de la salud, uno de los cuales atiende presencialmente al usuario y otro atiende a distancia. el primero es responsable de las decisiones/recomendaciones entregadas al paciente y el segundo es responsable de la calidad de la opinión que entrega y debe especificar las condiciones en las que se da dicha opinión, lo cual debe consignarse en la historia clínica. requiere autorización transitoria (decreto ) ( ) la telesalud y la prestación de servicios de salud en esta modalidad son estrategias seguras y efectivas para guiar, a distancia, el diagnóstico y el tratamiento del paciente hospitalizado y en estado crítico. sus ventajas generales se presentan en la tabla . asociaciÓn colombiana de medicina crÍtica y cuidados intensivos. amci ® tabla . ventajas generales de la telesalud decreto legislativo de .  facilita la viabilidad de aplicación modelos organizativos que favorecen la continuidad y la integridad asistencial y la atención centrada al entorno del paciente, aplicando conceptos de globalidad e interoperabilidad a las organizaciones sanitarias, dando lugar a nuevas formas de organización y trabajo en red.  mejora de la calidad asistencial, ya que facilitan el acceso y la disponibilidad de servicios asistenciales en condiciones de calidad.  mejora calidad de vida del paciente por la disminución de desplazamientos para la atención ya que permite la atención o monitorización remota con tic en su domicilio.  mejora la oportunidad y la resolutividad de la atención.  facilita la equidad en el acceso a los servicios de salud independientemente de la localización geográfica (acerca la atención especializada a toda la población).  mejora la atención integral y seguimiento tanto de los pacientes crónicos, como los de las enfermedades de baja prevalencia.  reduce los tiempos de espera (tanto en la realización del diagnóstico como en el tratamiento), evitando complicaciones por no atención oportuna.  posibilita realizar atención remota de mediana y alta complejidad en la baja complejidad, reduciendo el número de remisiones.  disminuye la posibilidad de infección cruzada entre usuarios de los servicios de salud y el personal de salud.  incide en la formación y competencia del talento humano en salud.  facilita la educación de pacientes en medicina preventiva y salud pública.  descongestiona servicios de urgencias y consulta externa.  contribuye a la reducción de movilidad de personas en la ciudad.  responde a las necesidades inmediatas en salud de la comunidad.  es un medio de racionalización de costos en salud. puede abarcar otros servicios de gestión administrativa como entrega de fórmulas o facturación. amci ® se recomienda la implementación de un modelo de cuidados críticos covid- liderado por intensivistas, en áreas habilitadas o adaptadas, con el beneficio preponderante de disminuir la mortalidad, tiempo de estancia y optimización de recursos. se recomienda una cobertura por intensivistas de al menos horas diarias y un cociente intensivista/paciente cercano a intensivista por cada - pacientes, basado en la alta complejidad de la enfermedad critica covid- con un alto porcentaje de pacientes en ventilación mecánica, largos tiempos de estancia y alto riesgo de mortalidad. fuerte a favor fundamento un intensivista es un profesional médico capacitado en medicina crítica y de cuidados intensivos conforme a los estándares establecidos por una institución de educación superior debidamente reconocida ante el ministerio de salud. este especialista debe liderar y tomar todas las decisiones con respecto al cuidado de los pacientes críticos, incluyendo admisiones y egresos, qué médicos consultar, estándares de atención, gestión de la calidad y seguridad, gestión humana y ética, interacción con la familia e implementación de un programa de investigación y de formación continua para mejorar capacidades y competencias del equipo de atención, control de conflictos, entre otras.( ) existe una enorme validez conceptual y una preponderancia de evidencia que sugiere que ser atendido por un especialista en cuidados críticos (intensivista) es "bueno" para los pacientes de la uci. la mayoría de los estudios demuestran el impacto positivo de un uci dirigida por intensivistas los modelos de personal médico de la uci más ampliamente estudiados difieren en el nivel al cual los intensivistas están involucrados en el manejo de los pacientes. las uci de alta intensidad son aquellas donde un intensivista de tiempo completo u obligatorio maneja a la mayoría de los pacientes diariamente. las uci de baja intensidad no tienen participación intensivista u ofrecen consultas intensivistas electivas. un metaanálisis mostró que un modelo de alta intensidad en comparación con uno de baja intensidad estuvo asociado con una menor mortalidad en la uci, menor mortalidad hospitalaria, y una reducción significativa en la duración de la estancia hospitalaria. un modelo de alta intensidad por la noche se asoció con menor mortalidad solo cuando durante el día era de baja intensidad. ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) asociaciÓn amci ® forzosa" por condición de riesgo siempre en consonancia con los términos establecidos en el decreto ley de ( ). según la normatividad vigente todo miembro del equipo de atención, especialista no intensivista o no especialista (enfermeras, fisioterapeutas/terapeutas respiratorias) y auxiliares de enfermería deben tener constancia de asistencia a acciones de formación continua y/o capacitación en atención covid- en cuidados críticos las cual puede ser parte de un programa institucional de capacitación liderado por el intensivista coordinador o titular del servicio o a partir de cursos respaldados por instituciones académicas acreditadas o los ofrecidos por la asociación colombiana de medicina crítica y cuidados intensivos (amci). las tablas y nos muestra como dentro de este proceso adaptativo por estado de emergencia y atribuido a un desequilibrio entre la oferta y la demanda, otras especialidades, profesionales de la salud y personal en formación pueden hacer parte de los equipos de atención bajo las siguientes premisas: . la supervisión, coordinación y liderazgo del intensivista es necesaria y . el intensivista establece los roles y competencias del th no intensivista y no normatizado, de acuerdo a sus perfiles, y delegada acciones asistenciales (vía aérea, accesos vasculares, reanimación cardiopulmonar, pronación), administrativas (ordenes médicas, notas clínicas) o de naturaleza humanitaria (comunicación con la familia, apoyo emocional al th, etc.). es necesario considerar los roles de las especialidades que formarán parte de la gestión asistencial y/o administrativa de pacientes con covid- en áreas críticas habilitadas o adaptadas de manera transitoria en colombia tabla . integridad de interdependencia (norma / ) integralidad e interdependencia (adaptiva) gfa integridad de interdependencia (norma / ) integralidad e interdependencia (norma / ) integridad de interdependencia (norma / ) obligatorio en gestión asistencial integralidad e interdependencia(norma / ) obligatorio gestión asistencial apoyo a tomas de decisiones(gfa) apoyo a tomas de decisiones (gfa) a:formacion continua + covid- (curso virtual); b: requiere capacitación covid- (curso virtual); c: gfa: grupo focal asistencial, sistema alerta-acción, rcp, pronación, accesos vasculares, ingreso a uci. d: gfad: grupo focal administrativo: consentimiento, notas de evolución. ts: telesalud. se recomienda la aplicación de la escala news- por parte de un equipo de respuesta rápida, para establecer el lugar de atención de pacientes con diagnóstico definitivo o sospecha de infección por covid- que encuentran en los servicios de urgencias o de hospitalización. se recomiendan escalas como el qsofa y el curb- para apoyar la decisión tomada con base en la escala news- . sin embargo, un qsofa mayor o igual a puntos (mínimo / ) y un crb- mayor o igual puntos tienen baja sensibilidad (alta incidencia de falsos negativos) para identificar pacientes que puedan requerir ingreso a uci. se recomienda el score de riesgo covid- -gram para identificar el riesgo de desarrollar un estado de la enfermedad crítico en pacientes con covid- y como herramienta complementaria a la escala news- en escenarios de alta demanda y escasez de recursos, de manera que la decisión de ingreso a uci se haga sobre aquellos pacientes que realmente se beneficiarán de la misma en términos de vidas salvadas y número de años salvados. la escala news fue construida y validada en paciente con infección por el virus de la influenza a/h n y se recomienda como una herramienta objetiva para decidir nivel de atención, incluyendo ingreso a uci o situaciones terminales que requieren acompañamiento familiar y medidas de cuidado paliativo. esta escala incluye variables fisiológicas que son: frecuencia respiratoria, saturación arterial de oxígeno (spo ), uso de oxígeno suplementario, presión arterial sistólica, frecuencia cardiaca (pulso), temperatura y nivel de conciencia. es de mencionar que esta escala no contempla la edad del paciente. en un reporte del acute medicine task force of the royal college, london, uk, recomendó la utilización de la escala news en los servicios de urgencias ( ) . un estudio que evaluó una base de datos con . signos vitales obtenidos de . pacientes demostró que esta escala tiene una buena capacidad para discriminar pacientes en riesgo para un desenlace combinado de paro cardiaco, ingreso no anticipado a uci o muerte dentro de las primeras horas de atención; de esta manera genera una gran oportunidad para el establecimiento temprano de una intervención clínica que cambie el pronóstico del paciente ( ) . la escala news- tampoco contempla la edad, pero sí incluye la presencia de hipercapnia bajo diferentes niveles de spo y oxígeno suplementario. esta escala es la que ha sido estudiada como herramienta para identificar pacientes en riesgo de desarrollar un estado de enfermedad crítica por covid- con un valor ≥ puntos ( / ) ( ) . con base en la escala news- se establece el grado de riesgo, el tipo de alerta, y la intensidad de monitoreo requerido; y de acuerdo con el puntaje arrojado, se define claramente el nivel de atención que requiere el paciente con diagnóstico de infección por covid- , incluso ingreso a uci (tabla ). esto se establece a través de una escala de puntuación así: -score puntos: manejo domiciliario bajo aislamiento y signos de alarma. -score punto: manejo domiciliario y seguimiento clínico en casa. -score - puntos: manejo en salas de hospitalización. -score - puntos: manejo en uci, área covid- . -score ≥ puntos sin condición extremadamente grave o irreversible y con alta posibilidad de recuperación: traslado a uci, área covid- . amci ® -score ≥ puntos con condición extremadamente grave y con datos de irreversibilidad o enfermedad terminal: no ingresa a uci y se traslada a salas de hospitalización con acompañamiento familiar y consulta a experto en bioética y cuidados paliativos. la ilustración establece un flujograma de conductas basado en el puntaje del news- . tabla . news- score. el qsofa con un valor ≥ puntos es otra herramienta recomendada para decidir qué pacientes que ingresan a uci. esta herramienta fue recomendada por la tercera definición de consenso (sepsis- ) para identificar pacientes con alto riesgo de muerte o estadía prolongada en uci entre aquellos con sospecha de infección ( ) . en este score, un punto es asignado para variables así: frecuencia respiratoria ≥ /min, presión arterial sistólica ≤ mmhg y escala de coma de glasgow (ecg) < . el score curb- y su versión simplificada, el crb- se utilizan para evaluar la severidad de enfermedad en personas hospitalizadas con neumonía adquirida en comunidad (nac). ambos scores han sido adoptados por la sociedad británica de tórax para predecir la necesidad de soporte respiratorio o vasopresor intensivo (srvi) en pacientes con covid- ( ) . el score crb- asigna un punto para variables así: confusión de reciente inicio, frecuencia respiratoria ≥ /min, presión arterial sistólica < mmhg o presión arterial diastólica ≤ mmhg y edad ≥ años. pacientes con un score ≥ puntos necesitan hospitalización ( ) . en un estudio clínico observacional realizado sobre los primeros pacientes ingresados en un hospital de noruega con diagnóstico confirmado de covid- , se evaluó la utilización de sistemas de score clínicos al momento del ingreso: news- , qsofa, crb- y sirs, con los puntos de corte previamente mencionados ( ) . la enfermedad se clasificó como enfermedad severa y enfermedad crítica. solo pacientes ( %) se clasificaron como enfermedad crítica. al evaluar los scores con sus puntos de corte, pacientes presentaron un qsofa ≥ [ con enfermedad severa ( %) y con enfermedad crítica ( %)], solo pacientes presentaron un crb- ≥ [ con enfermedad severa ( %) y con enfermedad crítica ( %)] y pacientes presentaron un news- ≥ [( con enfermedad severa ( %) y con enfermedad crítica ( %)]. la mediana del score news- para pacientes con enfermedad severa fue de . [riq: - ] vs . [riq: . - ] para pacientes con enfermedad crítica. los autores concluyen que el qsofa y el crb- se comportan similar y con una baja capacidad para la identificación de enfermedad crónica en pacientes con covid- ; por otro lado, los datos indican que el news- podría ser una herramienta más útil para identificar pacientes con riesgo de un curso más agresivo de la enfermedad ( % vs. %) ( ) . amci ® un estudio de cohorte retrospectivo realizado en un hospital en liverpool (uk), el puntaje qsofa se comportó como el más específico ( %; % ic: % - %) pero el menos sensible ( %; % ic: % - %) en comparación al puntaje sirs y la escala news como predictor de mortalidad hospitalaria en un grupo de paciente admitidos por sepsis al servicio de emergencias ( ) . otro estudio retrospectivo de un único centro comparó el rendimiento de sistemas de score, qsofa, curb- y crb- para predecir la necesidad de soporte vasopresor o respiratorio intensivo (svri) en pacientes ingresados con diagnóstico confirmado de covid- a un hospital de wuhan (china) ( ) . un total de pacientes ( . %) necesitaron svri durante su estadía en el hospital. la tasa de mortalidad hospitalaria en esta cohorte fue de pacientes ( . %). se evalúo el rendimiento de las escalas con análisis de curva roc (auc), puntos de corte óptimo, sensibilidad, especificidad y valores predictivos. el punto de corte óptimo del crb- para predicción de srvi fue de puntos, con una sensibilidad del % y una especificidad del . %. el valor auc del score crb- para predecir la necesidad de svri fue significativamente más alto que el del qsofa ( . ± . vs. . ± . , p= . ). los valores de auc fueron similares entre crb- y curb- para predecir svri ( . ± . vs. . ± . , p= . ). los autores concluyen que el crb- podría ser mejor que el qsofa para identificar paciente con covid- en riesgo de necesitar svri. su et al consideran que fue la inclusión de la edad ≥ años dentro del score crb- lo que le dio un mayor grado de superioridad sobre el qsofa ( ) . el crb- puede ser una herramienta de puntuación útil para covid- debido a su simplicidad en la aplicación, especialmente en emergencias y condiciones de escasez de recursos. finalmente, el score de riesgo covid- -gram fue descrito por un grupo de investigadores en covid- quienes reunieron datos de pacientes en hospitales en china ( ) . esta fue una cohorte retrospectiva multicéntrico en la que se recogieron un total de variables entre demográficas, médicas, clínicas (signos y síntomas), imagenológicas y de resultados de laboratorios. utilizando la metodología de regresión lasso (least absolute shrinkage and selection operator) construyen un modelo de regresión multivariable resultando en un score de riesgo predictivo para desarrollar enfermedad critica en pacientes con covid- confirmado al momento de la admisión. de las variables iniciales fueron predictores independientes estadísticamente significativos para el desarrollo de enfermedad crítica. estas variables fueron: anormalidad en los rx tx (or: . ; %ic: . - . recomendaciÓn se recomienda la aplicación de un algoritmo basado en una evaluación dinámica del score news que involucre una escala de evaluación funcional, para priorizar el ingreso a uci con transparencia científica y ética con equidad social, y de ser posible respaldado por un comité de priorización clínica (cpc) integrado por expertos de cuidado intensivo y un representante del comité de ética hospitalaria durante la pandemia por covid- . se recomienda un modelo determinado por prioridades para definir criterios de ingreso a uci, permite establecer rápidamente qué pacientes se benefician de ingreso uci y qué pacientes deben permanecer en servicios de hospitalización, o con acompañamiento familiar y cuales con medidas de cuidado paliativo. fuerte a favor page amci ® fundamento la pandemia covid- nos ha enseñado que la disponibilidad de camas de uci puede ser insuficiente y el plan estratégico diseñado para ampliar la capacidad de respuesta debe ir de la mano con la implementación rigurosa de un protocolo de triaje y de priorización de ingreso a cuidados intensivos, como medida extraordinaria para optimizar los recursos, mitigar y controlar los efectos de la pandemia sobre el balance oferta (efectiva y resolutiva) y la demanda. los protocolos de triaje y priorización están diseñados para asignar los limitados recursos de una manera justa y transparente donde, por definición, algunas personas serán excluidas del acceso a la atención orientado a aumentar la disponibilidad de camas de cuidados intensivos. sin embargo, es necesario enfatizar que la disponibilidad de camas no es un fin en sí mismo. la intención implícita y explícita de los protocolos de clasificación debiera ser el «bien público» de maximizar la supervivencia de la población. pero es incorrecto suponer que este bien público se logra al maximizar la supervivencia entre los que reciben cuidados intensivos. si bien muchos protocolos de triaje reconocen esto al tratar de excluir a los pacientes que no lo necesitan absolutamente (el «demasiado sanos») y los que tienen menos probabilidades de beneficiarse (él «demasiado enfermo»), no prestan suficiente atención a las diferencias entre grupos en términos de la duración de los cuidados intensivos necesarios para lograr resultados. si el objetivo del triage es mejorar la supervivencia de la población con un recurso escaso, entonces el recurso escaso no son camas, sino días de cama; no son ventiladores, sino tiempo de ventilación. de ello se deduce que el triaje no será efectivo si en la valoración, no se discrimina adecuadamente y se considera de manera equívoca que la gran mayoría de las personas que requieren cuidados intensivos tienen una probabilidad similar de supervivencia y una duración de estadía anticipada similar. ( ) de este modo, el ingreso a uci debe acogerse a los criterios habituales, científicos y éticos, bajo el rigor de "idoneidad clínica" tomando en cuenta parámetros como la gravedad de la enfermedad, la presencia de comorbilidades (severidad, clase funcional), potencial de recuperabilidad, deseo del paciente (o la familia), de equidad distributiva y el uso de las escalas validadas de severidad y de predicción de ingreso a cuidados intensivos. los pacientes con covid- tienden a progresar después del inicio de los síntomas dentro de los a días a una forma grave con síndrome de dificultad respiratoria aguda (sdra) o falla multiorgánica órgano. la identificación temprana y simple de pacientes que requieren respiración intensiva o el soporte vasopresor sería de gran valor durante el brote covid- . ( ) ( ) ( ) ( ) la implementación de un algoritmo "dinámico" que vincule uno o más de las escalas fisiológicas (news con o sin qsofa y/o crb- ≥ ), una escala de predicción de ingreso a uci (covid- -gram) y un puntaje de fragilidad (vipi) puede informarnos sobre el estado actual y evolutivo de la enfermedad y a priorizar el ingreso de pacientes a uci permitiendo un uso óptimo de los recursos y tomar decisiones éticas, transparentes y centradas en la dignidad de los pacientes y el bien público ilustración - . se recomienda no usar escalas de severidad de enfermedad (criterios objetivos) para definir el traslado de pacientes de uci hacia un nivel de menos complejidad de atención, ya que estas escalas no han sido validadas para este uso. se recomienda en la atención por covid- en cuidados intensivos utilizar los mismos criterios de egreso que se emplean para el traslado desde uci hacia una unidad de menor complejidad de pacientes sin infección por covid- . se recomienda contar con áreas de bajo nivel de complejidad asignadas solo a la atención de paciente con infección por covid- , las cuales serán las áreas hacia donde se realiza el de-escalamiento gradual de los pacientes basado en su evolución clínica. cuando se habla de criterios objetivos, se hace referencia a escalas de severidad de enfermedad que ayuden a tomar decisiones más racionales y no basadas en consideraciones tradicionales de resolución de cuadros clínicos. no hay una recomendación definida sobre el uso de escalas de severidad de enfermedad para definir el de-escalamiento de la atención para pacientes críticos. los sistemas de evaluación de severidad de enfermedad generales y específicos pueden identificar una población específica de pacientes en alto riesgo de deterioro clínico luego del traslado fuera de la uci( ); sin embargo, su valor para evaluar que tan preparado está un paciente individual para ser trasladado a un nivel inferior de cuidado no ha sido evaluado( ). los criterios que recomienda el colegio americano de cuidado intensivo( ) para definir el traslado desde uci a un nivel de menor complejidad (unidad de cuidados intermedios o sala de hospitalización) se basan en principios:  cuando el estado fisiológico del paciente se ha estabilizado y ya no es necesario monitoreo y tratamiento en uci.  cuando el paciente cumpla con los criterios de admisión del nivel de menor complejidad, teniendo en cuenta la disponibilidad actual del recurso, el pronóstico del paciente y la presencia de intervenciones activas en curso. específicamente para los pacientes con alto riesgo de muerte y reingreso a uci en quienes se decide no hacer intervenciones adicionales (alta severidad de enfermedad, inestabilidad fisiológica, soporte orgánico), el colegio americano de cuidado intensivo( ) siguiere pasarlos a una unidad de menor nivel de atención o a un hospital de cuidado agudo de largo plazo; siempre con un formato escrito donde se deje claro la decisión para reducir la tasa de reingreso a uci( ). específicamente en situaciones de pandemia y escasez de recursos, la sociedad española de medicina intensiva, crítica y unidades coronarias (semicyuc) recomienda la realización de la escala sofa al menos cada horas para identificar pacientes con evolución tórpida y progresiva a un fallo multiorgánico luego de la iniciación de tratamientos de soporte vital. esto permitirá la adecuación y reorientación de medidas terapéuticas a un objetivo más paliativo, incluyendo la transferencia del paciente a un nivel más bajo de complejidad y la consulta al servicio de cuidado paliativo para que brinde la atención respectiva( ). finalmente, se debe mencionar que no se ha establecido una diferencia en los criterios de egreso de pacientes con covid- para su traslado desde uci hacia una unidad de menos complejidad, con respecto a los utilizados para pacientes sin infección por covid- ( ). todo paciente hospitalizado en uci sea covid- o no, debe ser valorado diariamente para establecer en qué momento su condición clínica permite que sea trasladado fuera de la uci hacia una unidad de menor complejidad. en caso de tratarse de pacientes con sospecha o diagnóstico de covid- , este traslado puede hacerse a una unidad de cuidados intermedios dispuesta como área covid- o una sala de hospitalización con igual asignación( ); esto es lo que se conoce como de-escalamiento gradual de la atención hasta el final egreso del paciente a casa. se recomienda para pacientes con covid- que evolucionan hacia la mejoría, utilizar los criterios clásicos de de-escalamiento del nivel de atención (unidad de cuidados intermedios o sala de hospitalización) que propone el colegio americano de cuidado intensivo( ) . se recomienda aplicar la escala sofa al menos cada horas sumado al criterio de fragilidad y años de vida saludables salvados estos criterios podrían ser válidos en pacientes con fallo terapéutico a las medidas óptimas y orientar decisiones de deescalamiento de medidas y de nivel de complejidad de atención cuando la demanda potencial supera la oferta (capacidad y capacidades), estas decisiones deben ser tomadas idealmente en junta médica. amci ® se recomienda generar procesos administrativos más eficientes para el traslado de pacientes fuera de la uci hacia niveles de menor complejidad, estos ayudarán a liberar recursos para otros pacientes. se recomienda no utilizar el resultado de la rt-pcr positiva para decidir el egreso de la unidad de cuidados intensivos. la literatura no ha definido unos criterios de flexibilidad en uci para el traslado de pacientes con sospecha o diagnóstico confirmado de covid- ; y los criterios que definen la posibilidad de egreso de uci y que aplican a todos los pacientes, incluso aquellos ingresados por una condición crítica en relación con infección por covid- , están claramente definidos por el colegio americano de cuidado intensivo en su documento-guía del .( ). los principios fundamentales han sido revisados en los fundamentos de la pregunta . es importante implementar una estrategia de identificación temprana para aquellos pacientes con soporte vital avanzado que evolucionan progresivamente a fallo multiorgánico y pocas probabilidades de recuperación; el de-escalamiento de medidas y su posterior traslado fuera de la uci, liberará espacio para otros pacientes en situaciones de desborde de la demanda. la sociedad española de medicina intensiva, crítica y unidades coronarias (semicyuc) recomienda que en caso de complicaciones o que se prevea una mala evolución tanto clínica como funcional, se plantee el retiro terapéutico por futilidad y se inicie un manejo con medidas de soporte paliativo, tal como ha sido considerado en los fundamentos de la pregunta ( ) . para el grupo español es claro que, adecuar procesos administrativos más eficientes para traslado de pacientes fuera de la uci a los usualmente utilizados en condiciones normales de práctica clínica, ayudaría a ser más eficientes en la gestión de la demanda ( ) . finalmente, la persistencia de una rt-pcr positiva no es una contraindicación para el traslado del paciente fuera de la uci siempre y cuando se aseguren condiciones de aislamiento por contacto y aerosol en aquellas áreas de menor complejidad de atención; estas incluyen zonas de expansión o área del hospital específicamente acondicionadas para el manejo de pacientes estables o pacientes con limitación de esfuerzo y manejo paliativo de su condición ( ). recomendaciÓn se recomienda que la disposición final de cadáveres de personas con sospecha o diagnóstico de covid- se haga preferiblemente por cremación. en tal caso, las cenizas pueden ser objeto de manipulación sin que suponga ningún riesgo. amci ® se recomienda que la disposición final del cadáver se haga por inhumación en sepultura o bóveda individualizada cuando no se cuente con instalaciones para cremación en el territorio donde ocurrió el deceso o la disponibilidad de esta tecnología desborda la capacidad económica de las personas. se recomienda realizar siempre el aislamiento del cadáver en el lugar del deceso, siguiendo las recomendaciones del ministerio de salud y la protección social (msps). se recomienda que en los casos que se requiera necropsia médico legal y estuviese indicada la cremación, esta deberá contar con la orden del fiscal del caso. se recomienda que la institución establezca en sus procesos prioritarios un protocolo humanizado de despedida bajo estrictos criterios de bioseguridad. no existe una evidencia fuerte que recomiende hacer una disposición de cadáveres de pacientes fallecidos con sospecha o diagnóstico confirmado de covid- con un acto de cremación o con inhumación y sepultura en féretro; sin embargo, siempre y cuando la manipulación y manejo del cadáver hasta su disposición final se haga manteniendo todas las medidas de precaución para evitar la diseminación del virus y siguiendo la normatividad legal vigente, ambas formas son aceptadas. la infección por covid- es una enfermedad con gran variabilidad en presentación clínica, alta tasa de contagio y para la cual no existe en el momento un tratamiento definido. el riesgo de contagio al personal que ejecuta autopsias o procedimientos de tanatopraxia y la probabilidad de diseminación de la enfermedad por la manipulación de cadáveres no se conoce, pero se considera que puede ser alto, teniendo en cuenta que, en ausencia de la aplicación de un método de diagnóstico masivo, todo caso debe considerarse potencialmente positivo. por tanto, el manejo de cuerpos de personas fallecidas con diagnóstico confirmado, sospechoso o probable de sars-cov- (covid- ), debe realizarse con la mínima manipulación posible( ). el cadáver debe ser transferido lo antes posible al depósito y entregado al servicio funerario antes de horas luego del fallecimiento( ). el transporte, la cremación o inhumación, según sea el caso, se efectuará en el menor tiempo posible, con el fin de prevenir la exposición de los trabajadores y comunidad general al virus sars-cov- (covid- ). se debe evitar la realización de rituales fúnebres que conlleven reuniones o aglomeraciones de personas( ). el alistamiento del cadáver será realizado en el ámbito hospitalario del mismo sitio del deceso. las personas que accedan a la habitación donde se encuentre el cadáver, deben tomar las precauciones de transmisión por contacto y gotas, y para ello deben contar con todos los elementos de protección personal (epp) y seguir los procedimientos de amci ® bioseguridad de acuerdo con lo establecido en el protocolo del msps.( ) para hacer el alistamiento del cadáver, se debe cubrir todos los orificios naturales con algodón impregnado de solución desinfectante y se deberá envolver en su totalidad sin retirar catéteres, sondas o tubos que puedan contener los fluidos del cadáver, en tela antifluido o sábana; luego se deberá envolver en dos bolsas plásticas biodegradables que cumplan con las características técnico-sanitarias de impermeabilidad y resistencia a la presión de gases en su interior( ). se debe rociar el interior y el exterior de ambas bolsas con solución desinfectante de hipoclorito sódico que contenga . ppm de cloro activo( ) (exceptuando los casos asociados de covid- y muerte violenta). una vez el cadáver esté adecuadamente dispuesto en las bolsas, se podrá movilizar sin riesgo hacia el depósito de cadáveres siguiendo la ruta intrahospitalaria dispuesta para este traslado. luego el cadáver podrá será entregado al personal del servicio funerario para su depósito en ataúd o contenedor de cremación o inhumación y posterior traslado al sitio de destino final (horno crematorio y/o cementerio), luego de completar toda la documentación necesaria. cuando deba practicarse necropsia médico legal, el cuerpo será entregado a los servidores del sistema judicial quienes asumirán la custodia( ). si se han seguido correctamente todas estas indicaciones, se asume que no hay ninguna diferencia entre disponer del cuerpo enviándolo al crematorio o colocarlo en ataúd para llevarlo al tanatorio y realizar el entierro. si se opta por lo primero, las cenizas pueden ser objeto de manipulación sin que supongan ningún riesgo.( ) se recomienda que el trabajador de la salud conozca a través de la institución donde labora, los riesgos éticos, de salud y seguridad a que se expone por la atención en el paciente covid- , evitando así conflictos e incertidumbres que afecten la atención. se recomienda que las instituciones prestadoras de salud a través de los líderes de atención médica, guíen y orienten a los trabajadores, para ofrecer una mejor atención médica y menor daño emocional durante la pandemia. se recomienda dar a conocer las directrices institucionales sobre el nivel de atención (uci vs. sala médica); inicio del tratamiento de soporte vital (incluyendo rcp y soporte de ventilación); retirada del tratamiento de soporte vital; y derivación a cuidados paliativos (centrados en la comodidad) en la atención médica de los pacientes durante la pandemia, esto genera una atención caracterizada por alivio del sufrimiento, no abandono, respeto a amci ® los derechos y preferencias de los pacientes, igualdad moral de las personas y la equidad en la distribución de riesgos y beneficios en la sociedad. el apoyo de la práctica ética es necesario integrarlo al cuidado de la salud y al bienestar de la fuerza laboral del cuidado en salud. reconociendo los desafíos especiales a que se enfrentan al responder al covid- . esto forma parte del liderazgo en la atención médica y del servicio del deber cívico. berlinger n. el de abril del . en su artículo "respondiendo a covid- como un desafío regional de salud pública pautas preliminares para la colaboración regional que involucra hospitales". refiere que los trabajadores del equipo médico tienen el deber de conocer la gestión asistencial de los "desafíos éticos" previsibles durante la emergencia de salud pública (pandemia covid- ). que los desafíos éticos surgen cuando existe incertidumbre acerca de cómo "hacer lo correcto" es cuando los deberes o valores en los trabajadores entran en conflicto. estos desafíos afectan a la fuerza laboral (carga moral y emocional ante una decisión no prevista) en la atención médica. así como la operatividad en la atención médica (falta de epp y recursos que pueden limitar el buen desempeño por temor a infectarse).( ) los líderes de atención médica tienen el deber de guiar a los trabajadores de atención médica que experimentan condiciones laborales exigentes, mayor riesgo de daños ocupacionales, incertidumbre ética y angustia moral durante una emergencia de salud pública.( ) chih chen a, t. el de abril del . en su editorial ¿cómo deben prepararse los sistemas de salud para la evolución de la pandemia de covid- ? sugiere un apoyo emocional adecuado para el personal y horas razonables de exposición al riesgo para evitar el agotamiento, ya que los profesionales de la salud luchan por cuidar a los pacientes y proteger sus vidas y sus familias. se refiere que a medida que aumenta el número de casos, los médicos y los trabajadores de la salud en la primera línea deben reducir al mínimo su carga de trabajo clínico. las instituciones de atención médica deben reasignar al personal realizar tareas no clínicas, incluidos el papeleo y la recopilación de datos, tanto como sea posible. los hospitales deben tomarse su tiempo para capacitar al personal para implementar eficazmente las precauciones de contacto y los procesos de flujo. ( ) jick j.l. el de marzo del . en relación con la obligación de planificar la atención médica, considera que: los líderes de atención médica tienen el deber de planificar la gestión de los desafíos éticos previsibles durante una emergencia de salud pública. la planificación de los desafíos éticos previsibles incluye la identificación de posibles decisiones de triage, herramientas y procesos. en una emergencia de salud pública que presenta una enfermedad respiratoria grave, es posible que se deban tomar decisiones de clasificación sobre el nivel de atención (uci vs. sala médica); inicio del tratamiento de soporte vital (incluyendo rcp y soporte de ventilación); retirada del tratamiento de soporte vital; y derivación a cuidados paliativos (centrados en la comodidad) si el tratamiento de soporte vital no se iniciará o se suspenderá. es posible que también se deban tomar decisiones de clasificación en relación con la escasez de personal, espacio y suministros. el deber de cuidado fundamental requiere fidelidad al paciente (no abandono como una obligación ética y legal), alivio del sufrimiento y respeto a los derechos y preferencias de los amci ® pacientes. el deber de cuidado y sus ramificaciones son el enfoque principal de la ética clínica, a través de los servicios de consulta de ética clínica a pie de cama, el desarrollo de políticas institucionales y la educación y capacitación en ética para los médicos. deberes de promover la igualdad moral de las personas y la equidad (justicia en relación con la necesidad) en la distribución de riesgos y beneficios en la sociedad. estos deberes generan deberes subsidiarios para promover la seguridad pública, proteger la salud de la comunidad y asignar de manera justa recursos limitados, entre otras actividades. estos deberes y sus ramificaciones son el foco principal de la ética de la salud pública. no se puede emitir una recomendación a favor o en contra acerca del uso de los medicamentos y dispositivos "prototipos" utilizados en el manejo del covid- denominados de uso compasivo o fuera de etiqueta, se considera sin embargo que no pueden ser utilizados por fuera de ensayos clínicos o protocolos institucionales estandarizados de evaluación del perfil de riesgo/beneficio y bajo la aplicación de consentimiento informado. se entiende como uso compasivo la utilización, en pacientes aislados y al margen de un ensayo clínico. dichos medicamentos experimentales no han sido aprobados aún por la fda, ( ) y no se ha demostrado su seguridad y eficacia. es importante recordar que el medicamento médico puede tener efectos secundarios inesperados y graves, y que los pacientes deben considerar los posibles riesgos cuando procuran acceder a un producto médico experimental. hay que tener en cuenta que, para utilizar un medicamento bajo las condiciones de uso compasivo, se requerirá el consentimiento informado por escrito del paciente o de su representante legal, un informe clínico en el que el médico justifique la necesidad de dicho tratamiento. la regulación de la utilización de medicamentos por la vía del uso compasivo se ha realizado dentro de un texto legal sobre la realización de ensayos clínicos. una interpretación común del uso no indicado en la etiqueta y el uso compasivo de medicamentos es que, si el paciente murió, murió de la enfermedad, pero si el paciente sobrevivió, sobrevivió debido al medicamento administrado. recomendaciÓn se recomienda en la comunicación inicial con los familiares del paciente adulto con sospecha o diagnóstico de covid- críticamente enfermo incluir de forma clara y transparente los aspectos relacionados con el derecho al final de la vida que incluye: proporcionalidad en el tratamiento, adecuación del esfuerzo terapéutico, documento de voluntad anticipada, adecuación del esfuerzo terapéutico y la atención paliativa. situaciones estas que se pueden presentar durante la evolución hospitalaria y que requieren de una decisión conjunta entre el médico y el familiar del paciente. se recomienda dar una información específica, y adecuada a los familiares del paciente con sospecha o diagnóstico de covid- , para que firmen el consentimiento informado, generando esta información confianza y comprensión en el familiar. la información del consentimiento que recibe el familiar debe constar dentro de la historia clínica. fundamento el ministerio de salud y protección social del de marzo del a través del documento de "recomendaciones generales para la toma de decisiones éticas en los servicios de salud durante la pandemia covid- ". ante la posible circunstancia de pacientes sin capacidad para la toma de decisiones, por deterioro del estado general o requerimiento de aislamiento, en el cual no se puede contactar a su representante, conduzca la toma de decisiones teniendo en cuenta la prioridad de no hacer daño y la modificación en las condiciones de disponibilidad de los recursos en caso de presentarse deterioro. recuerde que esta situación debe preverse y debe ser informada a los representantes desde la admisión del paciente". durante toda la atención debe darse información sobre la posibilidad de que se presenten limitaciones de acceso a los soportes necesarios incluido al personal de salud , lo anterior puede limitar los derechos individuales o preferencias, esto debe ser informado al paciente y su familia, para que les permita entender que bajo la emergencia, puede presentarse una circunstancia que en condiciones habituales pudiera ser reversible de ser tratada pero en el contexto actual los recursos pueden verse trágicamente limitados, sin que esto implique abandono en el cuidado. el documento se refiere a la información que debe recibir el paciente o su familiar sustituto durante su evolución o fallecimiento. ( )la información durante la evolución también debe incluir: la información sobre el ejercicio de derechos al final de la vida incluyendo la adecuación de los esfuerzos terapéuticos y la suscripción de documentos de voluntad anticipada la consulta y revisión de existencia de este en todos los casos. se recomienda tener un consentimiento informado al ingreso hospitalario del paciente covid- , se deben tener en cuenta las circunstancias del paciente al ingreso hospitalario, si la capacidad para la toma de decisiones está limitada por su estado clínico o incapacidad mental. de ser estas las circunstancias se dará la información al familiar en primera línea de consanguinidad quien asume por el paciente el consentimiento de la información (consentimiento sustituto). se recomienda tener el consentimiento informado en situaciones de excepción o urgencia ante la pandemia por covid- , debe ser universal, en el que se informe el ingreso a la uci, o a cualquier otra área hospitalaria, realización de procedimientos, administración de tratamientos, posibles riesgos, beneficios durante su hospitalización. con esto se respeta el derecho a la autonomía personal en el paciente competente. en caso contrario el familiar tomará la información y asume el consentimiento a la información dada. es importante que el familiar esté informado de las decisiones que se vayan tomando durante la evolución hospitalaria (realización de procedimientos, inicios o cambios de tratamientos, movilización dentro del área hospitalaria. etc.) fuerte a favor fundamento el ministerio de salud y protección social, el de marzo del ha elaborado un formato de "consentimiento informado para acompañante de casos probable/confirmado de covid- ". en que se expresa: "que de manera detallada se me ha suministrado información completa, suficiente, con un lenguaje sencillo y claro. el profesional de la salud me ha explicado la naturaleza de la enfermedad, acerca del significado de caso sospechoso o confirmado del coronavirus covid- en cuanto a su presentación clínica, modo de contagio, medidas para contenerla, posibilidad de sufrir la enfermedad, complicaciones o muerte, mientras permanezca como acompañante del paciente". este documento se firma al ingreso por el acompañante o familiar quien asume las decisiones durante su estancia hospitalaria. ( ) el consentimiento informado en los pacientes covid- será un consentimiento sustituto para su ingreso a la uci y para los procedimientos que en la uci se realicen (colocación de tubo orotraqueal, diálisis, colocación de catéteres, reanimación cardiopulmonar, ecmo, etc.). previa información y autorización del familiar. en circunstancias normales el consentimiento debe ser firmado por el paciente quien en su autonomía acepta la información sobre su manejo y tratamiento. feld ad. recomendaciÓn se sugiere ante la pandemia del covid- , si es posible, que el grupo de expertos en bioética y/o comité de ética institucional sean consultados y estén informados por el médico responsable para la orientación o consejo en la toma de directrices ante decisiones difíciles. se sugiere en lo posible que el médico tratante no asuma solo la responsabilidad moral de la decisión y que la decisión sea institucional y documentada en la historia clínica e informada a los familiares. en caso de no contar con un comité de bioética y/o ética hospitalaria el médico responsable podrá tomar la decisión fundamentada en principios éticos y derechos del paciente o convocar a una junta médica u otro comité relacionado con su dilema o consultar un apoyo externo en bioética. débil a favor fundamento el ministerio de salud y protección social. ( ) el de marzo del en el documento "recomendaciones generales para la toma de decisiones éticas en los servicios de salud durante la pandemia covid- establece: "que en caso de que la institución cuente con un comité de bioética y/o Ética, con el servicio de bioética o Ética clínica, o consultoría clínico-ética, se debe definir una ruta de consulta para los casos que de forma concreta puedan superar las recomendaciones generales. de igual forma establece que en las circunstancias actuales que se viven el actuar ético es parte integral del profesionalismo del cuidado. teniendo en cuenta que los profesionales de la medicina serán los llamados a tomar decisiones de alto estrés moral, al tener que adherirse y promover conductas concretas basados en las circunstancias que les rodean para dar o retirar tratamientos de las personas infectadas, quienes esperan confirmación del diagnóstico y de las personas que acuden a la atención en salud por razones diferentes a la infección por covid- . partiendo de lo antes referenciado, se recomienda que las acciones emprendidas, busquen siempre poder responder a: a. no hacer daño b. beneficiar c. actuar con justicia sobre la persona en el contexto de la emergencia frente a la justicia sanitaria de la población d. mantener la integridad profesional" el comité de bioética y/o comité de ética institucional en tiempos normales o en tiempos de pandemia deberá mantener actualizadas las directrices de toma de decisiones. que apoyaran al médico responsable en la decisión. de no contar el médico con dicho comité o directrices institucionales y deba tomar una decisión que no permite interconsultar, deberá justificar en la historia clínica fundamentado en los principios éticos y derechos del paciente las razones que lo llevaron a tomar la decisión e informar y dialogar con los familiares del hecho. esto es importante que siempre quede documentado en la historia clínica la acción moral y ética de la decisión y el diálogo con la familia. en caso de que el médico responsable no cuente con un comité de bioética y/o comité de ética institucional, ni con el apoyo externo de expertos en bioética. y no quiera tomar la amci ® decisión a título personal podrá consultar a otro médico de la institución su decisión y entre ambos definir la acción a seguir. esta decisión conjunta debe ser documentada en la historia clínica e informada al grupo de trabajo y a los familiares como junta médica. si los familiares después de recibir la información no quedan satisfechos ellos tienen el derecho a la segunda opinión. los comités de bioética y/o comités de ética hospitalaria son entes administrativos, consultores, orientadores, asesores y consejeros de las situaciones que tienen que ver con el respeto y cumplimiento de los principios éticos, deberes y derechos de los pacientes, sus recomendaciones no son vinculantes, apoyan y orientan la decisión médica. aconsejando la mejor decisión ante una situación que genera un dilema moral o ético en el médico responsable. las decisiones médicas son tomadas por el consultor en bioética, quien es médico. los comités institucionales fuera de un comité de bioética y/o ética que podría dar apoyo al médico responsable y que serían otras instancias consultivas serian el comité de humanización, comité de historias clínicas, comité de bioseguridad, comité de infectología, comité de mortalidad hospitalaria, comité de paliativos o un comité de gerencia. pues todos los mencionados tiene que ver con el bienestar del paciente y la seguridad del médico ante una decisión. las decisiones especiales deben ser tomadas inicialmente a través de la realización de un comité (pueden ser los mencionados), en su defecto una junta médica. una vez se tenga la decisión esta debe ser consultada a la familia como una decisión institucional respaldada por el comité o junta médica realizada. se recomienda que todo ensayo clínico que se realice en la institución debe ser presentado, revisado y aceptado por un comité de investigación local o un comité de investigación externo nacional o internacional. se recomienda que todo paciente que se incluya en un ensayo clínico debe contar con un consentimiento informado el cual garantiza la aceptación voluntaria a participar y la comprensión de los objetivos, riesgos, beneficios, derechos y responsabilidades que tiene dentro de la investigación. se recomienda el consentimiento informado en todo ensayo clínico, el cual debe ser debe ser individual en tiempos de normalidad como en tiempos de pandemia por covid- . solo el comité de ética en investigación podrá establecer en tiempos de normalidad o de pandemia las condiciones de dispensa o excepción al requisito de obtener el consentimiento informado. refiere que el consentimiento informado tiene sus raíces en el código de núremberg de y la declaración de helsinki de y ahora es un principio rector para la conducta en la investigación médica. en el consentimiento informado para investigaciones clínicas es claro que los participantes deben entender ampliamente los componentes del consentimiento. ( ) thanh tam, n. et al. el de enero . mediante una revisión sistemática de pubmed, scopus y google scholar y revisando manualmente las listas de referencias para publicaciones hasta octubre de . realizó un metaanálisis de los resultados del estudio utilizando un modelo de efectos aleatorios para tener en cuenta la heterogeneidad. evaluó la proporción de participantes en ensayos clínicos que entienden los diferentes componentes del consentimiento informado. encontrando que los participantes en ensayos clínicos deben comprender los componentes fundamentales del consentimiento informado como: la naturaleza y los beneficios del estudio, la libertad de retirarse en cualquier momento y la naturaleza voluntaria de la participación, así como la comprensión de otros componentes, como la aleatorización y el placebo. la proporción de participantes en ensayos clínicos que comprendieron diferentes componentes del consentimiento informado varió de . % a . %. esto asegura que la toma de decisiones de los participantes es significativa y que sus intereses están protegidos. ( ) la red de américa latina y el caribe de cnb-unesco, ( de marzo de ) que agrupa a las comisiones y consejos nacionales de bioética cuya finalidad es la de asesorar sobre los problemas éticos relativos a las ciencias de la vida y la salud humana expresa su preocupación ante la realización de investigaciones biomédicas en relación con la pandemia de enfermedad infecciosa por coronavirus covid- . reconociendo lo siguiente en relación con el consentimiento informado: que la investigación con seres humanos durante las emergencias debe contar con garantías éticas mayores, no menores, que en las situaciones ordinarias. que en situación de excepción o emergencia los participantes deben seleccionarse en forma justa y proporcionar una justificación adecuada cuando se escogen o excluyen determinadas poblaciones, distribuyendo en forma equitativa las posibles cargas y beneficios de participar en esa investigación. "que se debe obtener el consentimiento informado individual de los participantes incluso en una situación de excepción o alarma, a menos que se cumplan las condiciones para la dispensa del consentimiento informado. las cuales el comité de revisión ética solo puede decidir dar la dispensa al requisito de obtener consentimiento informado: a) si no es factible obtenerlo; y si además los estudios: b) tienen un importante valor social y científico, c) solo suponen riesgos mínimos para los participantes, d) no implican agravio comparativo con otros grupos en situación o no de vulnerabilidad; y si e) se garantiza que no se privará a la población investigada de acceder en forma preferencial al beneficio derivado. de otorgarse un consentimiento informado amplio, éste debería ser única y exclusivamente para los procesos asociados con covid- ".( ) amci ® se recomienda considerar la transición del cuidado intensivo al cuidado paliativo en todo paciente con sospecha o diagnóstico de covid- sin mejoría a pesar de las intervenciones óptimas, con empeoramiento progresivo de su pronóstico vital y ante un evidente deterioro; aplicando medidas generales en control de síntomas ( manejo de secreciones -tratamiento del dolor -tratamiento de la disnea -sedación paliativa), así como apoyo espiritual, siempre acompañando al paciente y nunca abandonarlo en el final de la vida. fuerte a favor fundamento la sociedad española de anestesiología, reanimación y terapéutica del dolor en su documento: "marco Ético pandemia covid- " madrid, de marzo de refiere: la sedación paliativa en pacientes hipóxicos con progresión de la enfermedad no subsidiaria de tratamiento debe considerarse como una expresión de buena práctica clínica y debe seguir las recomendaciones existentes. si se prevé un período agónico no corto, se debe proporcionar una transferencia a un entorno no intensivo.( ) se recomienda la utilización de guías establecidas previamente a la pandemia por el ministerio de salud y sociedades científicas para orientar las decisiones que se tomen al final de la vida en pacientes con sospecha o diagnóstico de covid- . estas guías deben ser divulgadas al equipo de atención y aplicadas en los pacientes en casos de: adecuación del esfuerzo terapéutico (aet), orden de no reanimar (onr), consentimiento sustituto, voluntades anticipadas, cuidados paliativos. se recomienda fundamentar las decisiones del final de la vida individualizadas a cada paciente y a cada situación sin llegar a tomar decisiones apresuradas sin fundamento científico o ético, solicitando de ser posible una valoración por medicina paliativa para el manejo de síntomas. se recomienda indagar durante la hospitalización de todo paciente con sospecha o diagnóstico de covid- , si en tiempos de salud hizo válida su autonomía y realizó un documento de voluntad anticipada, teniendo en cuenta que esta será equivalente al consentimiento informado. amci ® terapéuticos y la suscripción de documentos de voluntad anticipada.( ) el inicio de sedación paliativa con reubicación del pacientede ser necesario el des escalonamiento por deterioro clínico. en situación de pandemia covid- cuando se refiere a situaciones del final de la vida se relaciona a la adecuación del esfuerzo terapéutico, la sedación paliativa la cual será la maniobra terapéutica que se utilizará en pacientes no recuperables y que no son candidatos a cuidados intensivos por covid- ,( ) que evolucionan desfavorablemente y tienen mal pronóstico a corto plazo, así como la disnea refractaria y la limitación del esfuerzo terapéutico. el delirium o síndrome confusional por fallo cerebral agudo, es un problema habitual en situaciones de alteración orgánica severa, y ha sido descrito como uno de los síntomas neurológicos presente en los pacientes que sufren infección por el covid- ( ). wilson c. de abril del . en su artículo "la crisis golpea al final de la vida" se refiere a que el brote de coronavirus está obligando a las personas a enfrentar dilemas en torno a la cantidad de atención médica que se debe brindar al final de la vida y apresurar decisiones controvertidas sobre rechazar ciertos tratamientos. dicen los expertos que esto ha alentado a más personas a tomar decisiones de tratamiento anticipadas relacionadas con la rcp y la ventilación,( ) haesen s. el de mayo de . en su artículo "dirigir a los ciudadanos a crear directivas anticipadas" las voluntades o directrices anticipadas son para las personas que quieran asumir plenamente su papel de ciudadanos responsables tomando decisiones proactivas. la decisión de redactar directivas anticipadas marca un cambio del enfoque actual de "aceptación" a un escenario de "exclusión voluntaria".( )al emitir una directiva de tratamiento anticipado, una persona autónoma puede expresar formalmente qué tipo de tratamiento desea y no desea recibir en caso de que se enferme o se lastime y no pueda decidir de manera autónoma sobre su tratamiento. ( ) ministerio de salud y protección social en su documento de voluntades anticipadas que es el documento en el que toda persona capaz, sana o en estado de enfermedad, en pleno uso de sus facultades legales y mentales y como previsión de no poder tomar decisiones en el futuro, declara, de forma libre, consciente e informada su voluntad sobre las preferencias al final de la vida que sean relevantes para su marco de valores personales.( ) se recomienda que el paciente crítico con covid- que no es candidato para ingresar o continuar recibiendo cuidados intensivos y que presente deterioro rápido con mal pronóstico a corto plazo, se le brinde una adecuación del esfuerzo terapéutico orientada a acompañamiento al final de la vida, alivio del sufrimiento y control de síntomas. dependiendo de la disponibilidad de recursos se sugiere dentro del plan de atención hospitalaria contar con un área destinada a la atención del final de vida con el recurso físico, humano y de procesos necesario. marzo del . refiere: en situación de adecuación terapéutica, retirada de medidas y/o mala evolución es adecuado derivar al paciente a un área de menor complejidad para establecer el plan de cuidados paliativos. consultar al servicio de cuidados paliativos para procurar la continuidad de cuidados de los pacientes en los que se haya acordado la limitación de tratamientos y aliviar su sufrimiento, incluyendo la sedación paliativa en los casos en los que sea precisa.( ) schmidhauser tf. el de abril . considera en su publicación que: los cuidados paliativos durante la pandemia de covid- deben adaptarse a un estilo de" cuidados paliativos de emergencia" ya que los pacientes pueden deteriorarse rápidamente y requieren decisiones rápidas y planes de tratamiento claros. estos deben ser seguidos fácilmente por los miembros del personal de salud que atienden a estos pacientes. además, los cuidados paliativos deben estar a la vanguardia para ayudar a tomar las mejores decisiones, atender a las familias y ofrecer apoyo espiritual.( ) se recomienda como estrategia de protección personal en las unidades de cuidado intensivo sin presión negativa y cubículos abiertos utilizar de forma continua el respirador n o fpp , adicional a otros elementos de protección para prevención del contagio por covid- . la atención segura en áreas crítica para todas las modalidades de atención se fortalece a través de las medidas de precauciones estándar en el manejo de todos los pacientes, establecidos en el "manual de medidas básicas para control de infecciones en prestador servicios de salud" ley de , por la cual se dictan medidas sanitarias. resolución de , numeral y numeral . epp. el respirador, n o fpp , puede utilizarse de manera continua por a horas, o desecharlo antes si está visiblemente contaminada o si está húmeda. luego de colocar la n se debe verificar prueba de ajuste antes de ingresar a la unidad para atención de pacientes con covid- de la siguiente manera: mascarillas sin válvula de exhalación: cubra la totalidad de la mascarilla con ambas manos y exhale con fuerza. si nota fugas de aire por sus bordes, reajuste la posición del respirador. mascarillas con válvula de exhalación: cubra el respirador con ambas manos e inhale con energía. deberá sentir una presión negativa dentro de la mascarilla. si detecta alguna pérdida de presión o entrada de aire, reajuste la posición del respirador. no se puede emitir una recomendación a favor o en contra acerca de la efectividad de bioseguridad del uso extendido, continuo o intermitente de los respiradores n ó ffp . sin embargo, se considera que puede ser una alternativa, bajo la adopción de un protocolo riguroso, cuando se debe optimizar el uso de los epp en el contexto de un acceso limitado. el reúso no está permitido en colombia. la duración máxima del uso continuo de la n es de a horas, siguiendo las recomendaciones del manual de medidas básicas para control de infecciones en ips de minsalud. pero en la vida real, ningún trabajador tolera a horas continuas con un respirador. por esto, su uso continuo en el sitio de trabajo dependerá de la necesidad de pausar para comer, para ir al baño, etc. en este caso, se guardará en una bolsa de papel para su nueva colocación, si tiene menos de horas, o desechará si está visiblemente contaminada o se torna húmeda. el reúso de la n dependerá de la casa del fabricante, de si contiene o no celulosa en su estructura del respirador. por ejemplo, la recomendación de desinfección para los respiradores sin celulosa es con peróxido de hidrógeno vaporizado al % por minutos (tabla y ). los respiradores n de uso industrial tienen mayor contenido de celulosa que los de uso médico por lo tanto para procesos de esterilización, solo los n de uso médico podrán ser esterilizados mediante de peróxido de hidrógeno vaporizado (sterrad®) ( - ) . ¿existe superioridad en términos de protección personal para el personal sanitario y de apoyo dentro de las unidades de cuidados intensivos con la utilización del overol frente a la bata manga larga con antifluido durante la atención del paciente con covid- ? no se puede emitir una recomendación a favor o en contra si los overoles ofrecen mayor protección por cobertura corporal frente a otros elementos como vestidos largos, batas y delantales. resulta intuitivo que su uso genera una mayor protección en especial en servicios cohortizados. sin embargo, su uso está asociado con una mayor dificultad para su colocación y retiro, lo que puede potencializar el contagio del usuario, la utilización debe hacerse bajo un protocolo supervisado y chequeado. las batas modificadas para ajustarse firmemente en el cuello pueden reducir la contaminación. en estudios ya descritos el uso de un respirador eléctrico purificador de aire con overol puede proteger contra el riesgo de contaminación mejor que una máscara n y una bata con un rr: . , intervalo de confianza (ic) del %: . a . pero fue más difícil su retiro con rr . , ic del % . a . . en un eca ( participantes), las personas con una bata larga tenían menos contaminación que aquellas con un overol. las batas pueden proteger mejor contra la contaminación que los delantales.( - ) los epp como batas y overoles deberían estar hechos de un material que cumpla con los requisitos mínimos de la asociación americana de químicos textiles:  tipo a: buena repelencia al agua, resistente a la penetración, pero mala permeabilidad al aire.  tipo b: buena repelencia al agua, buena permeabilidad al aire, pero poca resistencia a la penetración del agua.  tipo c: bata quirúrgica que tiene poca repelencia al agua y resistencia a la penetración de agua.  tipo d: hecho de fibras de polietileno de alta densidad, tela no tejida (tyvek®), tiene buena repelencia y resistencia al agua, mala permeabilidad al aire. no se puede emitir una recomendación a favor o en contra para el uso de respiradores elastoméricos como elementos de protección personal dentro de las unidades de cuidados amci ® intensivos. no hay evidencia que soporte la superioridad de los respiradores elastoméricos frente a los n , son más costosos, difíciles de utilizar y pueden implicar algún riesgo para el paciente. por tanto, su uso sólo debería considerarse frente a un desabastecimiento de los n y bajo la adopción de un protocolo institucional riguroso y bajo chequeo. los respiradores elastoméricos son respiradores ajustados a media cara o cara completa, esta última otorga protección ocular. su filtración está determinada por el filtro que se utilice, estos van desde partículas de nivel n a p . están hechos de material sintético o de goma que les permite desinfectarse, limpiarse y reutilizarse repetidamente. están equipados con cartuchos de filtro reemplazables. al igual que los respiradores n , los respiradores elastoméricos requiere entrenamiento adecuado para su correcta colocación y retiro. por eso es muy importante revisar el manual del usuario antes de su uso. los respiradores elastoméricos no deberían utilizarse en entornos quirúrgicos, debido al riesgo potencial de contaminación del campo quirúrgico, con el aire que sale de la válvula de exhalación. como recomendación de buena práctica, aprobado por la fda, debe colocarse una máscara quirúrgica encima de la válvula de exhalación para evitar este riesgo. solo se debe permitir el uso del respirador elastoméricos por clínica para evitar infecciones cruzadas, esto permitirá una protección esencial contra agentes infecciosos y la auto contaminación. aunque los cartuchos de filtro son finalmente desechables, están destinados a ser reutilizados hasta que ya no se puede respirar o se vuelven visiblemente sucio. generalmente se recomienda, en la mayoría de los casos, hacer recambios cada días. deben tener procedimientos de limpieza/desinfección actualizados y aprobados por su manufacturador.( , - ) recomendaciÓn se recomienda realizar la limpieza y desinfección de equipos biomédicos y de superficies las veces que sean necesarias y en el momento de egreso del paciente siguiendo los protocolos de cada institución. el desinfectante para este proceso debe ser de nivel intermedio o alto para superficies y equipos biomédicos y cumplir con las recomendaciones del fabricante según lo aprobado en el registro sanitario. fuerte a favor fundamento para la desinfección de las superficies ambientales hospitalarias y domiciliarias, la oms recomienda emplear un desinfectante que sea efectivo contra virus cubiertos (el coronavirus pertenece a esta categoría), específicamente, recomienda emplear alcohol etílico para la desinfección de algunos equipos biomédicos reusables (p. ej.: termómetros) y para las superficies, el hipoclorito de sodio o precursores de sodio como el dicloroisocianurato de sodio (nadcc) que tiene la ventaja de la estabilidad, la facilidad en la dilución y que no es corrosivo.( ) (tabla ). page se recomienda que el ingreso de un paciente a uci debe hacerse bajo un procedimiento estandarizado que incluya la coordinación y comunicación de los servicios vinculados, adecuación de la unidad de atención a las necesidades del paciente y la garantía de la bioseguridad del equipo multidisciplinario. se recomienda que cada institución establezca en sus procesos prioritarios el circuito del traslado que incluye el itinerario del traslado, el uso de ascensor, el número y la organización de los intervinientes sanitarios y no sanitarios (celadores, seguridad, limpieza), las medidas de protección empleadas por los mismos (epp, limpieza) y los recursos materiales necesarios durante el traslado. el traslado de pacientes con casos sospechosos o confirmados de covid- se puede presentar entre servicios a nivel hospitalario o entre instituciones con diferentes niveles de atención y deben tenerse las precauciones universales de un traslado seguro. una posición responsable es evitar el traslado de estos pacientes el máximo posible, a menos que sea imprescindible, teniendo en cuenta el riesgo/beneficio. considerar evitar traslados interinstitucionales solo por temas administrativos. el personal sanitario que realice el traslado debe contar con todos los epp, considerando este traslado como de alto riesgo de transmisión vírica. se debe utilizar mascarilla quirúrgica o n- , de acuerdo con el riesgo amci ® de aerosolización. hasta que la rt-pcr para sars-cov- este negativo se podrían retomar las prácticas habituales de traslado de los pacientes . ( ) ( ) ( ) capítulo . abordaje diagnóstico y covid- se recomienda en pacientes con diagnóstico o sospecha de infección por sars-cov- clasificar la enfermedad en leve, severo o paciente crítico, teniendo en cuenta los criterios de la clasificación por las fases y estadios de la enfermedad. se recomienda en pacientes con diagnóstico o sospecha de infección por sars-cov- , clasificados como críticos y que requieren de intubación orotraqueal realizar la clasificación por fenotipos ( o ), con el fin de proyectar una estrategia de ventilación mecánica. el covid- , es una enfermedad con una presentación clínica diversa, desde formas leves hasta presentaciones graves que incluyen el sdra, la mediana del período de incubación desde la exposición hasta el inicio de los síntomas es de aproximadamente a días, y el . % de los pacientes sintomáticos tendrán síntomas dentro de los . días después de la infección ( ) , que incluye fiebre, tos, disfagia, malestar general, mialgias, anorexia, náuseas, diarrea, anosmia y ageusia; la disnea se presentó entre los y días ( ) y puede representar progresión a covid- severo, que se manifiesta con hipoxemia, disfunción orgánica múltiple, documentación de arritmias cardíacas, rabdomiólisis, coagulopatía y choque ( ) . dentro del espectro de enfermedad, siddiki et al, proponen un enfoque estructurado por fases expresados en tres estadios (historia natural de la enfermedad), siendo el primero donde la patogenicidad viral es dominante, se incluye el periodo de incubación, síntomas leves, con multiplicación del sars-cov- centrándose principalmente en el sistema respiratorio gracias a la unión del virus con el receptor de la enzima convertidora de angiotensina (ace ), el hemograma puede revelar linfopenia y neutrofilia sin otras anormalidades significativas. el estadio es la enfermedad pulmonar establecida, neumonía viral, tos, fiebre con progresión en algunos casos a hipoxia con trastorno de los índices de oxigenación (pao /fio menor mmhg), hallazgos en imágenes de tórax (radiografía y/o tomografía) de infiltrados alveolares o vidrio esmerilado, mayor linfopenia y elevación de transaminasas ( a: sin hipoxemia, b: con hipoxemia). el estadio o fase de híper inflamación sistémica extrapulmonar se caracteriza por elevación de biomarcadores inflamatorios y estado protrombótico (il - , il- , il- , ftn -α, proteína c reactiva, ferritina y el dímero d), con presencia en las formas más graves de disfunción orgánica múltiple, lesión miocárdica (troponina y péptido natriurético de tipo b elevados), con fenómenos trombóticos, progresión a sdra y choque ( ) . amci ® la neumonía por sars-cov- , se característica por disociación entre la severidad de la hipoxemia y el mantenimiento relativamente bueno de la mecánica respiratoria, con compliance del sistema respiratorio en promedio de ml / cmh o; gattinoni, marini et al, proponen dos fenotipos de presentación de la insuficiencia respiratoria; el primero (tipo ) con una mecánica pulmonar adecuada, con baja probabilidad de reclutabilidad y con hipoxemia, relacionado al desbalance entre la perfusión y la ventilación; el segundo (tipo ) más acorde a las definiciones de sdra (csdra "covid- patient with sdra"), con una compliance pulmonar baja y reclutabilidad potencial( , ) ( ). se propone la siguiente clasificación clínica del covid- . ( , , y pacientes críticos de %, con tasa global de mortalidad de , %, siendo mayor entre los pacientes de a años con % y entre los mayores de años con . %, dentro del grupo de pacientes clasificado como crítico la mortalidad descrita fue del %( ). recomendaciÓn se recomienda en cuidado intensivo, realizar el diagnóstico de covid- del paciente sospechoso por medio de rt-pcr conociendo su alta especificidad, su variabilidad en relación con el tiempo y pérdida de rendimiento diagnóstico luego de la primera semana de inicio de los síntomas. se recomienda tomar la primera muestra para rt-pcr de hisopo nasofaríngeo o de cornete medio sobre hisopado oro faríngeo o de saliva, de ser negativo se puede repetir la prueba de a horas preferiblemente de tracto respiratorio inferior, esputo no inducido o en aspirado traqueal en paciente intubado. se recomienda el uso conjunto de rt-pcr e igm por elisa en pacientes con sospecha de covid- , primera rt-pcr negativa, que se encuentren entre la segunda y tercera semana desde el inicio de los síntomas, con el objetivo de mejorar la sensibilidad en la identificación de infección por sars-cov- . en cuidado intensivo, el diagnóstico de covid- se fundamenta con base en la presentación clínica compatible y factores epidemiológicos asociados con probabilidad de infección; el diagnóstico definitivo se realiza con pruebas de amplificación de ácido nucleico del virus (naat), la detección del genoma viral del sars-cov- se realiza por medio de reacción en cadena de la polimerasa por transcriptasa reversa (rt-pcr) dado a su especificidad del % ( , ) ; por lo cual todo paciente que cumple con la definición de caso sospechoso se le debe realizar rt-pcr, sars-cov- independientemente de si se encuentra otro patógeno respiratorio ( ) . las muestras para el diagnóstico por rt pcr se recolecta de las vías respiratorias superiores, nasofaringe, cornete medio u orofaringe; todos con alta especificidad. sin embargo, se sugiere recolectar los hisopos nasofaríngeos o de cornete medio por tener mayor sensibilidad ( ( / ) . en pacientes con neumonía severa a quienes se le realizó lavado broncoalveolar (bal) y rt-pcr entre los días y el % de las muestras fueron positivos, en pacientes no intubados con esputo no inducido el % de las muestras fueron positivas ( , ). wang et al, en un estudio de pacientes con covid- , las rt-pcr con tasas positivas más altas fue en muestras extraídas por bal ( %; de muestras) y esputo % ( de muestras) ( ) . para la detección del sars-cov- por rt-pcr en pacientes en cuidado intensivo, teniendo en cuenta la rigurosidad de aspectos de bioseguridad y aerosolización, se debe tomar la primera muestras en nasofaringe o cornete medio, si esta prueba es negativa se puede repetir en a horas, si este es el caso o existe más de días desde el inicio de los síntomas se prefiere una muestra del tracto respiratorio inferior, por esputo no inducido por personal de salud o por aspirado traqueal en pacientes intubados ( , ), aunque el rendimiento diagnóstico del bal es alto por lo general, se debe evitar la broncoscopia para minimizar la exposición de los trabajadores de la salud ( ) . la probabilidad de detección del arn de sars-cov- puede variar según la fase de la enfermedad, si bien una rt-pcr positiva confirma el diagnóstico de covid- , los reportes falsos negativos y la sensibilidad se ve influenciado por el tiempo desde la exposición e inicio de síntomas. kucirka et al, en un análisis de siete estudios evaluaron el rendimiento diagnóstico de la rt-pcr en relación con el tiempo desde el inicio de los síntomas o la exposición, con resultados expresados en tasa estimada de falsos negativos, siendo del % el día de la exposición, del % el día (estimado como primer día de síntomas, ic: % a %), % en el día (día desde el inicio de síntomas, ic: % a %) luego comenzó a aumentar nuevamente de % en el día (ic: a %) a % en el día (ic: a %) ( ) . la precisión y los valores predictivos de rt-pcr para sars-cov- no se han evaluado sistemáticamente, la sensibilidad de las pruebas moleculares está influenciada por múltiples factores como sitio y calidad de la muestra, técnica de procesamiento; probablemente las menores tasas de falsos negativos (sensibilidad entre y %) está entre el día y luego de inicio de los síntomas ( ) . a partir de aquí el rendimiento diagnóstico disminuye, por lo tanto, es importante que el intensivista valore estas consideraciones en el momento de tomar conductas, en cuanto tipo de aislamiento, tratamiento y pronóstico. amci ® las pruebas serológicas detectan anticuerpos contra el sars-cov- y ayudan a identificar pacientes que han tenido la enfermedad y algunos con la enfermedad activa, la seroconversión se ha descrito entre el día y , sin embargo, hay incertidumbre en la incidencia de la seroconversión ( ) . estas pruebas se usan principalmente en tamizaje poblacional y estudios de seroprevalencia; en cuidado intensivo el análisis de la igm por elisa contribuye a la detección de pacientes con infección reciente, además con el análisis conjunto con la igg se clasifica el estado de infección en agudo o convaleciente. las pruebas serológicas se realizan por diferentes técnicas como la inmunocromatográfica de flujo lateral, la inmunofluorescencia indirecta (ifi) y el ensayo de inmunoadsorción ligado a enzima (elisa) ( , ) . las pruebas serológicas no deben usarse como la única prueba para diagnosticar o excluir la infección activa por sars-cov- . la sensibilidad y la especificidad de muchas de estas pruebas serológicas son inciertas, así como su valor predictivo positivo. los anticuerpos detectables generalmente tardan varios días en desarrollarse. guo et al, documenta niveles de anticuerpos por elisa, con una mediana de detección de anticuerpos igm e iga de días (iqr, - ) y de igg de días (iqr, - ) después del inicio de los síntomas, con una probabilidad de resultados positivos de . %, . % y . % respectivamente; es probable que el rendimiento diagnóstico de igm por elisa sea mayor que la de rt-pcr después del quinto día luego de inicio de síntomas; cuando se combinan estas técnicas (elisa igm con rt-pcr) la tasa de detección positiva es del . % ( ) . zaho et al, en un estudio de pacientes con covid- , donde el , % estaba en condición crítica, la mediana del tiempo desde el inicio de los síntomas hasta la detección de anticuerpos (técnica elisa) fue de días para igm y días para igg; dentro de los primeros días desde el inicio de los síntomas solo el . % tenía anticuerpos detectables, entre los días a la sensibilidad de igm fue . % e igg de . %, luego de los días la sensibilidad igm e igg fue de . % y . % respectivamente; el uso combinado de rt-pcr y elisa igm presentó una sensibilidad del % entre los días a y del % entre los días a . ( ) la rt-pcr tiene especificidad del %, con adecuado rendimiento diagnóstico entre los días y luego del inicio de los síntomas con sensibilidad que varía entre el y %, con presencia ascendente de falsos negativos luego del día , por lo cual el diagnóstico debe tener consistencias epidemiológicas y clínicas (síntomas y hallazgos radiológicos compatibles con covid- ) donde una rt pcr negativa no excluye la enfermedad; la precisión y el tiempo para la detección de anticuerpos varían con la técnica utilizada, su uso es limitado en cuidado intensivo, sin embargo su identificación por técnica elisa en conjunto con rt-pcr mejora la sensibilidad y la probabilidad de falsos negativos, especialmente entre los días y desde el inicio de síntomas. faltan estudios que evalúen el rendimiento diagnóstico de las diferentes pruebas. amci ® se recomienda la medición de marcadores de severidad al ingreso a uci del paciente críticamente enfermo por covid- (hemograma, transaminasas, ldh, ferritina, troponina, dímero d y pcr) los cuales se han asociado con peor pronóstico en la enfermedad por covid- , logrando ofrecer intervenciones más tempranas. se recomienda no utilizar una periodicidad de rutina para la medición de seguimiento de biomarcadores de severidad en el paciente con sospecha o diagnóstico de covid- . en un estudio cohorte retrospectivo que evaluó pacientes diagnosticados con covid- desde el de enero de hasta el de marzo de , fang liu y colaboradores encontraron correlación en la elevación de il- y pcr con la gravedad clínica, lo que sugiere podrían usarse como factores independientes para predecir la severidad del cuadro, los pacientes con il- > . pg./ml o pcr> . mg/l tenían más probabilidades de tener complicaciones graves ( ) , así mismo en otro estudio multicéntrico retrospectivo de pacientes infectados se identificó resultados de laboratorio con diferencias significativas con elevación de glóbulos blancos, valores absolutos de linfocitos, plaquetas, albúmina, bilirrubinas, función renal, transaminasas, troponina, proteína c reactiva e interleucina (il ) en el grupo con desenlace de mortalidad contra los dados de alta ( ) . entre otros marcadores la troponina como lesión cardiaca (elevación de troponina por encima del percentil límite de referencia superior) se ha reportado en % a % de los pacientes con covid- en wuhan, china, en dos estudios retrospectivos por xiabo yang y colaboradores ( , ) . en una revisión sistemática de mayo , kermali m. y colaboradores exponen que existe evidencia a favor de los valores bajos de linfocitos y plaquetas y valores elevados de los biomarcadores il- , pcr, troponina, ldh, ferritina, proteína amiloidea a y dímero d, pueden relacionarse con la gravedad de la infección por covid- y su fuerte asociación con la mortalidad ( ) . estos resultados pueden usarse como un complemento en la práctica clínica para guiar a los médicos a identificar pacientes con mal pronóstico y la rápida implementación de medidas de soporte, monitorización y reanimación en la evolución de los pacientes críticos en la unidad de cuidados intensivos. solo en estudio, karmali et al, en , determinan en promedio entre a horas, la periodicidad en el seguimiento de estos, sin embargo, no se discrimina entre pacientes críticos y no críticos. consideramos que el seguimiento de estas pruebas debe estar ajustado al juicio clínico del médico intensivista tratante, según la evolución de los pacientes. se muestran el comportamiento de los biomarcadores mas frecuentes en la tabla . tabla . biomarcadores en pacientes críticos con sospecha o diagnóstico de covid- . tendencia de biomarcador en relación con la gravedad covid- pcr aumentada recomendaciÓn se recomienda la no medición de marcadores de inflamación o de severidad de forma rutinaria solo con el objetivo de iniciar un tratamiento específico o algoritmos terapéuticos en la enfermedad por covid- en pacientes críticos. el síndrome de liberación de citocinas o denominado "tormenta de citocinas" parece asociarse en pacientes con afecciones graves por covid- . la citocina proinflamatoria il- es la citocina mejor documentada en covid- correlacionada con la gravedad, el estado crítico del paciente, la carga viral y el pronóstico ( , , ) . se han descrito mayores niveles de citoquinas proinflamatorias (il- , il- , il- , factor estimulante de colonias de granulocitos, factor de necrosis tumoral e interferón gamma) asociadas a compromiso pulmonar severo en pacientes con infección por coronavirus, determinado por la rápida replicación del virus, infiltración masiva de células inflamatorias y trastorno severo de la inflamación ( , ) . igualmente, está asociada la presencia de linfopenia como biomarcador de mal pronóstico para covid- ( ) . hallazgos similares se encontraron en la pandemia de influenza a (h n ) de sin ser especificó su valor ( ) . las manifestaciones clínicas de la tormenta de citocinas incluyen síndrome de respuesta inflamatoria sistémica, hipotensión, síndrome de fuga capilar, insuficiencia renal, sdra, miocarditis, entre otras ( ) , algunos autores han determinado este cuadro como un síndrome de linfohistiocitosis hemofagocitica secundaria. es razonable pensar que, en pacientes con sospecha de tormenta de citocinas basado en los hallazgos de laboratorio, el manejo con inmunomoduladores puede resultar beneficioso, sin embargo, los resultados del manejo de la hiperinflación basado en pruebas diagnósticas han tenido resultados encontrados en pacientes con covid- . el uso de esteroides, inmunoglobulina endovenosa, inhibidores del receptor de citoquinas (tocilizumab) o inhibidores de janus kinasa, han disminuido los valores de los biomarcadores, días de hospitalización ( ) o necesidad de fracciones elevadas de oxígeno ( ) , sin embargo no han demostrado beneficio sobre la mortalidad y en algunos casos, si un aumento en la incidencia de infecciones bacterianas o fúngicas sobreagregadas ( ) . se recomienda realizar radiografía simple de tórax para todos los pacientes con sospecha o diagnóstico de covid- en uci. se recomienda realizar tac de tórax según disponibilidad de tecnología institucional, ante la incertidumbre diagnostica, teniendo en cuenta las condiciones clínicas, la tolerancia del paciente al traslado y los protocolos administrativos de seguridad. fuerte a favor fundamento se reconoce como el gold estándar diagnóstico de infección pulmonar por covid- a los estudios moleculares, sin embargo, estos presentan limitaciones: a) contaminación de las muestras b) errores en la técnica de la toma, c) muestra insuficiente para ampliación genética favoreciendo falsos negativos d) demora de reporte de los resultados. por lo anterior, se recomienda imágenes diagnósticas en la aproximación de paciente con sospechas de infección pulmonar por covid- ( , ) .  radiografía simple de tórax: ventajas: mayor accesibilidad que la tac de tórax y realización de la prueba a la cabecera del enfermo desventaja: baja sensibilidad en estadio temprano, después del día de inicio de los síntomas presenta aumento en el rendimiento diagnóstico  tac de tórax: ventaja: es ampliamente recomendada. alta sensibilidad en estadios tempranos. permite describir extensión, distribución, localización, densidades parenquimatosas, aplicables en clasificaciones y puntajes diagnósticos, pronósticos y de seguimiento en permanente evolución y mejoría. algunas asociaciones referentes como asociación china de radiología, en su recomendación de expertos propone clasificación tomografía en estadios temprana avanzada severo disipación desventaja: traslado del paciente hasta el tomógrafo (no todos los enfermos toleran el transporte) y tecnología no disponible en todos los niveles de atención page no se puede emitir una recomendación a favor o en contra para el uso de ecografía pulmonar a la cabecera del paciente crítico como herramienta diagnóstica o de pronóstico en con covid- . se puede considerar como una alternativa para la valoración imagenológica pulmonar en el paciente crítico con covid- cuando las condiciones del paciente no permitan su traslado. no se sugiere la utilización de la ecografía pulmonar para el seguimiento de lesiones pulmonares agudas en el enfoque del paciente crítico con covid- . puede utilizarse para determinar complicaciones asociadas a la enfermedad o en la inserción de dispositivos invasivos. en general los estudios de imágenes no representan un papel concluyente para el diagnóstico de covid- . la ultrasonografía en específico requiere estudios de validación, un programa de entrenamiento es operador dependiente, y se le atribuido limitaciones en la capacidad de discriminación en la cronicidad de las lesiones pulmonares. la ultrasonografía pulmonar puede servir como herramienta a la cabecera del paciente para mejorar la evaluación del compromiso pulmonar y reducir el uso de radiografías de tórax y tomografía computarizada ( ) , sin embargo no debe usarse para el diagnóstico inicial, pues éste se compone de criterios clínicos, radiográficos y microbiológicos que actualmente son el estándar de oro; la ecografía no los reemplaza debido a la baja especificidad en relación con el virus, se sugiere su uso como complemento en la valoración diaria del paciente, ojalá realizada por el mismo observador. la ecografía pulmonar es altamente sensible y puede revisar de forma rápida y precisa la condición pulmonar, creando un potencial para evaluar los cambios o la resolución con el tiempo, especialmente en la uci, escenario en el que cada vez se usa más para la detección de múltiples patologías pulmonares que se pueden demostrar junto con covid- , sin embargo, hasta la fecha no hay hallazgos específicos, ni patognomónicos que se relacionen con covid- en el examen ecográfico del paciente ( ) . la adopción de ultrasonido pulmonar puede reducir la necesidad de exposición a la radiación ionizante y, a su vez, reducir la cantidad de radiografías necesarias para la evaluación rutinaria del paciente, disminuyendo también la exposición de personal asistencial adicional como el uso de elementos de protección personal ( ) . es bien conocido el beneficio de la ecografía durante y después de la colocación de accesos venosos centrales para establecer la presencia o no de complicaciones inmediatas como neumotórax. a la fecha no hay publicaciones acerca de la utilidad del ultrasonido como herramienta para establecer pronóstico. se necesitan más estudios para evaluar la utilidad de la ecografía pulmonar en el diagnóstico y manejo de covid- ( ) . se recomienda no establecer un punto de corte en el valor de dímero d para el inicio rutinario de anticoagulación plena en el contexto de infección por covid- . se recomienda la administración de profilaxis antitrombótica según protocolo institucional independiente de niveles de dímero d en el paciente críticamente enfermo por covid- . los fenómenos inflamatorios inherentes a procesos infecciosos son considerados desde décadas previas, factores protrombóticos, no siendo una excepción la infección por covid- . en algunas publicaciones se hallan asociaciones con desenlaces cardiovasculares negativos ( ) y sugieren asociación entre niveles elevados de dímero d ( marcador de estado de trombosis ) y riesgo de embolismo pulmonar con or crecientes or de . a los días hasta . a los días de seguimiento ( ) . de igual manera, zhou et al, reportan asociación de dímero d mayor a mcg/ml y mortalidad ( ) sin embargo los estudios presentan limitaciones en su diseño, a pesar de ello algunos autores proponen anticoagulación como factor de protección en mortalidad sin precisar precisión en la dosis, tipo de heparina y selección de enfermos ( ) . finalmente la european heart journal en su entrega de farmacología cardiovascular desarrolla una propuesta en la cual combina un puntaje previo de riesgo de cid en uci a niveles de fibrinógeno; esta pudiera ser una herramienta para selección de pacientes a recibir anticoagulación sin embargo aún está en proceso de validación ( ) . por estos motivos, hasta el momento, no se tiene suficiente cuerpo de evidencia que permita hacer una recomendación basado en los niveles de dímero d como variable aislada para administración de anticoagulación terapéutica. se recomienda no utilizar de rutina la procalcitonina en un algoritmo diagnóstico, para diferenciar entre neumonía viral vs bacteriana o confirmar la presencia de una sobreinfección bacteriana en el paciente con sospecha o diagnóstico de covid- . se recomienda no medir de forma rutinaria la procalcitonina en pacientes con sospecha o diagnóstico de covid- como factor pronóstico. la procalcitonina es un biomarcador que ha sido incluido en algoritmos de diagnóstico y pronóstico durante los últimos años. schuetz et al, en , concluyen en una revisión sistemática de estudios, que la procalcitonina es segura dentro de un algoritmo, para guiar a los médicos tratantes entre iniciar o suspender antibióticos en neumonía adquirida en la comunidad; sin embargo en una revisión sistemática más reciente, kamat et al, reportaron una sensibilidad % [ic % %- %; i = . %], especificidad % [ % ic, %- %; i = , %]) para el inicio de tratamiento antibiótico para neumonía bacteriana, lo cual nos determina que la prueba es inespecífica para diferenciar entre infecciones virales vs bacterianas. en neumonía por sars-cov- , se han publicado algunos artículos evaluando el uso de la procalcitonina como prueba asociada al pronóstico de los pacientes. liu et al encontraron que la procalcitonina se asoció a mayor severidad de los cuadros de neumonía. hr, . ; % ci, . - . ; p= . . en este estudio también se tuvo un resultado similar con la proteína c reactiva y la il- . plebani et al, publican un metaanálisis, donde sugiere que los niveles elevados de procalcitonina se asocian a mayor severidad de la infección. (or, . ; % ci, . - . ). es importante mencionar, que en estos estudios se evaluaron otros biomarcadores de inflamación como interleucina , proteína c reactiva y ferritina; presentando todos ellos, aumento en sus valores y asociándose a severidad de la enfermedad; por tanto, se considera que, en particular, la procalcitonina elevada, no representa una diferencia en el pronóstico, comparado con otros biomarcadores de inflamación. hasta el momento no se han publicado estudios en infección por sars-cov- donde se evalué el papel de prueba diagnóstica para confirmar sobreinfección bacteriana o diferenciar entre neumonía viral vs bacteriana. ( , ( ) ( ) ( ) se recomienda no usar de forma rutinaria el uso de pruebas clínicas de laboratorios clínicos para determinar la resolución de la enfermedad crítica por covid- . se recomienda considerar la ausencia de dificultad respiratoria y fiebre por más de horas, requerimiento de oxígeno a baja concentración y bajo flujo, como indicadores clínicos de resolución de la fase crítica de la enfermedad por covid- . se recomienda no utilizar de forma rutinaria el uso de pruebas microbiológicas de erradicación viral, para determinar la resolución de la enfermedad en pacientes en uci con covid- . amci ® enfermedad está asociado tanto a la carga viral como a la respuesta hiperinflamatoria del huésped a la infección viral. en cuanto a la carga viral, en pacientes que tienen un curso leve de infección, el pico de la carga viral en muestras nasales y orofaríngeas ocurre durante los primeros - días tras el inicio de síntomas y prácticamente desaparece al día , mientras que en los que cursan con neumonía severa en uci, la carga viral es veces mayor y puede persistir la excreción viral hasta el día a ( ) . por esta razón, consideramos que en los pacientes en uci no es necesario confirmar la erradicación del virus o su negativización en muestra respiratorias, orina o heces, para determinar la mejoría clínica, curación o para el egreso del paciente crítico ( ) . en el contexto clínico, el pronóstico se ha asociado a la presencia de marcadores bioquímicos elevados, sin embargo, no existe evidencia que el seguimiento con estos marcadores iniciales de inflamación determine el momento exacto de la resolución de la enfermedad. varias organizaciones internacionales como el cdc de usa y el european centre for disease prevention and control ( mar ) ( , ), national centre for infectious diseases (ncid) singapore ( ), world health organization ( de marzo de )( ), han establecido criterios para resolución clínica y egreso hospitalario de los pacientes. estos criterios incluyen: ausencia de fiebre mayor a horas sin antipiréticos, mejoría de los síntomas respiratorios, ausencia de requerimiento de hospitalización por otras patologías, el resultado de dos ( ) rt-pcr para sars-cov- negativas, con intervalo de muestra mayor a horas. la utilidad de dichos criterios no ha sido evaluada en pacientes en cuidado intensivo. en uci, huang et al ( ) , describieron en pacientes a quienes se dio egreso, la ausencia de fiebre por días, mejoría radiológica y evidencia de erradicación viral, como criterios de alta. sin embargo, consideramos que deben primar los criterios clínicos sobre los paraclínicos, en el momento de definir el egreso de un paciente de cuidado intensivo, teniendo como principal indicador la ausencia de dificultad respiratoria y la mejoría en los índices de oxigenación, con requerimiento de oxígeno suplementario a bajos flujos y concentración ( ) . se recomienda la cánula de alto flujo, donde esté disponible, en pacientes con covid- a nivel del mar con hipoxemia leve (pao /fio < y > o sao /fio < y> ). en alturas superiores a los mts por encima del nivel del mar esta terapia se puede considerar en pacientes que no tengan hipoxemia severa (pafi< ). amci ® se recomienda en pacientes críticos por covid- el uso de la cánula de alto flujo en salas de presión negativa, donde estén disponibles, que garanticen la seguridad del recurso humano. si no se dispone de habitación con presión negativa se puede optar por habitación individual cerrada. se debe contar con todo el equipo de protección personal necesario para el personal sanitario y de apoyo. se recomienda colocar mascarilla quirúrgica por encima de la cánula nasal en el paciente con sospecha o diagnóstico de covid- y mantener una distancia mínima de metros con otros pacientes. se recomienda la intubación inmediata en pacientes críticamente enfermos con sospecha o diagnóstico covid- con índice de rox ([spo /fio ] / frecuencia respiratoria) < a las horas de iniciada la oxigenación con cánula de alto flujo teniendo en cuenta que el retraso en la intubación aumenta la mortalidad. se recomienda considerar la cánula de alto flujo en caso de agotamiento de ventiladores mecánicos. la cánula de alto flujo ofrece flujos de hasta litros/minuto, que aportan una fracción inspirada de oxígeno (fio ) constante que reduce el espacio muerto y produce una presión positiva que genera reclutamiento alveolar y puede redistribuir el líquido alveolar ( ) ( ) ( ) . se ha reportado que al generar aerosoles, aumenta el riesgo de contagio para el personal de salud ( , ( ) ( ) ( ) . hasta el punto de que se ha recomendado su uso en salas con presión negativa ( ) . recientes publicaciones han establecido que la cánula de alto flujo genera una corta distancia de dispersión de aerosoles con lo cual las medidas de distanciamiento, un adecuado equipo de protección personal y donde estén disponibles, realizarla en salas de presión negativa darían seguridad al uso de la cánula de alto flujo ( , ) . a pesar de que la experiencia en la actual pandemia ha sido escasa, basada en series de casos, estudios retrospectivos y de metodología limitada, ha resultado ser promisoria en cuanto a la mejoría en la oxigenación y la tolerancia por el paciente ( , , , , , ( ) ( ) ( ) ( ) ( ) . sin embargo, hasta el momento no se ha establecido que la cánula de alto flujo evite la intubación. la caf podría convertirse en un alto riesgo de mortalidad al prolongar la decisión de intubación y al favorecer la lesión pulmonar autoinducida (p-sili) por aumento del esfuerzo inspiratorio. por todo ello es necesario un estricto y estrecho monitoreo del paciente durante una o dos horas para definir si ha habido mejoría o no mientras se realizan estudios que demuestren que evita la intubación y genera desenlaces clínicos importantes como menor estancia en uci, menor estancia hospitalaria y menos días de ventilación mecánica. la terapia de oxígeno de caf podría ser considerada para pacientes atendidos en altitudes mayor a mts, que no tienen hipoxemia severa (pafi < ), la respuesta debe evaluar dentro de los a minutos posteriores a su inicio y los pacientes que no mejoran amci ® significativamente y progresa la dificultad respiratoria no deben mantenerse con esta terapia. el monitoreo del paciente con caf para la toma de decisión de éxito o fracaso de esta estrategia y considerar la posibilidad de intubación incluye la evaluación gasométrica, la oximetría de pulso, así como considerar los criterios para intubación: frecuencia respiratoria > por minuto, deterioro de conciencia, inestabilidad hemodinámica, pao /fio < (a nivel del mar), sao /fio < , índice de rox< , , spo < % ( , - , - , , ) . se recomienda crear o ajustar protocolos institucionales de sedoanalgesia basado en objetivos con escalas validadas. se recomienda el uso de analgesia multimodal que incluya analgésicos opioides, no opioides y bloqueos regionales en el paciente crítico por sospecha o diagnóstico de covid- . se recomienda sedación profunda con agentes como midazolam o propofol para mantener rass entre - y - en pacientes covid- con sdra severo, necesidad de uso de relajantes neuromusculares o posición prona. se puede considerar en planos de sedación superficial agentes como propofol o dexmedetomidina (coadyuvante) para mantener rass entre y - en pacientes seleccionados con sospecha o diagnóstico de covid- con sdra no severo. en la actualidad no se encuentra evidencia de alta calidad proveniente de ensayos clínicos, sino editoriales, series, reportes de casos y artículos de revisión de expertos ( ) ( ) ( ) . la creación y aplicación de protocolos de sedoanalgesia adaptados a cada institución ha mostrado disminución del tiempo en la uci y menores complicaciones ( , ) . es importante definir objetivos guiados por escalas, recomendándose sedación profunda o completa en situaciones especiales como ventilación mecánica invasiva por sdra severo, disincronía ventilatoria persistente, posición prona y bloqueo neuromuscular (bnm), como puede observarse en pacientes covid- con compromiso pulmonar severo. mientras que se debe procurar sedación ligera, cooperativa o no sedación en pacientes en ventilación mecánica invasiva en pacientes con sdra no severo, ventilación no invasiva y en el retiro de la ventilación ( , ) . los opioides han sido el pilar de la analgesia en dolor moderado a severo. el fentanilo es actualmente el más usado; el remifentanilo reduce el tiempo en ventilación en pacientes amci ® renales, hepáticos, ancianos y neurológicos; la hidromorfona se prefiere en el retiro de la ventilación y en pacientes extubados; y la metadona ha mostrado disminuir la tolerancia a opioides ( ) . se propone el uso de estrategias de analgesia multimodal asociando medicamentos no opioides como el paracetamol, ketamina a dosis analgésicas (< , mg/kg) en dolor somático, lidocaína en dolor visceral, y pregabalina en dolor neuropático ( ) . la sedación ligera o cooperativa son mejores con propofol en cuanto a tiempo de despertar y con dexmedetomidina para preservar funciones cognitivas y el impulso respiratorio, con menor desacondicionamiento ( ) . en sedación profunda, el propofol ha mostrado más fácil titulación y menos acumulación que el midazolam; sin embargo, su uso se ve limitado hasta horas y a dosis < , mg/kg/h, ante el riesgo de pris (síndrome relacionado con la infusión de propofol). el midazolam, sin dosis techo ni tiempo límite y de bajo costo, ha sido el más utilizado de los sedantes, disminuyendo su uso por su asociación con delirium y de retraso en los tiempos en ventilación; sin embargo, la pandemia covid- ha vuelto a aumentar su uso. la dexmedetomidina ha sido utilizada como adyuvante en sedación profunda, disminuyendo el consumo de sedantes, con menos efectos secundarios ( , ) . se muestran los medicamentos para sedoanalgesia y relajación neuromuscular que se pueden utilizar en los pacientes con covid- en la tabla . se recomienda iniciar una estrategia individualizada de ventilación mecánica ajustadas a las condiciones específicas de severidad en el paciente crítico con covid- . amci ® se recomienda la ventilación protectora en modos controlados por volumen o por presión que garanticen un volumen corriente < cc/kg de peso predicho teniendo como metas una presión meseta < cm h o y una presión de conducción < cm h o. se recomienda emplear fracciones inspiradas de oxígeno para lograr metas de saturación de acuerdo con la pao /fio entre y % en el paciente con sospecha o diagnóstico de covid- . en la paciente embarazada entre y %. se recomienda en el paciente crítico por covid- iniciar con peep individualizado a la severidad del compromiso pulmonar y ajustar el nivel de peep de acuerdo con la tabla de fio /peep. el estudio arma ( ) demostró que la ventilación con bajos volúmenes corrientes (vt) se asocia con reducción de: mortalidad (p= . ), en días libres de ventilación mecánica (p= . ) y días libres de falla orgánica (p= . ). una revisión sistemática posterior confirmó que el uso de bajos volúmenes se asocia con menor progresión a sdra ( ) . un metaanálisis que revisó estudios y meta-análisis en uci confirmó que la ventilación protectora era una de las tres intervenciones que mejora la sobrevida en pacientes con sdra ( ) . esto fue confirmado por landoni en un análisis de estudios multicéntricos con impacto en mortalidad en uci ( ) . recientes publicaciones han sugerido que en covid- puede haber dos fenotipos que se diferencian en la distensibilidad ( , ) . sin embargo, el mismo estudio arma demostró que "el beneficio de ventilación con vt más bajo fue independiente de la distensibilidad de las vías respiratorias, lo que sugiere que el vt más bajo fue ventajoso independientemente de la distensibilidad pulmonar". más aún, el uso de bajos vt se asoció con una reducción en las concentraciones de interleuquina lo cual explicaría el mayor número de días sin falla orgánica múltiple y sugeriría una reducida respuesta inflamatoria asociada a la ventilación protectora ( ) . con el tiempo la evidencia ha demostrado que la ventilación protectora, además de vt y presión meseta bajos, debe incluir presiones de conducción menores de cm h o ( ) ( ) ( ) . existe suficiente evidencia que demuestra que fio y pao altas se asocian con aumento en la morbimortalidad ( ) ( ) ( ) . en sdra el estudio arma y una más reciente publicación demostraron que el tener metas conservadoras de pao se asocia con mayor sobrevida ( , ) . hasta el momento, la literatura en ventilación mecánica ha demostrado que la mejor estrategia para ajustar el nivel de peep en sdra es la tabla de fio /peep ( , ) . amci ® se recomienda la ventilación mecánica protectora en sdra por covid- independiente del fenotipo de presentación. se recomienda la clasificación de fenotipos en sdra para pacientes con covid- para ajustar la toma de decisiones de manera individualizada en ventilación mecánica. el manejo ventilatorio en covid- tiene los mismos principios generales de los pacientes con sdra ( ) . sin embargo, la identificación de fenotipos podría impactar en la evolución y pronóstico ( ) . gattinoni ha postulado el desarrollo de un sdra típico (fenotipo h: con alta elastancia, alto cortocircuito, alto peso pulmonar) o una presentación atípica (fenotipo l: caracterizado por baja elastancia, bajo shunt, bajo peso pulmonar). ( ) . pelosi et al sugiere clasificar los pacientes con tres fenotipos similares ( ) . con base en tales fenotipos se han propuesto estrategias ventilatorias diferenciales para minimizar el daño inducido por el ventilador (vili) ( ): . el primer paso, en el fenotipo l, es revertir la hipoxemia aumentando la fio . . en el tipo l, hay varias opciones no invasivas: cánula nasal de flujo alto, presión positiva continua en la vía aérea (cpap) o ventilación no invasiva (niv). se debe evaluar el esfuerzo inspiratorio y, de estar disponible, medir la presión esofágica. la peep alta puede disminuir los cambios de presión pleural y detener el ciclo vicioso que exacerba la lesión pulmonar. sin embargo, la peep alta, en pacientes con distensibilidad normal, puede tener efectos hemodinámicos perjudiciales. en cualquier caso, las opciones no invasivas son cuestionables, ya que pueden asociarse con altas tasas de fracaso y retraso de la intubación. . la magnitud de las presiones pleurales inspiratorias puede determinar la transición del tipo l al h. la presión esofágica > cmh o aumenta el riesgo de vili y, por lo tanto, la intubación debe realizarse lo antes posible. los tipo l, si son hipercápnicos, pueden ser ventilados con volúmenes > ml / kg (hasta ml / kg). la posición prona debe ser usada solo en último caso, ya que las condiciones pulmonares son buenas. la peep debe reducirse a - cm h o, dado que la capacidad de reclutamiento es baja y el riesgo de falla hemodinámica aumenta. la intubación puede evitar la transición al fenotipo tipo h. . los pacientes tipo h deben ser tratados como sdra grave, incluyendo mayor peep, si es compatible con la hemodinamia, posición en pronación y soporte extracorpóreo. en el fenotipo se deben aplicar las estrategias de ventilación protectora convencional ( , ) . amci ® se recomienda en paciente con covid- considerar hipoxemia refractaria cuando no se obtienen las metas de oxígeno propuestas, a pesar de las maniobras ventilatorias recomendadas y cumple con los siguientes parámetros: pafi < , fio > , y peep apropiado, considerando la altitud. se recomienda considerar la utilización de ecmo, en sitios donde esté disponible y con alta experiencia para obtener resultados aceptables, en pacientes con hipoxemia refractaria luego de haber implementado ventilación protectora, relajación neuromuscular y posición prona. se recomienda administrar tromboprofilaxis en todos los pacientes con covid- con hipoxemia refractaria que no presenten contraindicaciones. la hipoxemia refractaria no es un concepto estático y absoluto, según la definición de berlÍn del sdra, se clasifica el sdra en leve, moderado y severo de acuerdo con la relación pao y fracción inspirada de oxígeno, con peep mayor de cms de h o. la hipoxemia severa es aquella que cuenta con una pafi menor de ( ) ( ) ( ) . la hipoxemia refractaria hace referencia a un estado de hipoxemia severa que a pesar de las diferentes estrategias ventilatorias no aumenta la pafi y tiene consecuencias en el estado ácido básico y metabolismo celular permitiendo una anaerobiosis ( , ) . para definir la hipoxemia refractaria deben coincidir varios escenarios , una pafi menor de , una fracción inspirada de oxígeno mayor de . , a pesar de un peep apropiado no se tiene en cuenta en la definición el ph ni la paco ni el tiempo transcurrido ( ) . algunas de las medidas terapéuticas no ventilatorias que se han empleado en sdra y covid- con hipoxemia refractarias son la oxigenación con membrana extracorpórea (ecmo) y la tromboprofilaxis o anticoagulación de rutina. la oxigenación extracorpórea a través de una membrana ha sido una estrategia controvertida en pacientes con hipoxemia refractaria de diferentes orígenes, en la epidemia de la influenza por el virus h n fue usada en pacientes con hipoxemia refractaria teniendo resultados aceptables ( ) . en el estudio eolia los resultados no mostraron mejoría en la supervivencia, aunque hay diferentes posiciones y estudios post hoc de este ensayo clínico con beneficios, su uso se limita a casos muy restringidos y en sitios de alta experiencia para obtener resultados aceptables ( ) . en sdra por covid- el ecmo se ha usado en hipoxemia refractaria entre un a . % en diferentes series con resultados variables ( , , ) . un tipo de pacientes hipoxémicos y con ventilación mecánica han presentado cuadros tromboembólicos pulmonares en estos casos la trombólisis de rescate con activador de plasminógeno tisular rtpa (alteplase) se ha recomendado con resultados alentadores en serie de casos, pero su evidencia es muy débil para ser recomendada( - ). dada la alta frecuencia de enfermedad tromboembólica reportada en covid- se ha reportado la utilidad de la tromboprofilaxis, especialmente en casos de dímero d o índice de sic elevado ( ) . amci ® se recomienda monitorizar sistemáticamente la oxigenación con los índices: pao /fio y sao /fio , y en donde esté disponible el monitoreo continuo con capnografía. se recomienda monitorizar de forma rutinaria la presión meseta y la presión de conducción como estrategia al pie de la cama para verificar la ventilación protectora. el sdra y covid- , es una condición dinámica que apenas se está caracterizando, hay varias presentaciones que no cumplen con todos los criterios de berlÍn ( , ) . gattinoni ha caracterizado en dos presentaciones el sdra en los pacientes con neumonía por coronavirus sars-cov- , una con alta compliance, mínima reclutabilidad; la otra con baja compliance, pulmones pesados y reclutabilidad, tal vez esta presentación sea el verdadero sdra ( , , ) . los pacientes que requieren ventilación mecánica por falla ventilatoria en covid- , son los que mayor mortalidad tienen al parecer por la lesión pulmonar inducida por la intubación tardía y el gran esfuerzo respiratorio con presiones transpulmonares oscilantes y muy negativas ( , , ) . la monitoria de estos pacientes soportados con ventilación mecánica tiene dos objetivos: el primero detectar el deterioro clínico para sugerir estrategias más avanzadas como el ecmo, y el segundo es evitar el daño pulmonar inducido por la ventilación mecánica. se debe tener presente la mayor posibilidad de contagio con el número de manipulaciones en el paciente, por esto nunca olvidar el perfecto uso de los elementos de protección personal y disminuir el número de contacto con el paciente. las metas que se buscan con la ventilación mecánica en el paciente con sdra por covid- son mantener una oxigenación adecuada teniendo en cuenta la altura sobre el nivel del mar con pao entre y mmh y metas de saturaciones reportadas entre y % a y % , mantener una ventilación adecuada evitando el espacio muerto , disminuir el trabajo respiratorio y protegiendo el pulmón del daño ocasionado por la ventilación mecánica y las repercusiones hemodinámicas ( ) .  el confort de los pacientes en ventilación mecánica es la principal señal de un uso adecuado del modo ventilatorio y los parámetros ventilatorios apropiados para la patología y demanda del paciente ( ) .  las curvas y bucles son herramientas indispensables para valorar la mecánica respiratoria del paciente soportado con ventilación mecánica, se puede diagnosticar amci ® las asincronías del paciente y el ventilador, el origen, tipo y frecuencia además de la respuesta al manejo. también se evalúa la resistencia de la vía aérea ( ) . variables fisiológicas:  es importante valorar la oxigenación del paciente, la literatura actual sugiere el monitoreo de la pafi es el más representativo y sencillo test para valorar la oxigenación y representa el shunt pulmonar, se debe hacer mínimo diariamente, o cuando se haga una intervención en el ventilador o paciente; en sdra por covid- la hipoxemia se relaciona directamente con mortalidad, debe mejorar con la ventilación mecánica ( , ) .  la medición de la paco indica la de la ventilación, la hipercapnia tiene relación directa con el espacio muerto en el paciente con sdra, y varios estudios la relacionan con la mortalidad. puede evaluarse directamente en los gases arteriales o relacionarla con el pco expirado por medios de la capnografía, gattinoni propone una forma de evaluarla al lado de la cama del paciente relacionando el etco /paco , cuando es < de sugiere un shunt elevado y mayor espacio muerto; áreas de pulmón ventiladas y no aireadas. otras tecnologías incluidas en el ventilador moderno como la capnografía volumétrica se está validando para evaluar el espacio muerto, la reclutabilidad y la titulación de peep ( , ) .  la saturación venosa mixta svo , refleja de manera subrogada la función ventricular, no todos los pacientes tienen catéter de arteria pulmonar para su medición por lo que se está reemplazando con el ultrasonido en la cabecera del paciente; recordar que el % de los pacientes con sdra cursan con falla ventricular derecha ( ) . monitoria de mecánica ventilatoria y protección pulmonar:  para evitar el daño pulmonar debe propender por un volumen corriente bajo ( - ml/kg de peso predicho) y presión plateau menor de cms h , para garantizar la ventilación con protección pulmonar ( , ) .  driving pressure ( presión cambiante de la vía aérea, presión diferencial o presión de conducción) es la presión plateau (presión pico en ventilación controlada por presión) menos peep, debe ser menor de cm h , está relacionado con aumento en la mortalidad en pacientes ventilados por que representa una medición indirecta del strain pulmonar porque relaciona el volumen corriente con la compliance del sistema respiratorio y este a su vez se relaciona con el volumen espiratorio pulmonar final ( ) .  la medición de la compliance del sistema respiratorio es necesaria y nos clasifica el paciente de acuerdo con su fenotipo para trazar el plan de manejo ventilatorio, cuando la compliance es baja, esto se puede hacer al lado de la cama del paciente con los ventiladores modernos ( , ) .  la construcción de la curva presión/volumen aún es una herramienta útil para ubicar el área de ventilación segura del paciente, evitando el atelectrauma y la sobredistensión pulmonar(estrés) , el peep se calcula dos puntos por encima del punto de inflexión inferior y el punto de inflexión superior nos indica hasta dónde podemos aumentar el volumen corriente este punto debe estar por debajo de cms h para evitar la sobre distensión, con los ventiladores modernos se puede construir esta curva ( , ) . amci ®  las curvas presión tiempo en pacientes ventilados con modos volumétricos pueden monitorizar la resistencia de la vía aérea, la compliance pulmonar, el trabajo respiratorio, las curvas de flujo puede también indicar si se presenta autopeep, resistencia aumentada de la vía aérea entre otras ( ) .  presión transpulmonar, en casos más complicados donde es más difícil obtener la meta de oxigenación a pesar del peep en aumento una opción es el catéter esofágico, para medir la presión transpulmonar en la inspiración y espiración y calcular así el stress pulmonar y evitar las presiones oscilatorias y sobre todo negativas para evitar el daño pulmonar. esta herramienta también ayuda a evaluar el trabajo respiratorio, y el diagnóstico de las asincronías que se presenten en el paciente ventilado ( , ) . se recomienda no utilizar de forma rutinaria la relajación neuromuscular en el paciente crítico con covid- con sdra. se recomienda utilizar la relajación neuromuscular en pacientes en posición supino o prono, que están fuera de parámetros de protección pulmonar (presión de conducción mayor y presión plateau mayor a ) con pafi menor y cuando ya no es posible limitar el volumen corriente. se debe considerar la utilización de protocolos estandarizados con el fin de disminuir la variabilidad, y según disponibilidad seleccionar el cisatracurio como primera opción, en caso de escasez se pueden utilizar otras opciones teniendo en cuenta su farmacodinamia y farmacocinética. el de marzo de , la organización mundial de la salud emite una serie de orientaciones para el manejo de la infección respiratoria aguda grave (irag) en pacientes con sospecha o diagnóstico de covid- . en el paciente críticamente enfermo con sdra moderado o grave (pao /fio < ) por covid- no está indicado de forma sistemática el bloqueo neuromuscular mediante infusión continua debido a que no se cuenta con evidencia suficiente que sustente mejoría en la supervivencia con respecto a una estrategia de sedación ligera sin bloqueo neuromuscular, se debe considerar su uso cuando se evidencia asincronía paciente-ventilador a pesar de la sedación, hasta el punto de que no se pueda limitar el volumen corriente de forma fiable, hipoxemia o hipercapnia que no mejoran con el tratamiento ( ) . recomendaciÓn se recomienda no utilizar de forma rutinaria oni en pacientes adultos que presenten sdra e infección por sars-cov- . fuerte en contra fundamento a la fecha (mayo de ) no contamos con estudios sobre el uso de óxido nítrico inhalado (oni) como tratamiento de pacientes con infección covid- . existe evidencia indirecta sobre el uso de oni en el síndrome de dificultad respiratoria aguda (sars-cov), y la infección por coronavirus en el síndrome respiratorio de oriente medio (mers-cov) ( ) . en cochrane realizó una revisión sistemática que incluyó ensayos de calidad moderada con pacientes adultos con sdra tratados con óxido nítrico inhalado. los resultados no mostraron ningún efecto estadísticamente significativo sobre la mortalidad (rr . , % ci . - . ). se mostró mejora transitoria en el índice de oxigenación a las horas (md (diferencia media) - , , ic del % - , a - , ) y mejoría en pao /fio a las horas (md , , ic del % , a , ). no se identificó diferencia significativa en los días libres de ventilación y finalmente se presentó aumento estadísticamente significativo en la incidencia de insuficiencia renal en pacientes con óxido nítrico inhalado (rr , , ic del % , )( ). amci ® en se realizó un estudio observacional que incluyó pacientes tratados de dos hospitales de beijing con oni como tratamiento para sars ( ) . en comparación con ningún tratamiento, oni mejoró la saturación arterial de oxígeno (spo ) de % a % (p< . ); se asoció a menor necesidad de oxígeno suplementario (p< . ) y menor necesidad y retiro de cpap y bial (p < . ). los cambios en radiografía de tórax mejoraron en de los pacientes que recibieron oni. sin embargo, debido a problemas graves de validez por pequeño tamaño de la muestra (n= , oni= , control= ), no aleatorización y no enmascaramiento en la asignación, se considera que este estudio cuenta con baja calidad metodológica, lo cual limita la interpretación de los resultados. en un estudio retrospectivo multicéntrico que incluyó pacientes con mers-cov en condición crítica en arabia saudita, se mostró que el manejo con ventilación no invasiva (niv) tenía mayor probabilidad de requerir óxido nítrico en comparación con los pacientes con ventilación mecánica invasiva ( , % vs , %, p a , ) ( ) . en una serie de casos en la que participaron pacientes con infección por mers-cov confirmada o probable, pacientes recibieron oni debido a hipoxemia refractaria. en el seguimiento a días, cinco de los pacientes continuaron vivos ( ) . los estudios sobre mers-cov se limitaron a una serie de casos y una cohorte retrospectiva con baja calidad de evidencia. en ambos estudios, los pacientes recibieron otras terapias de rescate (relajación neuromuscular, ventilación oscilatoria de alta frecuencia, ecmo y posición en prono), por lo tanto, se desconoce el efecto terapéutico clínico del oni en el tratamiento de la infección por mers-cov. a la fecha ( de mayo de ) tres ensayos clínicos evalúan el papel del óxido nítrico inhalado en pacientes con covid- y sdra leve/moderado, y como profilaxis para los trabajadores de la salud covid- (tabla ). tabla . comparación de ensayos clínicos que evalúan el papel del óxido nítrico inhalado en pacientes con covid- y sdra leve/moderado, y como profilaxis para los trabajadores de la salud covid- . no se cuenta con evidencia por el momento que respalde el uso de óxido nítrico inhalado en pacientes con covid- . los resultados de los ensayos en curso, así como ensayos clínicos de alta calidad son necesarios para apoyar su uso. sólo evidencia indirecta metodológicamente limitada de óxido nítrico en pacientes con sras mostró una mejor oxigenación, una menor necesidad de oxígeno suplementario y mejoría en la radiografía de tórax. en pacientes con sdra y mers-cov, no mostró un beneficio claro e incluso mostró un mayor riesgo de insuficiencia renal ( ) . otros estudios han evaluado el efecto tóxico asociado a su uso documentando metahemoglobinemia( ), inhibición de la agregación plaquetaria y formación de dióxido de nitrógeno ( ) . razones por la cuales guías recientemente publicada no recomienda su uso de forma rutinaria( ). recomendaciÓn se recomienda el uso temprano de la ventilación en posición prona, por al menos horas continuas, en pacientes con sdra por covid- con pao /fio < mmhg. la ventilación en posición prono como estrategia ventilatoria propuesta desde los años ( ), cuenta con evidencia que demuestra resultados positivos en cuanto a mejoría de mortalidad, mejoría en el trastorno de oxigenación y el reclutamiento alveolar en pacientes con sdra. los mecanismos por los cuales la posición prona conduce a la mejoría en el trastorno de oxigenación y del reclutamiento alveolar en los pacientes con sdra, incluyen ( ) ( ) ( ) ( ) : amci ®  mejoría de la relación ventilación/perfusión y mayor homogeneidad en la distribución de aire en los pulmones.  aumento del volumen de fin de espiración.  disminución del efecto compresivo del corazón en los pulmones.  mejoría del drenaje de las secreciones.  optimización del reclutamiento alveolar, con mejoría de la distribución del volumen corriente, a su vez, limita el desarrollo del daño alveolar pulmonar. los estudios coinciden en el efecto benéfico que esta terapia tiene en la mejoría de la oxigenación, el objetivo se centrará en evaluar las recomendaciones con respecto a:  beneficio de la terapia con respecto a la mortalidad al día , al día y a los meses.  beneficio de la terapia según el grado de severidad de sdra con respecto la relación pao /fio .  tiempo de terapia en posición prono con mayor beneficio.  número de sesiones de la terapia en posición prono.  recomendaciones según balance riesgo/beneficio en lo que respecta a los efectos adversos: retiro o desplazamiento no planeado de catéteres, obstrucción de tubo endotraqueal, neumonía asociada a ventilador, lesiones de presión. se eligieron artículos que con las características metodológicas consistentes ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . de estos ocho artículos se excluyó uno por corresponder a pacientes pediátricos ( ) . al evaluar los escritos de forma cronológica, se puede apreciar en los primeros artículos ( , ) , la dificultad para lograr enrolar (reclutar) el suficiente número de pacientes, de tal manera que algunos estudios fueron detenidos de forma prematura ( , , , ) . era entonces esperable que los resultados no fueran concluyentes, y que no lograran ser robustos, al no alcanzar el tamaño de muestra deseado, comprometiéndose la confiabilidad, el poder y corriendo el riesgo de obtener resultados falsamente negativos. sin embargo, se podía percibir en los diferentes estudios, una notoria mejoría en la oxigenación, sin repercusión en la mortalidad ( ) ( ) ( ) ( ) ( ) ( ) . por otro lado, es necesario tener en cuenta que los estudios iniciales ( ) ( ) ( ) ( ) no se realizaron con el uso de ventilación protectora asociada a la pronación. esto es un elemento pertinente, pues la ventilación protectora puede per se, brindar un efecto adicional en la mejoría de la oxigenación. otro efecto importante que debe analizarse es el bloqueo neuromuscular, cuya evidencia hoy sugiere su uso en criterios ya mencionados ( ) . con el tiempo y según la experiencia de cada centro, se fue utilizando con menor dificultad ( ) . sin embargo, no se tenía aún claro qué tipo de pacientes obtenían el mayor beneficio de la terapia, además que el tiempo de la terapia en posición prona, seguía siendo una incógnita. la evidencia reflejaba hasta el momento que los pacientes con sdra más severos y la terapia aplicada por más tiempo se asociaban con una tendencia a la reducción en mortalidad. para dirimir qué tipo de pacientes se beneficiarían más de la terapia, se tomó como base, la severidad del sdra según la relación pao /fio ( ) . a pesar de que los estudios iniciales( - ) enfrentaban el reducido tamaño de muestra, algunos análisis identifican beneficio de la mortalidad ( ) en pacientes con sdra más severo, constituyendo un probable umbral de beneficio cercano y por debajo de - mmhg ( ) . amci ® antes del estudio proseva ( ) en totalidad estudios reportan mortalidad al día ( , ) , un estudio mortalidad al día ( ) , uno estudio mortalidad al día ( )y uno mortalidad al día ( ) . sin embargo, al tomar todos los estudios, aún no se alcanzaba suficiente significancia estadística para reducir la mortalidad a los días y meses. con el estudio proseva -guerin et als -en el año ( ) , se alcanza más poder estadístico, demostrando un beneficio importante en pacientes con pao /fio menor de mmhg, ventilados con bloqueo neuromuscular y ventilación protectora en su totalidad, y logrando una reducción de la mortalidad al día y mantenida hasta el día con respecto al grupo control ( . % ( de participantes) versus . % ( de ) (p< . ). de tal manera que, según la evidencia disponible, en lo que al trastorno de oxigenación se refiere (según la relación pao /fio ), sugiere que el mejor candidato para esta estrategia ventilatoria es el paciente con sdra severo con una pao /fio menor de mmhg. es de primordial importancia detenerse a considerar por otra parte, el número de horas que se implementaría la terapia. el estudio inicial realizado por gattinoni et al. ( ) , llevó a los pacientes a un período corto de horas, sin encontrar resultados positivos en mortalidad. (resultados que no se pueden solamente atribuir a la calidad del estudio, sino también, al reducido número de horas de la ventilación en posición prono). mancebo et als (la abreviación latina para "otros "es et al.), y fernández et al. ( , ) por su parte, optan por ventilar un mayor número de horas ( - horas) obteniendo resultados (que sugieren una reducción en mortalidad) con tendencia a disminuir mortalidad. si bien diferentes metaanálisis sugieren no (la ausencia de beneficio) beneficio de la ventilación en prono ( , ) , los resultados son diferentes cuando se aborda la terapia con períodos mayores a horas ( ) . teniendo en cuenta, que el estudio con más poder estadístico ( ) postula horas de terapia en posición prono, la recomendación es la pronación por un tiempo mayor a horas, contemplando hasta las horas por sesión. otra pregunta que surge con frecuencia es el número de veces que se puede implementar la terapia. existen diferentes estudios en los cuales se indica el número de veces en promedio en que se llevó el paciente a posición prono ( , ) , otro protocolo en donde se estipula un número límite de días en los que se llevó a cabo la terapia( ), y otro estudios en los cuales no se precisó el número de sesiones de la terapia ( , ) . se podría entonces recomendar con respecto a la mayor evidencia disponible ( ) , pronar a los pacientes en varias sesiones ( en promedio) (depende de los criterios para continuar o suspender el prono que deben ser individualizados para cada paciente, en cada zona geográfica y en cada unidad de cuidados intensivos, es difícil saber si es el promedio para todos), y considerarla más veces si es necesario. en lo que respecta a los efectos adversos, tres estudios reportaron barotrauma y neumonía asociada a ventilador ( , , ) ; dos estudios desplazamiento de catéter central ( , ) y siete estudios reportaron extubación ( ) ( ) ( ) ( ) ( ) ( ) ( ) . sin embargo, los efectos adversos no alcanzaron significancia estadística para proscribir la terapia ( ) . recientemente fue publicado el consenso colombiano de sdra( ) en el cual se hace referencia a las recomendaciones para realización de ventilación prono. se presenta la lista de chequeo, y medidas que deben ser realizadas en la maniobra para lo cual se cuenta con la participación de terapia respiratoria, enfermería y médico. se recomienda implementar un protocolo de retiro de ventilación mecánica basado en la prueba de respiración espontánea y articulado con un protocolo de sedación y analgesia en el paciente críticamente enfermo por sospecha o diagnóstico de covid- . desde diciembre de , un número de casos de neumonía por síndrome respiratorio agudo severo sars-cov /covid- en wuhan china se identificaron, como causa de insuficiencia respiratoria aguda( ). el síndrome de dificultad respiratoria aguda (sdra) ocurre en el % de los pacientes hospitalizados y en el % de los pacientes admitidos a la unidad de cuidados intensivos (uci) en wuhan ( ) . mientras la ventilación mecánica es una intervención que potencialmente salva la vida, esta puede llevar a múltiples complicaciones y contribuir a la lesión pulmonar ( ) . es por todo esto que el retraso en el retiro de la ventilación mecánica puede aumentar el riesgo de amci ® infecciones, aumenta la sedación innecesaria, el trauma de la vía aérea y aumento en el costo de la atención de estos pacientes ( ) . el retiro de la ventilación mecánica es un proceso de tres pasos, el primero es conocido como preparación la cual depende de variables fisiológicas, criterios clínicos y predictores de weaning (destete), el segundo paso es el propio weaning, el cual consiste en la disminución del soporte ventilatorio entregado al paciente, con el objetivo de llevar al último paso que es la extubación( ), ilustración . por todo lo anterior, se recomienda realizar un proceso de retiro de ventilación mecánica invasiva adoptando un protocolo, seleccionando adecuadamente a los pacientes, ya que evidencia sugiere que la adecuada selección de los pacientes disminuye los días de ventilación mecánica, disminuye la estancia hospitalaria y la estancia en uci ( ) . se recomienda realizar el retiro de la ventilación mecánica siguiendo los pasos antes mencionados, iniciando con la preparación, la cual consiste en: preparación . asegurarse que la lesión pulmonar que llevó a la falla respiratoria esté resuelta. . adecuado intercambio de gas, definido como adecuados índices de oxigenación con peep (presión positiva al final de la expiración) a cmh o, y fio (fracción inspirada de oxígeno) < . . al igual que el proceso de intubación, es un proceso que genera aerosoles por lo cual se recomienda la aplicación de lidocaína dosis de , a , mg por kilogramo de peso, a minutos antes la extubación con el objetivo de disminuir el reflejo de tos ( ) y la exposición del personal de salud. se recomienda en el paciente con sospecha o diagnóstico de covid- a quien se considera realizar vmni, utilizarla en salas de presión negativa, donde estén disponibles, que garanticen la seguridad del recurso humano, con todo el equipo de protección personal necesario. se recomienda considerar la vmni en pacientes con covid- con hipoxemia leve (pao /fio < y > o sao /fio < y> ) en salas de presión negativas, donde estén disponibles que garanticen la seguridad del recurso humano, con todo el equipo de protección personal necesario. fuerte a favor se recomienda considerar la vmni en pacientes con covid- con hipoxemia leve (pao /fio < y > o sao /fio < y> ) y con historia de epoc o cuadro de edema pulmonar agudo asociado en salas de presión negativas, donde estén disponibles que garanticen la seguridad del recurso humano, con todo el equipo de protección personal necesario. se recomienda colocar doble filtro en el circuito del ventilador para reducir el riesgo de generación de aerosoles en vmni del paciente crítico con sospecha o diagnóstico de covid- . se recomienda la intubación inmediata en pacientes críticamente enfermos por covid- si se evidencia respiración toraco-abdominal, uso de músculos accesorios, frecuencia respiratoria > , hipoxemia (pao /fio < o sao /fio < ), fracaso ventilatorio (ph< . con paco > mmhg), hacor> . existe suficiente evidencia que demuestra que la vmni es una estrategia que reduce la mortalidad en pacientes críticos ( , , ) . además, reduce la necesidad de intubación. los números de casos necesarios a tratar (nnt) son: ocho para salvar una vida y para evitar una intubación ( ) . estos desenlaces son fundamentalmente en pacientes con epoc y edema pulmonar agudo ( ) ( ) ( ) ; también hay evidencia a favor, aunque menos fuerte, en pacientes inmunosuprimidos ( , ) . se ha planteado mayores tasas de éxito con interfaces faciales totales o con helmet ( , ( ) ( ) ( ) ( ) ( ) . por el contrario, en falla respiratoria hipoxémica hay evidencia en contra del uso de vmni ( ) . la experiencia previa con h n , sars y mers no apoya el uso de la vmni en falla respiratoria hipoxémica de origen viral ( ) ( ) ( ) ( ) ( ) ( ) . además, se ha cuestionado el uso de vmni en covid- por el riesgo de contagio al generar aerosoles ( ) . recientemente se demostró que la distancia de dispersión de aerosoles era menor de un metro ( ) . por otro lado, el fracaso de la vmni se ha asociado con alta morbimortalidad ( ) ( ) ( ) . ello obliga a evaluar estrictamente la posibilidad de éxito o fracaso. así, en falla respiratoria amci ® hipoxémica la escala hacor ha sido validada para este fin y un puntaje> contraindicaría la vmni ( ) . adicionalmente si se emplea la vmni el prolongar la decisión de intubación puede aumentar la mortalidad y es por ello necesario monitorizar estrictamente al paciente y evaluarlo para establecer, ojalá antes de dos horas, si el paciente responde a la vmni ( , , ) . las indicaciones para intubación en este caso son respiración toracoabdominal, uso de músculos accesorios, frecuencia respiratoria mayor de , hipoxemia (pao /fio < o sao /fio < ), fracaso ventilatorio (ph< . con paco > mmhg), hacor> o índice de rox (spo /fio )/frecuencia respiratoria) < ( , , , , , ) . recomendaciÓn se sugiere el uso de posición prono en pacientes no ventilados críticamente enfermos por covid- que no responden a la oxigenoterapia convencional de acuerdo con los protocolos institucionales, las condiciones de cada servicio y la tolerancia individual de cada paciente. débil a favor fundamento los pacientes con enfermedad por coronavirus (covid- ) están en riesgo de desarrollar un síndrome de dificultad respiratoria aguda (sdra) ( ) . en pacientes intubados con síndrome de dificultad respiratoria aguda grave, la posición prona (pp) temprana y prolongada (al menos horas diarias) mejora la oxigenación y disminuye la mortalidad ( , ) .debido a que las unidades de cuidados intensivos (uci) están sobrecargadas con pacientes con covid- , la pp en paciente despierto con respiración espontánea puede ser útil para mejorar la oxigenación y prevenir las transferencias hacia uci. un estudio describió la viabilidad del uso de la ventilación no invasiva y la cánula de alto flujo asociado a la pp estableciendo su tolerancia y su seguridad en pacientes con sdra moderado y severo( ); la pronación puede reclutar todas las regiones pulmonares y favorecer el drenaje de secreciones de la vía respiratoria, mejorando el intercambio gaseoso y la supervivencia en el síndrome de dificultad respiratoria (sdra) ( ) . en una comunicación corta proveniente de italia en el cual se incluyeron pacientes que son sometidos al pp asociada con el uso de ventilación no invasiva, se concluye que proporcionar niv en la posición prona a los pacientes con covid- y sdra en salas generales en un hospital en italia era factible. la frecuencia respiratoria disminuyó durante su implementación y la oxigenación mejoró durante una pronación posterior a su línea de base. si la intubación se evitó o se retrasó, queda por determinar ( ) . en otro reporte de caso, publicado recientemente por un grupo francés( ) de pacientes con covid- e insuficiencia respiratoria hipoxémica manejados fuera de la uci, el % amci ® fue capaz de tolerar pp durante más de horas. sin embargo, la oxigenación aumentó durante el pp solo un % y no se mantuvo en la mitad de los pacientes después del regreso a la posición supina. estos resultados son consistentes con los hallazgos de pequeños estudios previos de pp en pacientes no intubados ( ) . un ensayo clínico controlado que evalúe el uso del pp en pacientes no intubados puede ser un mecanismo para seleccionar pacientes que bien puedan beneficiarse de esta estrategia terapéutica. dada la evidencia débil que soporta el uso del pp en pacientes no intubados en términos de la disminución de la necesidad de entubación o ingreso a cuidados intensivos y la duda razonable de aumentar desenlaces deletéreos en aquellos pacientes en los cuales se retarde el tiempo de intubación, no se emite recomendación, a favor o en contra, del empleo de esta estrategia de manera rutinaria. en situaciones en las cuales hay limitación de recursos y de disponibilidad de camas en cuidados intensivos el uso de la pp asociada a vni o cánula de alto flujo podría ser una estrategia útil para mejorar la oxigenación en pacientes infectados con covid- e hipoxemia. se recomienda considerar la elevación de biomarcadores como la troponina i o t y el nt-pro-bnp en el paciente con covid- como indicadores de injuria miocárdica aguda, sin embargo, no reemplazan la ecocardiografía en el enfoque del paciente con sospecha de disfunción miocárdica. fuerte a favor fundamento la injuria miocárdica aguda asociada a covid- se reporta con frecuencia teniendo en cuenta los cambios en biomarcadores como la troponina y cambios electrocardiográficos, pero su impacto en la función cardíaca se desconoce y mucho menos su correlación con los cambios ecocardiográficos. los pacientes con covid- pueden desarrollar una serie de complicaciones cardiacas desde injuria miocárdica, arritmias, infarto, hasta miocarditis fulminante con falla cardiaca aguda y shock cardiogénico ( ) . la troponina i, se ha encontrado más elevada en pacientes con curso fatal por covid- ( ) . los niveles de nt-pro-bnp han sido reportados con elevación severa en pacientes con miocarditis y disfunción sistólica, con una disminución progresiva en relación a la mejoría de los pacientes, pero no parece tener un correlación significativa con el cambio de la fracción de eyección (fevi) ( ) . la evidencia clínica sugiere que la elevación de los biomarcadores es más relacionada al compromiso sistémico que el daño miocárdico directo, q. deng y colaboradores en un análisis retrospectivo de pacientes, reportaron niveles iniciales de troponinas normales casi en la mayoría, en el , % de los casos los niveles incrementaron significativamente, principalmente en los que fallecieron y solo pacientes tenían fevi menor al % y ninguno inferior al %, lo cual no sugiere una asociación entre las dos amci ® pruebas ( ) . en una publicación donde se compara el fenotipo de pacientes con covid- con un histórico de pacientes con sdra por influenza ( ) , se encontró que los primeros tenían mayor elevación de troponinas % vs %, pero en los parámetros ecocardiográficos contrario a lo que se esperaría los índices de rendimiento ventricular fueron mayores para el grupo de covid- : Índice cardiaco . vs . l/m/m ; fevi vs %; tapse vs mm, nuevamente aunque no fue uno de los objetivos del estudio, parece no encontrarse correlación entre los biomarcadores que sugieren injuria miocárdica y los parámetros ecocardiográficos, el cual constituye uno de los pilares de la exploración cardiaca. se recomienda considerar como marcadores iniciales de mal pronóstico en el paciente crítico con covid- con sospecha de disfunción miocárdica aguda la elevación persistente de troponina i, mioglobina o creatin kinasa; independiente de la fracción de eyección del ventrículo izquierdo evaluada mediante ecocardiografía. fundamento los pacientes con covid- admitidos a la unidad de cuidados intensivos presentan con frecuencia disfunción cardiaca primaria, que puede corresponder a cardiomiopatía por estrés o miocarditis viral, pero también pueden ser consecuencia del compromiso sistémico ( , ) . aunque parece que en los fenotipos cardiovasculares estudiados, el compromiso hemodinámico severo de la función sistólica izquierda y derecha es menor ( ) . en el estudio de deng y colaboradores con pacientes con covid- , la fiebre, la disnea, hipoxemia, la obesidad y niveles elevados de cpk, troponina y nt-pro-bnp se relacionaron significativamente con mayor severidad ( ) . en el subgrupo de pacientes con miocarditis frente a controles, el perfil clínico se describe con mayor edad, niveles de temperatura más elevados ( . ± . vs . ± . ; p: . ), mayor proporción de disnea ( , vs %), y de dolor torácico ( , vs , %). en los pacientes fallecidos el % tuvieron picos de elevación de troponina i y de nt-pro-bnp dentro la semana que precedió la muerte, el % presentaron alteraciones electrocardiográficas y solo el % presentaron fracción de eyección menor o igual al % ( ) . shi y colaboradores, enrolaron pacientes para describir el significado clínico del compromiso miocardio de pacientes con covid- en wuhan, pacientes fallecieron, de los cuales el % presentaron injuria miocárdica aguda ( ) . el área bajo la curva (auc) de la troponina i inicial para predecir muerte intrahospitalaria fue de , (ic %, de , - , ) con una sensibilidad y especificidad del %, el auc para mioglobina fue de . y para cpk-mb fue de , ( ). un punto de corte para el pico más alto de troponina i de . , tuvo un hazard ratio para mortalidad de . (ic %, . - . ; p= . ). en un análisis multivariado la edad avanzada, la respuesta inflamatoria y las enfermedades cardiovasculares subyacentes se asociaron con mayor riesgo de lesión miocárdica en pacientes con covid- . con la información disponible parece que los biomarcadores de lesión miocárdica aguda elevados amci ® al ingreso y de forma persistente pueden predecir el riesgo de mortalidad intrahospitalaria en los pacientes con sospecha o diagnóstico de covid- con afectación cardiovascular. se recomienda no realizar de forma rutinaria ecocardiografía en pacientes críticos con covid- . se debe practicar ecocardiografía en pacientes con sospecha o diagnóstico de covid- si presenta alguna de las siguientes condiciones: . síntomas y signos de insuficiencia cardíaca aguda de novo. . shock o deterioro súbito hemodinámico refractario a líquidos y/o vasoactivos con sospecha de origen cardiogénico. . sospecha de infarto agudo de miocardio o embolismo pulmonar para determinar intervenciones terapéuticas con un beneficio clínico. . cambios en el electrocardiograma, arritmias ventriculares o paro cardiorrespiratorio no explicados por otra causa. para nuestro conocimiento, en el momento no existe estudios clínicos que evalúen los criterios para realización de ecocardiograma en el paciente con covid- . las sociedades de ecocardiografía han recomendado realizar el ecocardiograma en el contexto clínico en el cual, la información obtenida proporcione un cambio en la conducta o se espere un beneficio clínico al realizar este procedimiento ( ) ( ) ( ) ( ) . igualmente, se recomienda realizar el examen a la cabecera del paciente y el escaneo debe ser dirigido a contestar preguntas específicas según el contexto clínico del paciente ( ) ( ) ( ) ( ) ( ) . ward et al, en su publicación describe cómo el uso del ecocardiograma limitado (dirigido) en la university of chicago medicine (ucm), aumentó significativamente durante la pandemia ( % frente a %, p < . ), posterior a la implementación de recomendaciones sobre el uso apropiado de la ecocardiografía en tiempos de pandemia ( ) . los pacientes con infección por sars -cov- pueden presentarse con comorbilidades cardiovasculares que potencialmente estén descompensadas y/o compromiso cardiovascular por covid- . en este último, podemos encontrar alguno de los siguientes fenotipos: falla cardiaca aguda en el marco de compromiso directo viral o secundario al estrés metabólico y liberación de citoquinas, síndrome coronario agudo, cor-pulmonar secundario a tep o por compromiso secundario al sdra ( , , , ) . las manifestaciones cardiovascular puede sospecharse en el marco de choque que no esté explicado por causas extracardiacas evidentes que no responde a líquidos, dolor torácico con clínica de síndrome coronario agudo, cambios electrocardiográficos y elevación de biomarcadores de lesión miocárdica, signos de falla cardiaca descompensada, deterioro súbito de la oxigenación, arritmias o paro cardiorrespiratorio ( , , ) . ante estas manifestaciones, el ecocardiograma podría ser útil para entender el origen de la descompensación aguda, al estar enfocado a amci ® responder preguntas acerca de la función ventricular global y segmentaria (en el abordaje de síndrome coronario agudo), compromiso del ventrículo derecho, alteraciones valvulares, derrame pericárdico, si existe una contribución cardiovascular al compromiso pulmonar, si en el marco del choque existe evidencia de componente cardiogénico y cómo podría guiarse/optimizarse el soporte hemodinámico de estos pacientes( - ). se sugiere en pacientes críticamente enfermos con covid- que cursan con shock y sdom, ajustar la monitoria a las condiciones clínicas del paciente y recursos disponibles. se puede considerar el cap para el monitoreo del gasto cardiaco, la valoración de la perfusión y orientar los elementos hemodinámicos del tipo de shock, el cap de gasto cardiaco continuo puede disminuir la exposición del personal de salud frente al catéter de medición convencional. se sugiere la utilización de la tdtp dependiendo de la disponibilidad del recurso para orientar el diagnóstico diferencial del sdra versus edema pulmonar cardiogénico en los pacientes con covid- . fundamento los pacientes con infección severa por sars-cov- , cursan con alto riesgo de falla renal y cardiovascular, con necesidad de un manejo restrictivo de líquidos, lo que justifica la monitoria estricta en uci ( , ) . el catéter venoso central es útil para la monitoria inicial de estos pacientes, sin embargo, su predicción a respuesta a volumen está limitada ( ) ( ) ( ) . la monitoría no invasiva tiene limitaciones en casos severos de inestabilidad hemodinámica, ventilación espontánea y en presencia de peep alto, lo que limita su uso para el cálculo de gasto cardiaco y la predicción de respuesta a líquidos ( , ) . la monitoria con cap puede ser considerada en pacientes con covid- que cursan con choque y doms, con el objetivo de realizar un diagnóstico definitivo de los componentes del choque, valorar la hipoperfusión, la función cardiaca y el estado de volemia ( , ) . igualmente, los pacientes con sospecha tep o compromiso del ventrículo derecho pueden beneficiarse de esta monitoria( ). richard et al, en su estudio determinaron los desenlaces asociados al uso de cap vs cvc en pacientes con shock, sdra, o ambos, sin evidenciar diferencias en mortalidad ( . % vs . % p =. ) o estancia hospitalaria. el uso de cap no garantiza la mejoría de desenlaces en pacientes con covid- , sin embargo, la presencia de una monitoria continua ayudaría a optimizar los recursos y disminuiría la interacción con el paciente, con menor exposición del equipo médico ( ) . la monitoria por tdtp puede utilizarse en pacientes con covid- que cursan con choque, buscando optimizar el manejo hídrico, valorar el agua extravascular pulmonar (evlw) y el índice de permeabilidad vascular pulmonar (pvpi) con el fin de establecer el diagnóstico amci ® definitivo del edema pulmonar: sdra vs cardiogénico ( ) ( ) ( ) . hu et al, en su estudio evaluaron los desenlaces del uso de evlw y la presión de cuña de la arteria pulmonar (pawp) como estrategias para el manejo de líquidos en pacientes sdra, no encontraron diferencias significativas en las tasas de supervivencia (p = , ). no obstante, en el grupo de evlw la duración de la ventilación mecánica y la estancia en la uci fueron significativamente menor (p < , ), al igual que el balance hídrico (p < . ), con mejoría significativa en los índices de oxigenación (p = . )( ). no se puede emitir una recomendación a favor o en contra para la utilización de un protocolo de ultrasonido rutinario a la cabecera del paciente (pocus). sin embargo, se podría considerar el uso en pacientes seleccionados, con los adecuados epp y desinfección de los equipos; donde el pocus pueda tener ventajas sobre otras modalidades de monitoria o en pacientes con limitaciones para monitoria invasiva que requieren evaluación del estado hemodinámico o determinación de severidad del compromiso pulmonar. las recomendaciones sobre la utilidad de pocus en pacientes con covid- están enfocadas principalmente en la evaluación de la severidad/progresión de la lesión pulmonar, diagnóstico de manifestaciones cardiovasculares, monitoria hemodinámica y en la guía de fluidoterapia ( , ( ) ( ) ( ) . en la valoración del compromiso pulmonar por covid- , el pocus ofrece una ventaja sobre otras modalidades de monitoreo, debido a la capacidad de enmarcar el compromiso pulmonar en una línea de tiempo según sus hallazgos: desde la aparición de un patrón de "líneas b", consolidaciones subpleurales con evolución a consolidaciones multilobares, irregularidades en el artefacto de la línea pleural y finalmente aparición de patrón de "líneas a" una vez inicie la recuperación, con adecuada correlación tomográfica ( ) ( ) ( ) ( ) . en la diferenciación del origen del choque, el pocus ha demostrado superioridad versus el concepto clínico al evaluar: función ventricular (incluyendo ventrículo derecho), vena cava inferior, líquido libre abdominal, lesiones aórticas, compromiso pulmonar y búsqueda de trombosis venosa profunda identificando tep ( , ) . en cuanto a la monitoria hemodinámica de pacientes con covid- , adicional a la función y gasto cardíacos, se recomienda variables dinámicas de respuesta a líquidos y ultrasonido pulmonar en el diagnóstico de sobrecarga hídrica ( ) . la variabilidad de gasto cardiaco calculado por pocus durante la elevación pasiva de miembros inferiores, identifica los respondedores a líquidos con sensibilidad del % y un lr (-): . [ %ci, . - . ]) ( ) . la variabilidad de la vena cava inferior muestra limitaciones en pacientes con aumento de presiones de cavidades derechas y respiración espontánea ( ) . en cuanto a otras modalidades de monitoría no invasiva, es importante conocer las limitaciones en el marco de compromiso hemodinámico severo, ventilación espontánea, alteraciones valvulares aórticas, entre otras ( ) . marik ( ) . el ultrasonido tiene limitaciones específicas como la necesidad de un operador experimentado y la adecuada calidad de las imágenes. adicionalmente, en los pacientes con covid- , esta modalidad de monitoreo requiere una mayor interacción con el paciente, mayor uso de epp en comparación con otras modalidades el pac( ). se recomienda perseguir al inicio de la reanimación del paciente críticamente enfermo con covid- , metas clínicas de fácil medición, como la presión arterial media (entre y mmhg) o el gasto urinario (mayor a , cc/k/h) y metas de perfusión como el lactato en sangre arterial (menor a mmol), la saturación venosa central de oxígeno (entre y %) y la diferencia veno arterial de co (menor a mmhg). se han reportado casos de disfunción ventricular como causa de choque asociado a covid- , sin embargo, no se ha descrito un manejo específico o cambios en las metas de reanimación para estos pacientes, las manifestaciones de hipoperfusión tisular son: alteración de la conciencia, oliguria, piel fría y moteada y pulso débil. a nivel de gases sanguíneos las metas de reanimación pueden ser globales como el lactato (en sangre arterial vn< mmol) y la diferencia veno arterial de co (pv-aco vn < mmhg) o regionales como la saturación venosa central de oxígeno medida en la sangre venosa tomada de un catéter central (vn: - %). se puede optimizar la perfusión, interviniendo los principales determinantes: fluidos para aumentar el volumen intravascular, inotrópicos para aumentar la fuerza de contractilidad, vasopresores para recuperar la presión de perfusión y transfusión de glóbulos rojos para aumentar la hemoglobina como transportador de oxígeno ( ) , las recomendaciones dadas en el documento anterior siguen siendo válidas e incluyen entre otras que todo paciente con covid- en estado de shock debe ser ingresado de forma inmediata a la unidad de cuidados intensivos, garantizando el aislamiento indicado, procurando recuperar la presión arterial media a valores > mmhg, para ello la utilización de un catéter venoso central en los pacientes que no responden al manejo inicial y el procedimiento debe ser realizado por el médico con mayor entrenamiento, idealmente guiado por ecografía si hay disponibilidad y las competencias, así mismo las estrategia de control estricto de fluidos para no generar efectos deletéreos relacionados a la sobrecarga de volumen, es lo más indicado. se recomienda en la reanimación inicial de pacientes en estado de shock con sospecha o amci ® diagnóstico de covid- , guiar la fluidoterapia con el uso de índices clínicos como el tiempo de llenado capilar, temperatura de la piel y depuración de lactato; en fases avanzadas donde el monitoreo clínico es insuficiente utilizar medidas dinámicas como la variabilidad de presión de pulso (vpp), la variabilidad de volumen sistólico (vvs), la respuesta a la maniobra de elevación pasiva de piernas o la prueba de oclusión teleespiratoria de acuerdo a los recursos disponibles y la experiencia. fuerte a favor fundamento sobre los objetivos de intervención y el tipo de agente vasoactivo a utilizar en pacientes con covid- y shock no existe una evidencia directa y las recomendaciones se basarán en evidencia indirecta de pacientes críticos con diversos tipos de shock en especial el séptico y vasopléjico. la falla circulatoria aguda asociado a sdra en covid- se presenta con una frecuencia del - % y en la admisión a urgencias la hipotensión y un lactato ≥ es infrecuente( , ). la baja sensibilidad del qsofa y crb- para predecir la severidad del covid- y la necesidad de intervenciones de terapia intensiva refleja lo infrecuente del shock en esta condición. factores como la vasoplejia, fuga capilar asociada al estado hiperinflamatorio, altos requerimientos de peep y disfunción cardiaca pueden ser generadores o contribuyentes del shock y deben ser considerados en el abordaje diagnóstico, en su interpretación para lograr tomas de decisiones adecuadas. en pacientes con sdra, una reanimación óptima de líquidos debe tomar en cuenta aspectos como tiempo (oportunidad), tipo (cristaloides balanceados y/o no balanceados o coloides) y volumen (ni mucho, ni poco) con el objetivo de disminuir la mortalidad, el tiempo de vm y de cuidados intensivos, sin que ello afecte los índices de oxigenación, de perfusión tisular y la morbilidad asociada con su uso inadecuado. la administración agresiva de líquidos puede empeorar la oxigenación y la disfunción ventricular, lo que potencializa un mayor tiempo de ventilación mecánica e incluso la mortalidad. la evidencia ha demostrado que una estrategia conservadora de fluidos (balance - +/- amci ® en una revisión sistemática y un metaanálisis de rct (n = . ) una terapia dirigida al aclaramiento temprano de lactato frente a una terapia guiada por la saturación venosa central de oxígeno (svo ), se asoció con una reducción significativa de la mortalidad (rr , ), menor estadía en la uci (dm , días), y menor duración de la ventilación mecánica (dm - , horas). pero se debe resaltar que un nivel alto de lactato no siempre indica hipovolemia; también puede ser causada por uso de adrenalina, agonistas beta o por disfunción mitocondrial, insuficiencia hepática e isquemia mesentérica ( ) . por otra parte, el llenado capilar (crt), una prueba técnicamente fácil y accesible, realizada cada minutos se asoció con una reducción no significativa de la mortalidad (hr , ) en comparación con la medición de lactato sérico cada horas. dado el potencial beneficio sobre mortalidad, duración de estancia en uci y la duración de la ventilación mecánica, así como su accesibilidad, sugerimos utilizar parámetros dinámicos de temperatura de la piel, tiempo de llenado capilar y / o medición de lactato sobre parámetros estáticos para evaluar la capacidad de respuesta a la fluidoterapia en pacientes con covid- y shock( ). se recomienda en pacientes adultos con covid- y estado de shock, escoger la norepinefrina como el vasopresor de primera línea y a la vasopresina el de segunda. si no se cuenta con norepinefrina el uso de vasopresina o epinefrina serían la primera elección; la dopamina no se recomienda por el mayor riesgo de arritmias. se recomienda iniciar dobutamina frente al aumento de la dosis de norepinefrina en pacientes en estado de shock con evidencia de disfunción cardíaca e hipoperfusión persistente a pesar de la reanimación inicial. fuerte a favor fundamento sobre los objetivos de intervención y el tipo de agente vasoactivo a utilizar en pacientes con covid- y shock no existe una evidencia directa y las recomendaciones solo pueden basarse en evidencia indirecta de pacientes críticos con sepsis y sdra. en pacientes en shock séptico los agentes vasoactivos para alcanzar una pam de - es un objetivo razonable. una presión media más alta puede incrementar . veces el riesgo de arritmias cardiacas y no está exento de riesgo de isquemia en las extremidades ( ) . para aproximarnos a la escogencia de los vasoactivos en shock séptico, basados en su perfil de riesgo/beneficio, la guía scandinavian society of anaesthesiology and intensive care medicine (ssai ) task force for acute circulatory failure( ), la revisión sistemática de cochrane database con rct con un n: ( ) y el ensayo clínico controlado de honarmand k et al con un n: , ( ) amci ®  la noradrenalina es el agente vasoactivo más ampliamente estudiado con el menor riesgo a priori de efectos no deseados, razón por la cual se sugiere usar como el agente vasoactivo de primera línea en pacientes con covid- y shock.  si la noradrenalina no está disponible la vasopresina o epinefrina se muestran como la mejor alternativa. los factores que determinan la elección entre vasopresina y epinefrina pueden incluir disponibilidad y el perfil de seguridad de estos agentes. con la vasopresina, la isquemia digital puede ser una preocupación y con epinefrina, la taquicardia, la isquemia miocárdica y el exceso de producción de lactato.  el uso de dopamina se ha asociado un . veces mayor de riesgo de aparición de arritmias frente a la norepinefrina y un posible aumento del riesgo de mortalidad. por ello la dopamina no debe utilizarse en pacientes con covid- y shock donde haya disponible de norepinefrina o las alternativas señaladas  en shock distributivo la adición de vasopresina a las catecolaminas evidenció baja certeza de reducción de la mortalidad (rr , ; ic del %: , a , ), alta certeza de una reducción de la fibrilación auricular (rr , ; ic del %: , a , ) y certeza moderada de un mayor riesgo de isquemia digital (rr , ; ic del %: , a , ). en vista de estos hallazgos se plantea la vasopresina como un agente vasoactivo de segunda línea a ser utilizado si la pam objetivo no se ha alcanzado con norepinefrina en pacientes con covid- y shock. no existe evidencia directa en pacientes con covid- y shock, para establecer una recomendación sobre cuál es el agente inotrópico óptimo. en una guía de práctica clínica de que evalúa el agente inotrópico óptimo en pacientes con insuficiencia circulatoria aguda (shock), no se identificaron rct que comparen dobutamina versus placebo o ningún tratamiento. con base en una justificación fisiopatológica, sugerimos agregar dobutamina, más que no suministrar ningún tratamiento, en pacientes con covid- y shock con evidencia de disfunción cardíaca e hipoperfusión persistente a pesar de la reanimación con líquidos y altas dosis de norepinefrina. el uso de dobutamina en estado de shock, incluso en pacientes con covid- con shock, debe ser investigado. recomendaciÓn se recomienda no suspender la medicación estándar para falla cardiaca en pacientes con sospecha o diagnóstico de covid- , especialmente los iecas, ara-ii, y b-bloqueadores, si la condición clínica permite continuar el uso de esta medicación, ya que no se ha podido confirmar una asociación nociva. amci ® previos alrededor de la relación independiente de la edad avanzada, enfermedad cardiovascular subyacente (enfermedad coronaria, insuficiencia cardíaca y arritmias), tabaquismo activo y epoc con muerte por covid- ( ) ( ) ( ) ; esos mismos reportes sugieren que las mujeres son proporcionalmente más propensas a sobrevivir a la infección por covid- que los hombre; existen además consideraciones especiales desde el punto de vista cardiovascular, que se deben tener en cuenta al decidir cualquier terapia en paciente afectados por covid- y existe la hipótesis de un efecto nocivo de la terapia estándar para falla cardiaca en estos pacientes ( ) . en un estudio observacional que incluye paciente con infección por covid- , se evaluó la relación entre la enfermedad cardiovascular subyacente, y la asociación entre la terapia farmacológica cardiovascular y la mortalidad ( ); respecto a los factores de riesgo cardiovascular, el . % de los pacientes tenían hiperlipidemia, el . % tenía hipertensión, el . % tenía diabetes mellitus, y en relación a los medicamentos, los sobrevivientes usaron más comúnmente ieca y las estatinas que los no sobrevivientes, mientras que no se encontró asociación entre la supervivencia y el uso de ara ii; respecto a los otros medicamentos incluidos b-bloqueadores, antiplaquetarios e insulina no se encontraron diferencias significativas; sin embargo, y teniendo en cuenta el impacto sobre mortalidad del uso de betabloqueadores en la falla cardiaca, consideremos que la decisión de continuar su uso debe basarse en el análisis clínico de cada paciente y su estabilidad clínica. se recomienda establecer un protocolo de reanimación ajustado al contexto del paciente con covid- con una organización administrativa ajustada a la pandemia que incluya las siguientes estrategias:  desarrollar una estrategia de prevención del paro cardiorrespiratorio en el paciente con sospecha o diagnóstico de covid- basada en la detección oportuna.  formar un equipo multidisciplinar formado en rcp con líderes médicos, de en enfermería y en terapia respiratoria, los cuales deben educar a todo el equipo de trabajo en la identificación de signos de alerta temprana, cambios abruptos de variables clínicas, técnicas de monitoreo, interpretación de paraclínicos y de alarmas de monitoreo.  entrenamiento del personal sanitario a través del uso de la simulación clínica en manejo de crisis, epp, y procesos de atención fundamentales en la atención del paro cardiaco en el paciente con sospecha o diagnóstico de covid-  promover la comunicación asertiva, planeación y retroalimentación de las intervenciones realizadas antes y después de un evento de paro cardiaco (briefing y debriefing), con el fin de establecer modificaciones que conllevarán a mejoras en la atención de futuros eventos. amci ® el pronóstico y sobrevida de un paciente con sospecha o diagnóstico de covid- que presenta paro cardiaco depende de la prevención a través del reconocimiento oportuno de las causas reversibles de éste, la no presencialidad y un ritmo de paro no desfibrilable lo hacen de mal pronóstico. la prevención va a depender del nivel de entrenamiento del equipo previamente, y en época de pico de pandemia cuando el talento humano especializado disminuya, se hace necesario que en los equipos de trabajo estén liderados por especialistas en la disciplina para que puedan guiar al equipo. los nuevos procesos de atención del paciente, la alta contagiosidad del virus, y el uso de nuevos medicamentos hacen que se requiera un entrenamiento del personal para estandarizar los procesos y disminuir el error médico. la crisis de covid- está ejerciendo una presión sin precedentes sobre las personas, los equipos y los sistemas organizacionales, conllevando a errores médicos que van desde la infección cruzada por el personal sanitario con la posibilidad de cometer errores en la atención. cada día trae nuevos desafíos: picos en volumen y gravedad, escasez de equipos y estrés en los médicos sobrecargados, que se manifiesta según la experiencia de wuhan en insomnio y depresión. se propone implementar una estrategia antes, durante y después del trabajo clínico, denominada circle up covid- desarrollada por center of medical simulation dirigida a convertir equipos de trabajo muchas veces insustituibles , en eficientes , seguros, fuertes y que se apoyan mutuamente incluyendo en mejora de la salud psicológica . impactando en el rendimiento del equipo , y promoviendo el bienestar y la resiliencia( - ) se recomienda establecer un protocolo de reanimación ajustado al contexto clínico del paciente con covid- que incluya las siguientes modificaciones:  implementar criterios de selección e inicio de maniobras de rcp en la atención del paro cardíaco basados en la bioética y en el pronóstico de supervivencia a corto y largo plazo de los pacientes.  promover la prevención del paro cardiaco mediante la detección oportuna del riesgo y definir intubaciones programadas.  asegurar la correcta protección con los epp necesarios al abordar el paro cardiaco y la intubación que son procedimientos generadores de aerosoles.  priorizar el manejo de la vía aérea antes del inicio de las compresiones torácicas, haciendo énfasis en la reducción de la exposición de aerosoles (código azul protegido).  utilizar filtro de alta eficiencia contra virus para todas las estrategias de ventilación (bolsa mascarilla con cierre hermético y en el circuito del ventilador).  promover la realización de la intubación por el operador de mayor experticia con uso de videolaringoscopio si está disponible y considerar el acceso supraglótico solo si el intento de intubación es fallido. amci ®  en caso de parada cardiaca en ventilación mecánica iniciar el masaje cardiaco evitando las desconexiones del circuito del respirador.  en caso de paro en posición prono si el paciente se encuentra vigil retornar rápidamente a la posición supino y si está en ventilación mecánica es razonable realizar compresiones en la espalda. fuerte a favor fundamento se hace necesario el entrenamiento en el manejo de los procedimientos generadores de aerosoles y se sugiere que el que realiza la intubación orotraqueal debe ser el más experto. el riesgo de aerosolización es de . en el proceso de intubación orotraqueal, el de compresiones torácicas es de , , ventilación mecánica no invasiva de , , ventilación manual pre-intubación de , , succión después de intubación , . se ha descrito que si no se ha capacitado previamente en el retiro de los epp existe más riesgo de auto contaminación y aumenta ésta sin un líder supervisor al retirarlo. según revisión de cochrane sobre ropa y equipo de protección para los trabajadores sanitarios para evitar que se contagien con el coronavirus y otras enfermedades altamente infecciosas da a conocer que la capacitación presencial, la simulación por ordenador y la capacitación por vídeo dieron lugar a menos errores a la hora de quitarse el epp que la capacitación impartida solo como material escrito o una conferencia tradicional ( ) ( ) ( ) ( ) ( ) ( ) . se recomienda no considerar la existencia de manifestaciones neurológicas específicas o típicas atribuidas a la infección por sars-cov- . se recomienda realizar la valoración neurológica integral del paciente con diagnóstico o sospecha de sars-cov- teniendo en cuenta manifestaciones frecuentes relacionadas a covid- : disgeusia, anosmia, cefalea, vértigo, confusión, delirium, alteración de estado de consciencia, eventos cerebrovasculares, ataxia, polineuropatías inflamatorias y convulsiones. una revisión sistemática realizada por asadi-pooya et al. entre diciembre y marzo de mostró que el % de los pacientes con covid- presentaron sintomatología neurológica. analizaron cinco artículos (n= ) donde eran retrospectivos, y era prospectivo, encontrando cefalea entre el y el %, vértigo entre el y el %, confusión en el %, alteración del estado de consciencia en el %, eventos cerebrovasculares en un %, ataxia en un , % y convulsiones en un , % ( ) . amci ® menor a participantes donde recolectarán información sobre el compromiso neurológico en pacientes con covid- con un seguimiento hasta febrero de ( ) . la hiposmia y la disgeusia de aparición súbita son manifestaciones clínicas muy prevalentes en pacientes con covid- evidentes aun en ausencia de sintomatología respiratoria alta. lechien et al publicaron un estudio multicéntrico que incluyeron hospitales europeos reclutando pacientes infectados con covid- levemoderado donde , % presentaron alteraciones relacionadas con el olfato y , % presentaron alteraciones relacionadas con el gusto, donde la anosmia se presentaba antes que cualquier otro síntoma en el , % de los casos y el , % de los casos no presentaba rinorrea u obstrucción nasal, la recuperación del olfato fue presente en el % de los pacientes y las mujeres fue el grupo poblacional más afectado (p= , ) ( ) . otras manifestaciones clínicas son las alteraciones de la agudeza visual, y dolor tipo neuralgia ( ) . respecto a las patologías psiquiátricas; la serie de mao comenta que el , % de los pacientes tiene clínica de alteración del estado de consciencia, concepto que se aproxima a la defunción de delirium. severance et al. encontraron que pacientes con sintomatología psicótica aguda presentaron niveles elevados de inmunoglobulina g para coronavirus del tipo hku , nl y oc con diferencias estadísticamente significativas respecto a los individuos controles (n= ) (p< , ). donde la respuesta inmune para nl fue asociado con el espectro-esquizofrenia (or: . , ci . - . , p= . ) pero no se correlaciona con desórdenes afectivos ( ) . aún no se ha descrito una correlación directa de este trastorno psicótico con covid- . los síntomas musculares se han observado en pacientes infectados por covid- incluyendo la miopatía del paciente en estado crítico (miopatía difusa no necrotizante con degeneración grasa de fibras musculares), la miopatía necrotizante (ligada a falla orgánica múltiple) y la miopatía de filamentos gruesos( ). se recomienda considerar como predictores clínicos neurológicos de alerta para sospechar covid- : anosmia, disgeusia, delirium y alteraciones neuromusculares inespecíficas sin otra causa aparente de explicación. se recomienda no establecer de rutina predictores neurológicos específicos de mal pronóstico en el paciente críticamente enfermo con covid- . se deben tener en cuenta los factores de riesgos generales de mal pronóstico para la población general como la edad avanzada, las comorbilidades cardiovasculares y el tabaquismo. page la proteína spike (s) del covid- es reconocida por la enzima convertidora de antígenos (eca ) de la célula huésped cuyo papel es el punto de entrada molecular a tejidos pulmonares, gastrointestinales y neuronales ( ) . la forma como el covid- ingresa al sistema nervioso central es desconocido, pero se especula que inicialmente invade terminales nerviosas periféricas y después llega al sistema nervioso central a través de una ruta guiada por sinapsis nerviosas con un patrón ascendente (ruta dada por el coronavirus hev y el oc- ) ( , ) . en modelos de roedores el covid- ingresaría al cerebro a través del nervio olfatorio, atravesando la lámina cribiforme propagándose por el tálamo y el tallo cerebral; explicándose por la expresión de los receptores de la enzima convertidora de antígenos (eca ) en la superficie de las mucosas nasales, las neuronas y la glía ( , , ) . una segunda forma de ingreso es a través de la vía hematológica mediante arterias cerebrales atravesando la barrera hemato-encefálica utilizando las células inflamatorias como un modelo similar al del caballo de troya ( , , ) logrando una ubicación definitiva en células neuronales y endoteliales del lóbulo frontal como lo demostraron en estudios post-mortem descritos por paniz -mondolfi et al, lo que explicaría los cambios comportamentales de paciente ( ) . la tercera forma de acceso al sistema nervioso central es mediante el drenaje del sistema linfático cerebral invadiendo ganglios linfáticos hiliares y mesentéricos con sintomatología gastrointestinal asociada ( ) . una vez ha logrado ingresar tiene la capacidad de infectar macrófagos, microglía, y astroglía los cuales secretan factores proinflamatorios como interleuquina , interleuquina , y factor de necrosis tumoral alfa ( ) . esta condición se exacerba con el desencadenamiento de la tormenta de citoquinas liderada por la interleuquina , interleuquina , interleuquina e interferón gamma ( ) . de esta forma los coronavirus siendo neurotrópicos ocasionan múltiples manifestaciones clínicas ya mencionadas, así como encefalitis, parálisis flácida, incluyendo la asociación con guillain-barré ( ) . los síntomas que harían sospechar la presencia de neurocovid- son la náusea, el vómito y la anorexia, estos síntomas pueden ser el reflejo del compromiso del virus en el área postrema del piso del cuarto ventrículo que hace parte del complejo vagal dorsal de la médula oblonga. sin embargo, estos síntomas pueden enmascararse como una respuesta inespecífica relacionada con un compromiso gastrointestinal ( ) . como se mencionó previamente la anosmia y la disgeusia son síntomas significativos para sospechar en covid- ( ) ; esto es debido a una lesión directa sobre el nervio olfatorio (i par craneal), y la lesión de alguno de los tres nervios encargados de registrar el sentido del gusto como lo son el vii, ix y x pares craneales, así como el compromiso del núcleo solitario y del tálamo como zona de relevo; de hecho el núcleo del tracto solitario es muy cercano al centro respiratorio que podría ocasionar disnea de origen central ( ) , otros núcleos como el núcleo dorsal motor del vago y el núcleo ambiguo están relacionados con funciones cardiovasculares a tener en cuenta ( ) . respecto a los factores de riesgo destaca el tabaquismo el cual aumenta la posibilidad de neuroinfección debido a interacciones funcionales entre el receptor nicotínico de acetilcolina y el receptor eca el cual está sobreexpresado en pacientes fumadores ( ) . amci ® en pacientes con infecciones severas vs infecciones no severas ( . % vs . %, p = . ), incluyendo eventos cerebrovasculares ( , % vs , % p= , ), alteración del estado de consciencia ( . % vs . %; p< . ) y lesiones musculoesqueléticas ( , % vs , % p< ) ( ) . la encefalopatía que se manifiesta como una alteración aguda o subaguda del estado de consciencia presentándose en pacientes con comorbilidades, factores de riesgo cardiovasculares, edad avanzada y deterioro cognitivo previo ( , , ) . así como aquellos individuos con hipoxemia la cual induce metabolitos anaerobios en el sistema nervioso central, edema celular, intersticial e isquemia ( ) . los eventos cerebrovasculares pueden ser desencadenados por cuadros de hipoxia, inmovilización, un incremento de la respuesta proinflamatoria o por predisposición a la hipercoagulabilidad ( ) . respecto a este último rubro tanto la edad (hazard ratio , /por año % ic , - , ) y la coagulopatía definida como como un tiempo de protrombina mayor a segundos, o tiempo de tromboplastina mayor a segundos (hr , % ic , - , ) fueron considerados predictores independientes de complicaciones trombóticas ( ) ; otros trabajos reportan incremento del conteo plaquetario y niveles elevados de dímero d ( ) . de hecho, un scoping review realizado por wilson y jack muestra que la presencia de eventos cerebro vasculares es un factor de riesgo de mal pronóstico para pacientes infectados por covid- ( ) . mao et al. reportaron eventos cerebro vasculares en pacientes con covid- , los factores de riesgo más relevantes fueron los clásicos factores de riesgo cardiovasculares (diabetes, hipertensión y edad avanzada), así como una presentación sistémica severa, teniendo como un denominador común el compromiso estructural de grandes vasos ( ) . la presencia de convulsiones (clínicas o subclínicas) puede ser una manifestación de eventos cerebro vasculares, meningoencefalitis o hipoxia cerebral. siendo los factores de riesgo más importantes en su exacerbación las alteraciones electrolíticas como hipocalcemia, las reacciones adversas a medicamentos y la epilepsia como comorbilidad de base ( , ) . otros tipos de coronavirus como lo son el e, , y oc se han aislado de líquido cefalorraquídeo (lcr) de pacientes con esclerosis múltiple sugiriendo posiblemente sean agentes etiológicos en la exacerbación de brotes de esta patología sin embargo aún no se ha documentado la asociación entre covid- y ésta condición ( ) . otras patologías en las cuales se ha asociado la presencia de esta familia de microorganismos es la enfermedad de parkinson, la esclerosis lateral amiotrofia, la neuritis óptica y la encefalitis aguda diseminada ( , ) . múltiples trabajos han documentado la asociación entre polineuropatía y coronavirus. la mayoría de ellos consideran que existe una estrecha relación entre una polineuropatía autoinmune exacerbada por la infección por coronavirus o bien un compromiso nervioso periférico inducido por bloqueo neuromuscular, alteraciones hidroelectrolíticas o disvitaminosis( ). amci ® se recomienda que todo paciente con acv isquémico se considere sospechoso de infección por covid- si presenta: sintomatología asociada sospechosa de infección por covid- , contacto cercano con individuos con sintomatología infecciosa, allegados con viajes recientes, si la historia clínica es atípica, si la información suministrada no es clara, si presenta deterioro del estado de alerta inexplicable, y si al examen físico presenta hallazgos compatibles con una infección por covid- . se recomienda que todo paciente con acv isquémico de quien no se pueda recibir información se considere sospechoso de covid- ya que el evento cerebro vascular es una complicación que se ha reportado en pacientes con infección por covid- . hay una gran evidencia que correlaciona la presencia de infección por covid- con factores de riesgo cardiovasculares. esto se demostró en un metaanálisis que incluyó estudios de china (n= ) donde las comorbilidades más frecuentes fueron hipertensión ( , %, % ic: - , %), diabetes ( , %, % ic , - , %), enfermedad cardiovascular ( . %, % ic: . - . ) y patologías respiratorias ( . %, % ic: . - . %). al comparar severidad vs no severidad la presencia de hipertensión tuvo un or de . ( % ci: . - . ), y la enfermedad cardiovascular un or de . ( % ci: . - . ) respectivamente ( ) . existieron pocos casos de hipercoagulabilidad en pacientes sin factores de riesgo cardiovasculares ( ) . la cohorte de wuhan (n= ) publicada por wang et al. mostró otros factores de riesgo que podrían eventualmente estar asociados a estado de embolia y trombosis como shock en , %, arritmias , % y miocarditis en un , %. situaciones que conllevarían a hipercoagulabilidad, lesión endotelial y eventualmente la aparición de acv. ( ) . en este orden de ideas existe una estrecha correlación entre la presencia de factores de riesgo cardiovasculares que ocasionarían acv y que podrían eventualmente estar relacionados con la patogenia de covid- . ante esta inquietud khosravani et al. publicaron en stroke un informe especial donde realizan ciertas recomendaciones para el abordaje de los pacientes con acv en el contexto de la pandemia por covid- de una forma rápida, eficaz y segura para los diferentes profesionales de la salud. surge así el código stroke protegido el cual consiste en:  usar elementos de protección personal y una mascarilla al paciente  ejecutar protocolo de aislamiento de contacto y gotas  ejecutar protocolo de aerosoles si el paciente está sometido a ventilación mecánica no invasiva, manejo de aspiración de secreciones o maniobras de reanimación cardiopulmonar básica y avanzada  si el paciente presenta deterioro del estado de alerta con necesidad de soporte ventilatorio alto con fracción inspirada de o mayor a % se recomienda intubar temprano y proceder con el transporte. se debe proceder con el código stroke protegido si el paciente presenta alguna de las siguientes condiciones:  si el paciente presenta sintomatología sospechosa de covid- (fiebre, tos, dolor torácico, disnea, cefalea, mialgias, emesis)  si existe algún contacto cercano con sintomatología infecciosa  si el paciente o alguno de sus allegados ha presentado viajes recientes amci ®  si el paciente es covid- positivo  si refiere al interrogatorio una historia clínica atípica o poco clara  si el paciente o alguno de sus acudientes es incapaz de suministrar información  si el paciente presenta deterioro del estado de alerta  si al examen físico se encuentran signos compatibles con patologías diferentes a covid- por último, estas son las recomendaciones en el momento de realizar el traslado a saber:  no apresurarse dentro de la sala de reanimación o dentro de la unidad de cuidados intensivos y mantener la calma  designar un líder para el traslado del paciente y para supervisar el uso adecuado de los elementos de protección personal  limitar el número de personas encargadas del transporte  evitar la contaminación con otras áreas del hospital( ). se recomienda no realizar neuroimagen de rutina en pacientes críticos por covid- con cefalea y anosmia, dado que no existe una evidencia concluyente que demuestre una estrecha correlación entre estos síntomas y hallazgos imagenológicos. fuerte en contra fundamento en la mayoría de los casos la cefalea es un síntoma no específico que no es característico de irritación meníngea la ocurrencia de cefaleas aisladas en ausencia de otros síntomas sugiere un mecanismo benigno más que un compromiso de sistema nervioso central ( ) . sin embargo, el covid- al tener una capacidad neuroinvasiva, se han reportado casos de encefalitis virales con o sin necrosis hemorrágicas de compromiso temporal mesial y talámico que ameritarían estudio de imagen diagnóstica ( ) . un estudio retrospectivo publicado por kandenmirli et al. evaluó pacientes en hospitales infectados con covid- de los cuales requirieron manejo en uci donde el % de ellos (n= ) presentaron sintomatología neurológica. la resonancia magnética cerebral fue realizada en % (n= ) de estos pacientes. el % tuvieron hallazgos agudos, el % tuvieron alteraciones corticales de la captación de señal en el modo flair, pacientes tuvieron anormalidades en la señal flair en la sustancia blanca profunda y subcortical, pacientes con lesiones en el lóbulo frontal, en el lóbulo parietal, en el lóbulo occipital, en el lóbulo temporal, en la corteza de la ínsula, y en el giro cingular ( ) . amci ® un paciente presentó trombosis de seno venoso y otro presentó un infarto en el tercio medio del territorio de la arteria cerebral media. en % de los pacientes no se encontraron hallazgos que sugieran compromiso intracraneal de covid- . una correspondencia escrita por helms et al. reportaron pacientes con sdra y covid- en dos unidades de cuidados intensivos en francia entre marzo y abril del , el % presentaron signos neurológicos como delirium evaluados mediante cam-icu ( %), agitación ( %), signos del tracto cortico espinal ( %), síndrome disejecutivo ( %). de ellos se realiza resonancia magnética en pacientes encontrando alteraciones en la perfusión en un %, un realce de leptomeninges en un %, y un acv isquémico en un %; dos pacientes asintomáticos presentaron áreas isquémicas con hiperintensidad focal ( ) . ante estos hallazgos los principales diagnósticos diferenciales son las patologías autoinmunes, encefalitis, convulsiones e hipoglucemia. los pacientes con compromiso frontal bilateral poseen hipoxemia que ocasionan hipoperfusión fronto temporal. las microhemorragias corticales (y no) son consecuentes de la ruptura de la membrana hematoencefálica resultando en este patrón mencionado. el estado postictal muestra un compromiso simétrico de la sustancia blanca. de esta forma hay que considerar otras condiciones como las comorbilidades cardiovasculares, las reacciones adversas a los medicamentos, e hipoxia inducida por sdra que ocasionarían patrones imagenológicos de confusión, que ponen en duda la estrecha relación entre covid- y hallazgos de resonancia magnética ( ) . en este orden de ideas necesitamos más datos para determinar cuáles son los hallazgos imagenológicos relacionados con neurotropismo y qué patrones pueden encontrarse directamente relacionados con la presencia de convulsiones, hipoxia o el desencadenamiento de una tormenta de citoquinas( , , ). se recomienda la valoración neurológica completa en los pacientes con sospecha o infección por covid- . se recomienda la monitorización electroencefalográfica continua por al menos h o según la consideración del especialista en neurociencias en el paciente en estado crítico con sospecha o infección por covid- en quien se sospeche crisis epilépticas o estatus no convulsivo. las manifestaciones neurológicas en pacientes con infección por covid- pueden estar presentes en el % de los casos ( , , ) . en el paciente en estado crítico con infección por covid- se han observado complicaciones vasculares que puede causar ataque cerebrovascular agudo isquémico o hemorrágico con tazas de entre el al % por lo que el examen neurológico debe realizarse para documentar la presencia de déficit amci ® neurológico focal que haga sospechar esta patología ( , , ) . es conocido que los pacientes en unidades de cuidado crítico tienen patologías que aumenta el riesgo de crisis o estado epiléptico, entre las cuales se encuentran ( ) ( ) ( ) ( ) :  pacientes sin patologías neurológicas hospitalizados en unidad de cuidado intensivo  hemorragia subaracnoidea  hemorragia intracraneal  trauma cráneo encefálico moderado a severo  infección del sistema nervioso central  tumor cerebral  encefalopatía hipóxico-isquémica algunas de estas patologías se presentan más frecuentemente en pacientes por covid- por lo que la monitorización electroencefalográfica continuar es requerida en este grupo de pacientes. el virus puede producir descompensación de paciente con epilepsia conocida, crisis por fiebre, crisis producidas por el estado crítico del paciente o las patologías subsecuentes que se han observado en esta infección ( ) . el tiempo de monitorización requerido debe ser entre a horas para lograr una sensibilidad de a %( , ). se recomienda realizar tomografía cerebral simple ante las manifestaciones neurológicas focales que nos hagan sospechar ataque cerebrovascular isquémico o hemorrágico. la resonancia cerebral puede ser necesaria como estudio complementario para determinar otros diagnósticos diferenciales en los pacientes con sospecha o infección por covid- . las manifestaciones neurológicas en pacientes con infección por covid- pueden estar presentes en el % de los casos ( , , ) . en el paciente en estado crítico con infección por covid- se han observado complicaciones vasculares que puede causar ataque cerebrovascular agudo isquémico o hemorrágico con tazas de entre el al % por lo que el examen neurológico debe realizarse para documentar la presencia de déficit neurológico focal que haga sospechar esta patología ( , , ) . la tomografía cerebral hace parte de la valoración inicial del ataque cerebrovascular isquémico y del estudio para determinar la presencia de otros diagnósticos diferenciales como ataque cerebro vascular hemorrágico en paciente con dicha patología puede ser necesario realizar angiotac con extensión a tórax en caso de sospecha de oclusión proximal o estudios endovasculares en pacientes que sea indicado ( ) . la realización de resonancia cerebral usualmente documenta alteraciones en los pacientes con covid- en el - % de los casos evaluados no relacionada con el acv. se recomienda la realización de punción lumbar en el paciente con sospecha o diagnóstico de covid- con base en los reportes de casos disponibles:  paciente con crisis epilépticas de novo.  paciente con alteración del estado de conciencia persistente a pesar de encontrarse metabólicamente compensado, descartado ataque cerebrovascular u otra causa de encefalopatía.  paciente con manifestaciones como mielitis, neuropatía craneal múltiple o sospecha de polineuropatía desmielinizante aguda. las capacidades neurotrópicas de los coronavirus en general han sido expuestas desde la infección por sarscov. en cuanto al sars-cov- , asadi-pooya y colaboradores( ), han descrito, la posibilidad de ingreso al sistema nervioso central tras el ingreso por la mucosa nasal o por una gran viremia en el torrente sanguíneo. se han descrito procesos inflamatorios asociados (encefalitis) y en previamente con el sars y el mers hasta lesiones desmielinizantes (encefalomielitis aguda diseminada). estas primeras manifestaciones en con el sars fueron presencia de crisis epilépticas de novo, en quienes excluyendo otras causas tanto por neuroimagen además de pruebas microbiológicas en lcr, les fue descubierto el sars cov mediante pcr rt. en la presente pandemia, takeshi moriguchi y colaboradores ( ) describieron el primer caso de encefalitis asociado a sars-cov- , en un hombre joven en japón, que, tras días de clínica respiratoria, desarrollo crisis epilépticas y alteración del estado de conciencia, sin antecedente conocido de epilepsia. se realizo imagen por resonancia cerebral, demostrando hiperintensidades a nivel temporal y lcr que mostró pleocitosis linfocitaria, se descartaron otros virus ( herpes, herpes zoster) y se le realizó pcr rt para sars-cov- siendo positiva. inclusive en este paciente los primeros hisopados faríngeos fueron negativos, pero los hallazgos en tomografía de tórax hicieron sospechar la infección por sars-cov- . otro caso descrito de encefalitis hemorrágica aguda, fue también descrito en la revisión de ahmad y colaboradores ( ) . donde una mujer joven presentó cuadro respiratorio de fiebre de días de evolución y compromiso del estado de conciencia. en esta paciente se aisló el sars-cov- en hisopado faríngeo y ante el compromiso severo del estado de conciencia, se realizó resonancia que mostró compromiso hemorrágico bitalámico, en regiones temporales e ínsula, apoyando que se tratara de una diseminación tras neuronal probablemente con puerta de entrada mucosa olfatoria y siguiendo la diseminación por el los tractos del primer nervio hasta la corteza entorrinal, que es la vía propuesta de infección descrita inclusive por grupos como el de montalvan ( ) y natoli ( ) . amci ® precisamente, en la revisión sistemática de montalvan ( ) se describen caso de mielitis en contexto de paciente con infección por sars-cov- , en quien se documentó la infección en lcr. se recomienda la medición de ácidos nucleicos por rt-pcr para sars-cov- en líquido cefalorraquídeo en los pacientes con sospecha o confirmación de infección por sars-cov- que realice crisis epilépticas de novo, en quien se descarte otras causas de estructuralidad (acv, tumores) o causas metabólicas (alteraciones hidroelectrolíticas, hipoglicemia, uremia etc.). se recomienda la medición de ácidos nucleicos por rt-pcr para sars-cov- en líquido cefalorraquídeo en pacientes con sospecha o confirmación de infección por sars-cov- con alteración franca del estado de conciencia, en quien se haya descartado como causante hipoxia, ataque cerebrovascular, alteración hidroelectrolítica o estatus no convulsivo mediante imágenes y electroencefalograma. se recomienda la medición de ácidos nucleicos por rt-pcr para sars-cov- en líquido cefalorraquídeo en pacientes con sospecha de encefalitis, mielitis o síndrome de guillain barré. en pacientes críticos, se ha recomendado búsqueda activa de compromiso del snc por sars-cov- , sobre todo en pacientes con crisis epilépticas que no sean sintomáticas a trastornos metabólicos, además de hallazgos imagenológicos y curso clínico. las descripciones hechas por asadi y takeshi ( , ) , muestran pacientes con cursos tórpidos, en los que el común denominador es una alteración del estado de conciencia persistente a pesar que otras variables (metabólicas, vasculares e infecciosas diferentes al sars-cov- ). de igual forma pacientes que realicen en contexto de la enfermedad, un cuadro de debilidad generalizada aguda, con arreflexia e incluso compromiso de nervios craneales como los descritos por zahra sedaghat( ). page amci ® se recomienda en los pacientes críticos con compromiso pulmonar por covid- , un manejo orientado de fluidos frente a una estrategia liberal, ajustando el balance de fluidos de acuerdo con la evaluación clínica y/o a la capacidad de respuesta a volumen para garantizar la perfusión renal. se recomienda ajustar la intensidad de la monitoria en el paciente crítico con covid- al grado de severidad de la enfermedad para alcanzar tempranamente metas de reanimación que se reflejen en menor riesgo de lesión renal aguda. se recomienda evitar el uso de medicamentos nefrotóxicos teniendo en cuenta la farmacocinética y farmacodinamia individual, así como las interacciones farmacológicas en el paciente crítico con covid- . el manejo de la volemia en los pacientes críticos ha cambiado considerablemente en las últimas décadas orientándose a un manejo titulado, evitando la administración empírica de altos volúmenes de líquidos durante la fase de reanimación de los pacientes siendo el flujo sanguíneo el determinante primordial del aporte tisular de oxígeno; los principales componentes de este deben optimizarse y balancearse para evitar la disoxia tisular. en estados de bajo flujo, los mecanismos compensadores neurohumorales producen una redistribución del flujo a lechos no esplácnicos y a nivel renal una redistribución corticomedular convirtiendo el tejido medular renal en una zona vulnerable a la lesión. de igual forma, el flujo sanguíneo renal se ve reducido de forma refleja en presencia de hipoxemia y/o hipercapnia. diversas fuentes de información nos han indicado cómo orientar adecuadamente el manejo del estado de perfusión tisular en los pacientes críticos con o sin una condición de shock, siendo la estimación aproximada del estado de volumen del paciente el pilar fundamental sobre el cual se basará toda la estrategia de restablecimiento o mantenimiento de la volemia del paciente con el fin de mantener la perfusión adecuada. aunque históricamente se prioriza la normalización del volumen en la reanimación de un paciente inestable, hemos comprendido la importancia que tiene el tiempo para lograr las metas en el pronóstico general del paciente y evidencias como el trabajo de ospina y cols. soportan el uso temprano de vasopresores para lograr de forma temprana metas de perfusión mientras buscamos la normovolemia. los pacientes con enfermedad pulmonar asociada a covid- deben mantenerse normovolémicos para preservar el flujo sanguíneo renal siendo la evaluación del estado de volumen un verdadero reto clínico. la fiebre, el aumento de pérdidas insensibles, la baja ingesta o las pérdidas por el tracto digestivo, pueden hacer que un paciente con covid- tenga hipovolemia, situación que puede desencadenar daño renal de no ser revertida apropiadamente y a tiempo. del otro lado del espectro, la administración liberal de líquidos además del potencial de empeorar la lesión pulmonar en presencia de una membrana alveolo capilar seguramente alterada, puede producir por sí misma un incremento en el riesgo de desarrollar lesión renal como lo demostró el estudio de grissom. desde el punto de vista de las metas amci ® hemodinámicas que se deben tener con un paciente con covid- , se recomienda seguir los lineamientos de la campaña sobreviviendo a la sepsis, orientadas a mantener un óptimo estado de volumen, unas presiones de perfusión en un rango que permita la regulación de los flujos regionales en los distintos órganos y un gasto cardíaco dentro de unos rangos establecidos para una perfusión sistémica óptima. el examen clínico sigue teniendo vigencia absoluta para una adecuada aproximación al paciente, por ello debemos buscar los indicadores clínicos tradicionales de hidratación (piel, mucosas, enoftalmos, edema, etc.), el llenado capilar como lo describe hernández y cols en el estudio andrómeda-shock, el estado de alerta, las funciones cognitivas y la rata urinaria son entre otros unos marcadores aceptables para hacernos una idea del estado de adecuación de la perfusión periférica. la oliguria como marcador de perfusión renal está presente en / parte de los pacientes al ingreso a uci sin tener en sí sola una implicación pronóstica, sin embargo, la persistencia de esta en el tiempo es un indicador de alerta y obliga a una evaluación más detallada de las diversas variables que pudieran ocasionarla. debemos recordar que la administración de cargas de líquidos solamente está justificada cuando hay una respuesta cardiovascular a dicha administración, situación que se puede prever con una prueba de elevación pasiva de las piernas o con métodos más invasivos como la variabilidad de la onda de pulso, del volumen sistólico y/o del gasto cardíaco, entre otros. el esfuerzo respiratorio del paciente, los volúmenes utilizados en las estrategias de ventilación protectora y las arritmias frecuentemente presentes en los pacientes con compromiso pulmonar limitan el rendimiento diagnóstico de diversos dispositivos utilizados para la evaluación del estado de volumen y el gasto cardíaco, situación que debemos conocer y manejar( , - ). se recomienda no utilizar de forma rutinaria la administración de tratamientos específicos antivirales para el paciente crítico con covid- , con lesión renal aguda o crónica se recomienda no utilizar de rutina remdesivir en los pacientes con falla renal crónica y debe suspenderse en los pacientes que desarrollan lesión renal aguda con tfg < ml/min. las intervenciones farmacológicas en los ensayos clínicos deben ajustarse a la farmacocinética y farmacodinamia específicas de cada molécula. la incidencia de falla renal reportada por criterios de kdigo, en los estudios chinos fue de . % de los pacientes críticamente enfermos y de . % en los pacientes con covid- y amci ® sdra. sin embargo, otros estudios han demostrado que hasta un %de pacientes con covid- que ingresan a la uci pueden presentar falla renal aguda. la falla renal en los pacientes con covid- es multifactorial como se ha descrito en preguntas anteriores por lo que se recomienda la toma diaria de creatinina sérica y el seguimiento continuo del gasto urinario y otros parámetros de la función renal como hematuria, proteinuria, tasa de filtrado glomerular, nitrógeno ureico en sangre, dímero d. los medicamentos que se emplean en el manejo de la infección covid- que actualmente incluyen oseltamivir, lopinavir/ritonavir, ribavirina, y la cloroquina o hidroxicloroquina son metabolizados principalmente en el hígado, aunque en la orina se encuentran metabolitos derivados de oseltamivir, ribavirina y de la hidroxicloroquina. por esto en ninguno de los estudios realizados en torno a la infección por sars-cov- se ha realizado ninguna recomendación en cuanto a la modificación de su dosis. la hidroxicloroquina por su parte se metaboliza a cloroquina, que a su vez se metaboliza a monodesetilcloroquina y a bisdesetilcloroquina. este medicamento no es dializable en las diálisis intermitentes y la única recomendación en los pacientes con falla renal, es hacer seguimiento electrocardiográfico estrecho para vigilar la prolongación del qtc. el favipiravir es un inhibidor de la polimerasa dependiente de rna que se encuentra en fase experimental para el tratamiento de la infección por sars-cov- . la eliminación de este medicamento se realiza por vía renal y en los pacientes con falla renal en estadios leves a moderados se ha encontrado una concentración dos veces mayor en el riñón que sus niveles en sangre; sin embargo, esto no se ha asociado con ningún evento adverso por lo que la recomendación actual es no disminuir la dosis en pacientes con falla renal. el remdesivir se elimina por vía renal por lo cual no se recomienda administrar en pacientes con falla renal y los pacientes que desarrollan falla renal con el medicamento durante los estudios han sido retirados de los ensayos clínicos. no se cuenta con estudios que evalúen el remdesivir en una tfg < ml/min. en la tabla se describen algunas intervenciones farmacológicas propuestas en medio de la pandemia para el manejo del covid- y su relación con la tasa de filtración según tfg. recientemente , izzedine et al publicaron una carta editorial en el kidney international may , donde alertan sobre el posible efecto nocivo de la hidroxicloroquina en la aparición de falla renal aguda al inhibir la autofagia celular que es un proceso importante en la remodelación de los túbulos renales, siendo estas células de alto recambio, pudiendo todo esto contribuir a la aparición de falla renal aguda( , , - ) se recomienda aplicar las indicaciones tradicionales de terapia de soporte renal en pacientes críticamente enfermos con covid- . se recomienda el inicio de terapia de soporte renal en pacientes críticos con covid- con hipercalemia severa o acidosis metabólica severa, refractarias a pesar del manejo médico óptimo o cuando el balance positivo de fluidos es deletéreo, con mayor requerimiento de oxígeno suplementario y que no responde a diuréticos. se recomienda el inicio temprano de terapia de soporte renal dentro de las primeras horas de una indicación absoluta, asegurando previamente una adecuada reanimación de la perfusión tisular. en ausencia de trastornos hidro-electrolíticos y severa sobrecarga de volumen, el tiempo de inicio de diálisis es controversial. amci ® se recomienda en pacientes críticos con covid- que requieren soporte renal, las modalidades de terapia continua o extendida si cursa con inestabilidad cardiovascular, de acuerdo con la disponibilidad institucional. se debe considerar en pacientes críticos con covid- , que requieren inicio de soporte renal preferir la vía femoral para inicio de la terapia para disminuir el riesgo de contaminación por proximidad, la siguiente vía se establecerá de acuerdo con la evolución y condiciones del paciente. en pacientes diagnosticados con covid- se puede presentar la insuficiencia renal aguda como parte de su enfermedad. en estudios observacionales de usa y china la ira se reportó entre un y % de los pacientes. la enfermedad renal en pacientes con covid- se puede manifestar como ira, hematuria o proteinuria, y conllevan un mayor riesgo de mortalidad. la ira se asocia con cambios hemodinámicos y liberación de citocinas, pero no se descarta citotoxicidad directa por el virus. en un estudio realizado en nueva york con pacientes covid- positivos se diagnosticó lesión renal aguda en % de ellos, siendo leve con aumento de creatinina dos veces por encima del nivel basal en % de los pacientes, moderada en % de los pacientes, y severa con más del triple de la creatinina basal en %. hubo hematuria en el % de los pacientes y proteinuria en el %. se requirió terapia dialítica en el % de todos los pacientes con diagnóstico de ira, y el % de los pacientes que requirieron diálisis estaban en ventilación mecánica. la ira fue notada dentro de las primeras horas de admisión a uci en el % de los pacientes y se relaciona con la severidad de la enfermedad. existen además predictores independientes: edad, raza negra, diabetes, hipertensión, enfermedad cardiovascular, ventilación mecánica, y uso de vasopresores. la terapia dialítica debe instaurarse precozmente una vez realizado el diagnóstico, idealmente dentro de las primeras horas, después de asegurado que se ha completado el proceso de reanimación correspondiente. las indicaciones te trr en pacientes críticos con ira en covid- no difieren del paciente crítico general y se debe considerar ante: manifestaciones severas de uremia, sobrecarga de volumen, trastornos ácidos básico, refractarios, hipercalemia severa con manifestaciones cardiovasculares. pero no hay datos clínicos que respalden el inicio temprano vs tardío en esta población particular. pero un planteamiento válido es que la sobrecarga de volumen en pacientes que desarrollan sdra es perjudicial, dificultando el soporte ventilatorio óptimo, por lo cual se puede considerar un umbral más bajo para el inicio de trr con esta indicación específica: sdra + sobrecarga de volumen + infección covid- ( ) . amci ® el paciente debe ser dializado en el cubículo de cuidado intensivo o en la habitación de aislamiento en los casos en que esté disponible y siempre evitar traslado a unidades con otros pacientes. la crrt es la modalidad preferida para este tipo de pacientes, pero debe quedar claro que esto depende de las facilidades de la institución que albergue al paciente y de la experticia de los profesionales. el acceso vascular en el paciente crítico general debe ser en su orden: vena yugular interna derecha, venas femorales comunes, vena yugular interna izquierda, y debe ser colocado por el médico encargado del paciente si está capacitado para ello, para evitar exposiciones innecesarias del personal de la salud. sin embargo, por precaución por el riesgo de contaminación, recomendaciones de expertos basadas en seguridad sugieren la utilización el catéter femoral. el tipo de catéter recomendado es un catéter doble lumen transitorio. sería ideal el monitoreo a través de cámaras del procedimiento para evitar el contacto prolongado del personal de enfermería durante el procedimiento de diálisis. en algunos casos específicos y de acuerdo con la disponibilidad, la diálisis peritoneal puede ser una alternativa. en los casos de crrt el líquido efluente no es contaminante para el personal de la salud. para terminar, es importante hacer énfasis en que en algunos hospitales ha habido escasez de insumos y esto puede llegar a convertirse en un serio problema. se recomienda para casos de fuerza mayor:  un litro de solución salina al . % con cloruro de potasio a necesidad  un litro de dextrosa al % en agua con meq de bicarbonato de sodio  un litro de solución salina al . % con gr de cloruro de magnesio  un litro de solución salina al . % con gr de cloruro de calcio esto nos da una solución de cuatro litros que contienen: meq/l de sodio, . meq/l de bicarbonato, . mmol/l de magnesio y . mmol/l de calcio, más una cantidad variable de potasio. esta solución se puede usar como líquido dializante en pacientes en terapias de reemplazo renal continuo. especial cuidado se debe tener en el proceso de anticoagulación, pudiéndose usar heparina no fraccionada, hbpm, y citrato en los centros donde se tenga experiencia ( , , ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . se sugiere no utilizar de rutina la trrc más hemoperfusión en el paciente crítico con covid- . débil en contra page amci ® se puede considerar en el paciente crítico con covid- con lesión renal aguda en quien se considere inicio de trrc, considerar la utilización de filtros de fibra hueca con propiedades adsortivas o asociado con cartuchos para hemoperfusión directa. el síndrome de liberación de citocinas (tormenta de citocinas) es un importante determinante en la transformación de infección por covid- de leve a moderado y progresión de la lesión de un órgano como el pulmón con neumonía y sdra a compromiso sistémico con inestabilidad hemodinámica, cid y fom. los pacientes afectados de tormenta de citocinas se encuentran con niveles altos de il- especialmente, además de il- , tnf, que se relacionan con pobres pronósticos y mayor mortalidad. la asociación entre la lesión alveolar y renal (eje pulmón-riñón) es evidenciada en estudio del por panitchote y cols con pacientes con sdra secundaria a neumonía y sin enfermedad renal preexistente que desarrollaron lesión renal aguda en el % con aki en el % de ellos. recientemente en estudio en china zhou, con pacientes afectados de covid- se encontraron como indicadores de mal pronóstico pacientes con altos niveles de dímero d, il- , troponina i, dhl, ferritina y choque séptico. las terapias de depuración extracorpórea han sido utilizadas como tratamiento en pacientes con lesión severa por covid- ; dentro de estas se cuentan la crrt, hemoperfusión aislada, intercambio plasmático (tpe), plasmafiltración y adsorción (cpfa) y crrt + hemoperfusión. dentro de los beneficios de la crrt se cuentan la estabilidad hemodinámica, estabilidad del medio interno, depuración de toxinas pequeñas y medianas, incluidos mediadores inflamatorios, cuando se utiliza terapia convectiva, además de permitir soporte nutricional. la asociación de este procedimiento con membranas especiales (an + metilsulfonato y polietilamina oxiris) permiten hacer adsorción de citocinas (il- ) y endotoxinas, por periodos de h por días consecutivos para manejo de tormenta de citocinas. la hemoperfusión aislada o asociada a crrt, también permite la remoción de il- , utilizando cartuchos ha , con procedimientos de a horas de duración por días consecutivos ( , ( ) ( ) ( ) ( ) ( ) ( ) . no se puede emitir una recomendación a favor o en contra sobre el uso rutinario de la plasmaféresis como opción terapéutica en la fase de inflamación del paciente con covid- . amci ® el coronavirus covid- puede inducir el síndrome respiratorio agudo severo (sars), que conduce a la disfunción inmune, la liberación excesiva de citoquinas inflamatorias, y a una serie de reacciones en cascada de activación de citoquinas, que resultan en lesiones alveolares difusas, formación de membrana hialina, exudación de fibrina y otras manifestaciones de lesión del pulmón. en casos severos, la tormenta de citoquinas sistémicas invade el sistema circulatorio, lo que lleva a una inestabilidad hemodinámica, shock y mods ( ) . los niveles de il- , il- , tnf-a y otras citoquinas inflamatorias en pacientes con covid- grave son significativamente más altos, lo que puede estar relacionado con un mal pronóstico ( ) . por lo tanto, la plasmaféresis se puede usar con seguridad y efectividad en pacientes con covid- grave, para eliminar mediadores inflamatorios de gran peso molecular. la seguridad depende como todas las terapias extracorpóreas de un personal de la uci entrenado, preparado y capacitado para aplicar las intervenciones en forma óptimas ( ) . las terapias extracorpóreas de soporte de órganos pueden representar una parte importante de la respuesta y los médicos y otros profesionales de la salud deben estar familiarizados con estas terapias sofisticadas. se debe hacer un llamado a la acción, para crear conciencia sobre las diferentes técnicas extracorpóreas, cada una con criterios específicos y modalidades de prescripción, entrega y monitoreo( , ). se recomienda no utilizar de forma rutinaria el uso de un tratamiento específico dirigido a pacientes con infección por sars-cov- /covid- comparado con el manejo estándar para mejorar desenlaces clínicos fuertes. actualmente no existe una terapia dirigida que se a efectiva para el manejo del virus; un número alto de estudios han surgido en los últimos dos meses, la mayoría sin el rigor metodológico suficiente para tomar decisiones adecuadas con respecto al manejo del amci ® paciente con infección por sars-cov- . el conocimiento en la estructura del virus y el mejor entendimiento en la fisiopatología de la enfermedad genera un sinnúmero de potenciales fármacos que han sido ensayados para el manejo de la enfermedad. en tiempos de pandemia, con una patología catastrófica en términos de vidas humanas y costos hospitalarios; es importante encontrar soluciones a desenlaces importantes como mortalidad, días de estancia en uci y en el hospital, aumento en los días libres del ventilador, disminución de complicaciones mayores debido a la enfermedad entre otros. hasta el momento no se ha documentado ninguna terapia específica que pueda impactar sobre estos desenlaces; pero la calidad de los trabajos, tampoco dejan claro sin él no usar ningún tratamiento específico mejora los desenlaces al menos al disminuir el número de complicaciones. este nuevo beta-coronavirus es similar al coronavirus del síndrome respiratorio agudo severa (sars-cov) y del síndrome respiratorio del medio este (mers-cov); por lo tanto, varias moléculas que habían sido evaluadas en este tipo de enfermedad rápidamente se abrieron paso a ensayos clínicos en paciente con covid- . estos ensayos principalmente observacionales, aleatorios pero abiertos con un número pequeño de pacientes no han permitido sacar adecuadas conclusiones y es frecuente como ver las diferentes guías de las principales sociedades del mundo cambiar de forma frecuente sus recomendaciones; no existes evidencia de estudios clínicos aleatorios y controlados que midan desenlaces fuertes, la premura de un tratamiento efectivo ha sacrificado el rigor metodológico que una investigación requiere. una estructura viral y replicación conocidas generan posibles dianas para que diferentes fármacos puedan ser investigados, antivirales tipo arbidol el cual inhibe la fusión de la membrana en la envoltura viral a algunos receptores; antimaláricos como la hidroxicloroquina y la cloroquina, las cuales inhiben la entrada viral y endocitosis por múltiples mecanismos, así como los efectos inmunomoduladores demostrados en el huésped; antivirales que impiden la replicación como el lopinavir o darunavir inhibiendo las proteasas o la ribavirina, el remdesivir o el favipiravir que actúan como análogos de nucleótidos o fármacos que actúan modulando la respuesta específica del huésped como el tocilizumab el cual se une al receptor de la il- inhibiendo el punto de acción de esta; los corticosteroides con múltiples efectos en la modulación del sistema inmunológico del paciente o los fármacos para evitar la respuesta secundaria a esta cascada inflamatoria como son los anticoagulantes. por último, se han buscado estrategias con el fin de mejorar la inmunización pasiva del huésped en el uso del plasma de pacientes convalecientes o el uso de inmunoglobulinas enriquecidas entre otros tratamientos propuestos para esta enfermedad. como vamos a ver más adelante, actualmente no existe un tratamiento específico con el nivel de evidencia suficiente para recomendar de manera generalizada; tampoco existe suficiente evidencia del manejo del soporte básico sin el uso de fármacos dirigidos, que demuestre que esta estrategia se deba implementar de manera sistemática en todos los pacientes; por lo tanto, a continuación trataremos de resolver las inquietudes con respecto a los diferentes medicamentos que han sido usados en la pandemia del sars-cov- /covid- . se recomienda no utilizar antimaláricos tipo hidroxicloroquina (hcq) o cloroquina (cq) para el manejo de pacientes con infección por sars-cov- /covid- . para la fecha no hay un adecuado sustento bibliográfico que soporte el uso de antimaláricos en la prevención o manejo de pacientes con infección por sars-cov- tanto leve, moderada como severa. los mayores estudios no muestran utilidad clínica y tendencia a mayores eventos cardiovasculares con el uso de antimaláricos en pacientes con infección por covid- comparado con no darlo. su utilidad se deriva principalmente de resultados en estudios preclínicos e in vitro; como los presentados por wang y cols donde evaluaron medicamentos de manera in vitro contra el covid- , siendo el remdesivir y la cq efectivos de manera in vitro contra el nuevo coronavirus( ); liu y cols, donde la hcq fue efectiva en inhibir la infección por sars-cov- in vitro que junto con su potencial antiinflamatorio tenía potencial para el uso clínico ( ) y yao y cols, donde la hcq fue más potente que la cq para inhibir el sars-cov- in vitro y fue recomendado para el uso en humanos en dosis de mg dos veces al día por el primer día, seguido de mg dos veces al día por días más mantendría la concentración efectiva del fármaco en el tejido pulmonar ( ) . los pocos estudios clínicos, son de baja calidad y no han mostrado mejoría ni eficacia en el uso de antimaláricos para el manejo de paciente adultos con covid- , algunos estudios iniciales con pocos pacientes con resultados favorables ( ) e incluso con recomendaciones para uso en las primeras versiones de guías internacionales para la amci ® hcq y cq, encontrando superioridad en estudios observacionales, de pocos pacientes, sin comparadores para inhibir la exacerbación de la neumonía, hallazgos de las imágenes pulmonares, promover una conversión negativa al virus y acortar el curso de la enfermedad; la cq tuvo un efecto notable tanto en términos de resultado clínico como de eliminación viral ( ) ; considerando la hcq y la cq como un tratamiento costo efectivo ( ) . estudios posteriores con un mayor número de pacientes no han logrado reproducir los estudios preclínicos iniciales; mahévas y cols, evaluaron la efectividad de la hcq en pacientes admitidos a cuatro hospitales en francia, con neumonía por covid- quienes requieren oxígeno, pero no se encontraban en uci, comparado con una población con manejo estándar; la hcq se usó a dosis de mg día en las primeras horas a la admisión, este estudio no soporte el uso de la hcq en pacientes admitidos al hospital con covid- que requieren oxígeno al no reducir de forma significativa la admisión a la uci, el sdra o muerte en el día después del ingreso ( ) ; por el contrario, se han reportado efectos secundarios frecuentes (prolongación del intervalo qt, hipoglucemia, cambios en el estado mental, alteraciones gastrointestinales y retinopatía); silvia borda y cols, evaluó la seguridad y eficacia de dos dosis de cq en pacientes con covid- severo en un estudio aleatorizado, doble ciego fase iib en pacientes adultos hospitalizados con infección por sars-cov- , los pacientes fueron expuestos a dosis altas de cq ( mg dos veces al día por días) o dosis bajas ( mg dos veces al día en el día y una vez al día por días), los hallazgos preliminares de este estudio sugieren que la dosis más alta de cq no debe recomendarse para pacientes críticos con covid- debido a sus posibles riesgos de seguridad, especialmente cuando se toman simultáneamente con azitromicina y oseltamivir; estos hallazgos no pueden extrapolarse a pacientes con covid- no severo ( ); tang y cols, evaluaron la eficacia y seguridad de la hcq con el manejo estándar en un estudio multicéntrico, abierto, aleatorio y controlado en china, pacientes con covid- positivo se incluyeron en el análisis de intención a tratar ( en el grupo de hcq y en el grupo estándar), la hcq fue administrada a dosis de mg día por tres días y mantenimiento con dosis de mg día (duración del tratamiento: dos a tres semanas en pacientes con enfermedad leve a moderada o enfermedad severa respectivamente); la administración de hcq no resultó en una significativa mayor probabilidad de conversión negativa comparado con el tratamiento estándar, los efectos adversos fueron mayores en el grupo de hcq ( ) . con todo esto la hcq y la cq, si se usan deberá ser bajo estudios experimentales aprobados con una estricta monitorización y vigilancia clínica de la frecuencia cardíaca y el intervalo qt, los niveles de glucosa, la función hepática y renal, y el cribado clínico de trastornos mentales y visuales en pacientes que reciben estos fármacos. debe evitarse hcq/cq en pacientes con enfermedades cardiovasculares subyacentes. nuevas evidencias con un mayor número de pacientes podrían sacar la hcq y la cq inclusive de estudios clínicos; barbosa y cols, evaluaron en un estudio cuasialeatorio comparativo el uso fuera de registro de la hcq en pacientes positivos por el sars-cov- , el pronóstico primario fue la necesidad de escalar el soporte ventilatorio, cambio en el conteo de linfocitos o cambio en el índice de neutrófilos/linfocitos, un total de pacientes fueron incluidos, en el brazo de hcq. la administración de hcq fue asociada con la necesidad de aumentar el nivel del soporte ventilatorio comparado con aquellos que no recibieron hcq al día del estudio, no hubo beneficios en la mortalidad, reconstitución inmunológica y riesgo de intubación ( ) . el estudio con un mayor número de pacientes proviene de la ciudad de new york; geleris y cols, examinaron la asociación entre el uso hcq y la intubación o muerte en un centro médico de ny, se analizaron . amci ® estaban más enfermos en términos de oxigenación, en este estudio observacional la administración de hcq no fue asociada con una disminución en el riesgo compuesto de intubación o muerte ( ) . se recomienda no utilizar antimaláricos tipo hidroxicloroquina (hcq) o cloroquina (cq) en combinación con azitromicina (az) para el manejo de pacientes con infección por sars -cov- /covid- . fundamento para la fecha la evidencia no favorece el uso combinado de los antimaláricos en combinación con la azitromicina; por el contrario, la combinación de estos dos medicamentos puede ser deletérea, inclusive con un aumento reportado en la mortalidad y la aparición de arritmias ventriculares de novo; estudios iniciales fueron promisorios, gautret y cols, evaluaron inicialmente el efecto de la hcq en la carga viral respiratoria en conjunto con el uso de azitromicina, la presencia del virus al día fue el pronóstico primario; pacientes con tratamiento mostraron una significativa reducción en la carga viral al día de la inclusión comparado con los controles; la azitromicina adicionada a la hcq fue significativamente más eficiente en la eliminación viral ( ) ; nuevamente gautret y cols, realizaron un estudio observacional, no controlado, no comparativo de pacientes tratados con la combinación de hcq más azitromicina, presentando una mejoría significativa en disminución de la carga nasofaríngea del virus y una menor tiempo de enfermedad ( ) ; luego million y cols, evaluaron la combinación de hcq y az en un estudio retrospectivo de . pacientes con sars-cov- tratados con hcq ( mg tres veces al día por días) + az ( mg en el día , seguido de mg al día por los próximos días), el pronóstico fue mortalidad, empeoramiento clínico (ingreso a uci) o persistencia viral; la administración de hcq+az en combinación antes de que aparecieran las complicaciones del covid- es segura y asociada a una baja mortalidad en los pacientes ( ) ; soportado además por estudios in vitro que demuestran que la combinación de hcq y az tienen efectos sinérgicos para el sars-cov- a concentraciones compatibles con las que se obtienen en pulmones humanos ( ) . otros estudios por el contrario no han encontrado resultados positivos, es así como, magagnoli y cols, en un análisis retrospectivo de pacientes confirmados con infección por sars-cov- en centros de veteranos de los eeuu, un total de pacientes fueron evaluados (hcq, n= ; hcq+az, n= ; no hcq, n= ), en este estudio no hubo evidencia que el uso de la hcq tanto sola o en combinación con la az, redujo el riesgo de ventilación mecánica en pacientes hospitalizados con covid- ; una asociación con un aumento en la mortalidad fue identificada en pacientes tratados con hcq sola ( ) . nuevamente los estudios con un mayor número de pacientes se encuentran en la ciudad de new york; rosenberg y col, describieron la asociación entre hcq, con o sin az en el pronóstico de pacientes hospitalizados con covid- ; un estudio de cohorte multicéntrico retrospectivo en pacientes hospitalizados con covid- en hospitales de ny, los pacientes recibieron ; en pacientes hospitalizados en el área metropolitana de ny con covid- , el tratamiento con hcq, az o ambos, comparado con ningún tratamiento, fue no significativamente asociado con diferencias en la mortalidad hospitalaria ( ) . recomendaciÓn se recomienda no utilizar de forma rutinaria el uso rutinario del lopinavir/ritonavir para el manejo de pacientes con infección por sars-cov- /covid- . en la actualidad no existe evidencia a favor o en contra en el uso del tratamiento con antirretrovirales con lopinavir/ritonavir en el manejo de pacientes adultos hospitalizados con covid- ; no se observó ningún beneficio con lopinavir/ritonavir más allá de la atención estándar. se está en espera de cierre de diferentes ensayos futuros que confirme o excluyan el uso de lopinavir/ritonavir en el paciente covid- . en diciembre de , un nuevo coronavirus, designado sars-cov- , ha causado una pandemia ( , , , ) ; cuando hablamos de enfermedad producida por covid- hablamos de enfermedades que van desde las enfermedades leves autolimitantes del tracto respiratorio hasta neumonía rápidamente progresiva, neumonía grave, falla multiorgánica y muerte. hasta este momento no existen agentes terapéuticos específicos para las infecciones por coronavirus. después de la aparición del síndrome respiratorio agudo grave (sars) en , entre los fármacos aprobados se identificó lopinavir, un inhibidor del aspartato proteasa tipo del virus de inmunodeficiencia humana (vih), que tiene actividad inhibitoria in vitro contra el sras-cov, el virus que causa el sars en los seres humanos y el ritonavir combinado con lopinavir para aumentar su vida media plasmática a través de la inhibición del citocromo p ( ) . se comenzaron estudios evaluando la respuesta antiviral in vitro de la combinación de lopinavir/ritonavir y ribavirina en pacientes con sars; comparados con pacientes tratados con ribavirina sola, que sirvieron como controles históricos; el pronóstico adverso (sdra o muerte) fue significativamente más bajo en el grupo de tratamiento comparado con los controles históricos ( . % vs . %, p = . ) al día del inicio de los síntomas; una reducción adicional en el uso de esteroides y de infecciones nosocomiales fue vista en el grupo de tratamiento con una disminución en la carga viral y aumento en el conteo de linfocitos ( ) ; del mismo modo, el lopinavir tiene actividad, tanto in vitro como en modelo animal, contra el coronavirus del síndrome respiratorio de oriente medio (mers-cov) ( ) . estos estudios previos son el soporte inicial para el uso del lopinavir/ritonavir en la epidemia del covid- ; cao y cols, en mayo de publicaron en china, un estudio controlado, aleatorizado en pacientes hospitalizados con prueba ; los efectos adversos gastrointestinales fueron más comunes con el lopinavir-ritonavir, pero los eventos adversos serios fueron más común con el grupo control; el tratamiento con lopinavir-ritonavir fue suspendido en pacientes ( . %) secundario a los eventos adversos ( ) . otro estudio hung y cols, en hong kong, evaluaron en un trabajo multicéntrico, prospectivo, aleatorizado, fase la eficacia y seguridad de la terapia combinada por días de lopinavir mg y ritonavir mg cada h, ribavirina mg cada h y tres dosis de millones de ui de interferón beta- b en días alternos en pacientes con covid- comparado con lopinavir/ritonavir cada h (grupo control); el resultado primario fue tiempo en la negativización de la pcr viral en el hisopado nasofaríngeo en paciente con covid- ; pacientes fueron ingresados, en el grupo de combinación y en el grupo control; en el grupo de intervención de forma significativa se negativizo la prueba de pcr de forma más rápida ( días [iqr - ]) que el grupo control ( días [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ; hr . [ic % . - . ], p = . ); los eventos adversos fueron similares entre los grupos; ningún paciente murió durante el estudio ( ) . por último, un pequeño estudio de zhu y cols en china, con pacientes con sars-cov- ; evaluaron de forma retrospectiva los efectos antivirales y seguridad del lopinavir/ritonavir y el arbidol (antiviral aprobado en china y rusia para el sasr y la influenza), pacientes en el grupo de lopinavir/ritonavir y en el grupo de arbidol; los pacientes lopinavir/ritonavir presentaron un mayor tiempo para la negativización de la prueba de pcr viral (p < . )( ). se recomienda no utilizar de forma rutinaria remdesivir como antiviral para el manejo de pacientes con infección por sars-cov- /covid- . se debe considerar su uso en escenarios de estudios de investigación clínica aprobados. actualmente no hay disponibilidad del medicamento en el país (colombia) por lo cual no se incluye en los protocolos de manejo de paciente con covid- . en estados unidos el primer paciente con covid- mostró una mejoría significativa de sus síntomas con horas de tratamiento con remdesivir ( ) , lo que abrió la puerta a un nuevo tratamiento para el sars-cov- ; el remdesivir (gs- ) es un análogo de los nucleótidos que inhibe la rna polimerasa; con un amplio espectro antiviral, puede inhibir la replicación de múltiples coronavirus en las células epiteliales del sistema respiratorio ( ) ; estudiado ( ) . por último, un estudio publicado por antinori y cols, en milán, italia; de manera prospectiva (compasional) incluyó pacientes con neumonía por sars-cov- mayores a años bajo ventilación mecánica o con una saturación de oxígeno ≤ % al aire ambiente o un puntaje del national early warning score ≥ ; el pronóstico primario en cambio en el estado clínico en una escala ordinal de categorías ( = no hospitalizado de regreso a sus actividades diarias normales; = muerte); de los paciente ingresados, se encontraban en uci y en un piso de hospitalización de enfermedades infecciosas; un curso de días de remdesivir fue completado por pacientes ( %) y suspendido en , de os cuales ( . %) se descontinuo por eventos adversos; a los días, ( . %) pacientes de piso fueron egresados, permanecían hospitalizados y uno murió ( . %), en la icu ( . %) fueron egresados, ( . %) pacientes murieron, ( . %) aún se encontraban en ventilación mecánica y ( . %) estaba con mejoría pero aún hospitalizado; la hipertransaminasemia y la injuria renal aguda fueron los eventos adversos más frecuentes reportados ( . % y . %, respectivamente); los datos sugieren que el remdesivir puede beneficiar a pacientes con neumonía por sars-cov- hospitalizados por fuera de la unidad de cuidado intensivo ( ). page se recomienda no utilizar de rutina la ivermectina para el manejo de pacientes con infección por sars-cov- /covid- . para la fecha no se cuenta con la suficiente evidencia para emitir una recomendación para el uso de la ivermectina en pacientes con covid- , en estudios iniciales in vitro, caly y cols, demostró como la ivermectina, una droga autorizada por la fda como antiparasitario tiene un efecto antiviral de amplio espectro de manera in vitro, con una reducción significativa de la replicación viral en modelos experimentales ( ) , un estudio aún sin publicar, observacional multicéntrico de casos y controles (n: casos y n: controles), realizado entre el de enero y de marzo de , incluyó pacientes diagnosticados con covid- confirmados por laboratorio, la dosis fue de mcg/kg de ivermectina más la terapia médica de soporte en comparación con terapia médica sin ivermectina, el resultado principal fue la medición de supervivencia; en pacientes que requirieron ventilación mecánica, la mortalidad fue menor en el grupo de ivermectina ( , % y , % respectivamente) y las tasas de mortalidad global fueron más bajas con ivermectina ( no se puede emitir una recomendación a favor o en contra sobre el uso compasivo o rutinario de tocilizumab en pacientes con infección por sars-cov- /covid- . en pacientes individualizados se ha reportado desenlaces clínicos favorables. se puede considerar su uso en pacientes que cumplan con todos los siguientes criterios: ver tabla . el tocilizumab (tcz) un anticuerpo monoclonal humanizado igg k, el cual se puede unir de manera específica a los receptores solubles de membrana para la il- (sil- r and mil- r) y ha sido ampliamente usado en el tratamiento de enfermedades autoinmunes, tales como la artritis reumatoide, la enfermedad de still del adulto o vasculitis de grandes vasos ( ) . un primer estudio de xiaoling xu y cols, describió en china, pacientes tratados con tocilizumab, se documentó una mejoría en los síntomas, en los requerimientos de oxígeno y en los hallazgos imagenológicos de la tomografía de tórax; los niveles promedio de il- antes de la terapia fueron de . pg./ml; todos los pacientes recibieron lopinavir y metilprednisolona antes de la terapia. se trata de una serie con una muestra pequeña de pacientes en donde solo ( %) pacientes estaban en condición crítica ( ) . posteriormente luo y cols, en china, reportan el uso de tocilizumab en pacientes, con mejoría en el aumento de la pcr en todos los pacientes, excepto uno, y una disminución de la il . el nivel sérico de il- tendió a aumentar inicialmente y luego disminuyó en pacientes. los niveles medios de il- antes de la terapia fueron de . pg./ml; % de los pacientes recibieron metilprednisolona; el % fallecieron. es otra serie pequeña, con pacientes ( , %) en condición crítica ( ) . roumier y cols, en francia, estudiaron pacientes que recibieron tcz, observando que se redujo la necesidad de ventilación mecánica en comparación con los controles ( amci ® . ); en este estudio, no hubo diferencias en la reducción de la mortalidad y pacientes ( %) se encontraban en uci ( ) . klopfenstein y cols, también en francia, en un estudio retrospectivo de casos y controles, encontraron que pacientes que recibieron tcz (n= ), a pesar de tener más requerimiento de oxígeno, con resultados biológicos más pobres (mayor linfopenia y un nivel de pcr superior) al inicio del estudio que los pacientes sin tcz (n = ), presentaron el objetivo combinado (ingreso a uci y mortalidad) menor que los pacientes sin la terapia ( % vs %, p = . ); es otra serie, que disminuye la necesidad de ventilación mecánica ( % vs %, p = . ) de manera significativa ( ) . luego rimland ca y cols informan los primeros datos de pacientes con covid- tratados con tcz en los estados unidos, de ellos en ventilación mecánica; la pcr y el fibrinógeno mejoraron rápidamente, pero no hubo mejoría en otros marcadores o resultados clínicos. sólo a seis pacientes les tomaron niveles previos de il y de ellos dos tenían niveles bajos ( ) . en otro estudio mikulska y cols, próximo a salir en jama, en pacientes con sdra moderado a severo, hay mayor disminución de la mortalidad con el tratamiento combinado de tocilizumab y esteroides, en relación con cada una de estas terapias. por último, no todos los estudios han mostrado resultados positivos; kimmig y cols, de chicago (eeuu), en de los pacientes críticos con covid- , que recibieron tcz, se asoció con una mayor incidencia de infecciones bacterianas secundarias, incluida la neumonía asociada al ventilador ( . % vs. . % p = . ) ( ) . posterior a la recomendación hay nueva evidencia publicada y estudios aún sin publicar que puede soportar el uso de los inhibidores de la il- en pacientes con sars-cov- /covid- ; morena y col, en un estudio de tocilizumab como uso "off-label" en el tratamiento de neumonía por sars-cov- en milán, italia, este estudio abierto, prospectivo, describe las características clínicas y el pronóstico de pacientes con covid- confirmado y severo tratados con tcz iv, todos los pacientes, presentan niveles plasmáticos elevados de il- (> pg/ml) y saturación de oxígeno < % al aire ambiente, pacientes ( %) se encontraban con sistema de alto flujo de oxígeno y en ventilación invasiva, a los días luego del tratamiento se observa una caída dramática en la temperatura corporal y la pcr, con un incremento significativo en el conteo de linfocitos (p < . ); a los días del tratamiento, pacientes ( %) mostraron una mejoría en la severidad del cuadro; fueron dados de alta; ( %) mostraron empeoramiento de su cuadro clínico y de estos, murieron ( %). la mortalidad fue significativamente asociada con el uso de ventilación mecánica al inicio ( . % vs % de los pacientes en soporte de oxígeno no invasivo, p = . ), el efecto adverso más frecuente reportado fue la elevación de las enzimas hepáticas ( %), trombocitopenia ( %) e infecciones bacterianas serias e infecciones fúngicas en un ( %); los autores concluyen que el tcz ejerce un rápido beneficio sobre los marcadores inflamatorios y la fiebre, aunque no se consiguió un impacto clínico sobre el pronóstico, el riesgo aumentado de infecciones severas no es despreciable ( ) . capra y col, describieron pacientes en un hospital de italia con neumonía por covid- y falla respiratoria sin soporte ventilatorio y al menos uno de los siguientes: frecuencia respiratoria ≥ respiraciones/min, saturación ≤ % o pao /fio <= mmhg, los pacientes recibieron la terapia estándar para el momento (hidroxicloroquina, lopinavir y ritonavir) y fueron considerados el control; pacientes recibieron tzc con días de la admisión más el manejo estándar, los pacientes en el grupo de tratamiento mostraron de manera significativa una mayor sobrevida comparado con los pacientes control (hr para muerte, . ; % ic], . a . ; p = . ), ajustado para las características clínicas de base; de pacientes en el grupo de tcz y de en el grupo control murieron; % y . % de los pacientes que se dieron de alta en el grupo de tcz y en el control se recuperaron; la función respiratoria mejoró en el . % de los amci ® pacientes con tcz que aún se mantenía hospitalizados, donde el % de los controles empeoro y requirieron ventilación mecánica, dando al tcz un espectro positivo en términos de curso clínico y sobrevida en pacientes con covid ( ). guaraldi y col; evaluaron el papel del tcz en reducir el riesgo de ventilación mecánica invasiva en pacientes con neumonía severa por covid- quienes recibían tratamiento estándar para el momento (hidroxicloroquina, azitromicina, antirretrovirales y heparinas de bajo peso molecular) en un estudio retrospectivo, observacional en bologna, reggio emilia y módena, italia; el tcz fue dado a dosis de mg/kg de peso corporal de forma iv (con un máximo de mg) en dos infusiones separadas h o mg sc administradas en dos dosis simultáneas, una en cada muslo ( mg en total), cuando la formulación iv no se encontraba disponible; el pronóstico primario fue la combinación de ventilación mecánica invasiva o muerte; de pacientes ingresados, ( %) tenían neumonía severa por covid- y fueron incluidos, ( %) de pacientes en el grupo estándar requirieron ventilación mecánica, comparados con ( %) de pacientes tratados con tcz (p = · ; [ %] de pacientes tratados iv y [ %] de pacientes tratados sc); ( %) pacientes en el grupo estándar murieron, comparado con ( %; p < . ) pacientes con tcz ( [ %] del grupo iv y [ %] sc); luego de ajustar para sexo, edad, centro de reclutamiento, duración de los síntomas y puntaje de sofa, el tratamiento con tcz fue asociado con una reducción en el riesgo de ventilación mecánica invasiva o muerte (hr . , % ic . - . ; p = . ); ( %) de pacientes tratados con tcz fueron diagnosticados con nuevas infecciones en comparación con ( %) de pacientes en el grupo estándar (p < . ) ( ) . campochiaro y col, en un solo centro evaluó la eficacia y seguridad del tcz en pacientes con covid- severo, se diseñó un estudio retrospectivo en pacientes con características de hiper-inflamación (definida como una elevación tanto en la pcr, ≥ mg/l, normal < mg/l o ferritina ≥ ng/ml, normal < ng/ml en presencia de un incremento en la dhl > u/l), acompañado de un compromiso respiratorio severo, definido como hallazgos típicos en la radiografía y/o tomografía, la presencia de una saturación de oxígeno ≤ % al aire ambiente o una pao :fio ≤ mmhg ingresados a la uci, comparando pacientes con tcz iv al manejo estándar, pacientes fueron incluidos de los cuales fueron tratados con tcz; los pacientes se encontraban con alto flujo o ventilación mecánica no invasiva, a días de seguimiento, % de los pacientes con tcz experimentaron mejoría clínica comparado con un %del tratamiento estándar (p = . ); la mortalidad fue % en el grupo de tcz y % en el grupo estándar (p = . ); la incidencia de infección y trombosis pulmonar fue similar en ambos grupos ( ); somers y col, evaluaron un estudio observacional en pacientes con neumonía por covid- severo que se encontraban en ventilación mecánica, evaluando como pronóstico la probabilidad de sobrevida posterior a la extubación; pacientes fueron incluidos, recibieron tcz y no; en los modelos ajustados, el tcz, fue asociado con una reducción del % en el riesgo de muerte [hr . % ic . a . ]; aunque el tcz, fue asociado con un incremento en la proporción de pacientes con superinfecciones ( % vs. %; p < . ), no hubo diferencias en la mortalidad a días entre los pacientes tratados con tcz con o sin superinfecciones [ % vs. %; p= . ] ( ). price y col, también publicaron un estudio observacional de pacientes hospitalizados con covid- , los pacientes recibieron tcz si cumplían criterios del síndrome de liberación de citoquinas, se evaluaron pacientes; de los cuales ( %) recibieron tcz, estos pacientes que recibieron ventilación mecánica la sobrevida fue del % ( % ic, - ), luego del tcz pocos eventos adversos fueron reportados y tanto la oxigenación como los biomarcadores de inflamación mejoraron ( ) y por último, en un estudio preliminar con datos aún sin publicar perrone y col, evaluaron la eficacia del tcz en pacientes con neumonía por covid- , en un estudio amci ® multicéntrico fase en italia; se utilizó tcz, a dosis de mg/kg iv, una o dos administraciones con horas de diferencia; y casos fueron disponibles para un análisis de intención a tratar, pacientes murieron; las tasas de letalidad fueron de . % ( . % ic, . - . , p = . ) y . % ( . % ic, . - . , p < . ) a y días; el tcz redujo la tasa de letalidad a días pero no a días comparado con las esperadas sin presentar una toxicidad significativa; la eficacia fue más evidente en los paciente que no requerían ventilación mecánica ( ). recomendaciÓn se recomienda no utilizar de rutina bloqueadores de interleuquina- (anakinra) en pacientes con infección por sars-cov- /covid- . aunque su ventaja está en el perfil de seguridad, su vida media corta ( horas) y porque las infecciones oportunistas son raras ,no hay suficiente evidencia para emitir una recomendación sobre el uso de este medicamento; cavalli y cols, de milán, italia, realizaron un estudio de cohorte retrospectivo en pacientes con sdra moderado a severo con hiperinflamación (pcr ≥ mg/dl, ferritina ≥ ng/ml o ambos), manejados con ventilación mecánica no invasiva fuera de la uci y que recibieron tratamiento con hidroxicloroquina y lopinavir, los pacientes que recibieron anakinra, mg/kg dos veces al día intravenosa (n= , dosis alta) o mg dos veces al día subcutánea (n= pacientes, dosis baja) fueron comparados con una cohorte retrospectiva que no recibió anakinra (tratamiento estándar); la duración del tratamiento se prolongó hasta el beneficio clínico sostenido (reducción del % en la pcr, y una pafi > , durante al menos días consecutivos) o hasta la muerte, bacteriemia, o efectos secundarios (alt > veces valores de referencia); el tratamiento con anakinra a bajas dosis se interrumpió después de días debido a la escasez de efectos sobre la pcr y el estado clínico. a los días, el tratamiento con dosis altas de anakinra se asoció con una reducción en la pcr y mejoría en la función respiratoria en de pacientes ( %); en el grupo estándar, ocho de pacientes ( %) mostraron mejoría respiratoria a los días. en días de seguimiento, la sobrevida fue del % en el grupo de dosis altas de anakinra y del % en el grupo estándar (p = . ). se trata del primer estudio que demuestra seguridad y mejoría en los pacientes covid- , pero en el contexto fuera de la uci ( ) . huet y cols, de parís, francia, realizaron el estudio llamado ana-covid- en el que compararon pacientes tratados con anakinra subcutánea mg dos veces al día durante h, luego mg diarios durante días, con pacientes históricos; su criterio de inclusión fue tener una saturación de oxígeno del % o menos con un soporte de mínimo de l / min de oxígeno. la admisión a la uci por ventilación mecánica invasiva o muerte se produjo en ( %) pacientes en el grupo de anakinra y ( %) pacientes en el grupo histórico (hr . amci ® pacientes en el grupo histórico tuvieron un aumento en las aminotransferasas hepáticas ( ). recomendaciÓn se sugiere que la terapia con interferón sólo sea considerada en pacientes con formas graves de infección por covid- en el marco de un estudio clínico. estudios preliminares muestran que el virus del covid- induce una expresión muy débil de interferones en las células infectadas, lo que obstaculiza la respuesta inmune innata temprana a la infección y sugiere que el uso de interferón (ifn) exógeno para estimular la inmunidad antiviral ( ) . zhou q y col, en china, en un estudio observacional de pacientes con covid- con gravedad mixta proporcionó evidencia de muy baja calidad que la adición de interferón-α a la terapia con umifenovir no afecta el tiempo de eliminación viral o la duración en la estancia hospitalaria cuando se comparó con el umifenovir solo ( ) . hung if y cols, de hong kong, realizaron un ensayo multicéntrico, en adultos con covid- , en los que pacientes recibieron una combinación de lopinavir/ritonavir y tres dosis de millones de unidades internacionales ( · mg) de interferón beta- b en días alternos (grupo de combinación) y pacientes recibieron lopinavir/ritonavir (grupo control); la terapia de combinación fue segura y superior al control, para aliviar los síntomas y acortar la duración de la eliminación del virus y la estancia hospitalaria; se trata de un estudio fase , en pacientes con covid- leve a moderado (ningún paciente con ventilación en el grupo de combinación), en el que el ifn se administró en los primeros días de inicio de los síntomas y con el uso de un análogo de nucleósido oral (ribavirina), que no está en nuestras guías ( ) . se necesitan estudios de ifn solo o combinado en pacientes críticos con covid- . los efectos adversos de los ifn tipo i pueden limitar su uso para una intervención generalizada, como se propone en el brazo ifn-β con lopinavir/ritonavir del ensayo solidaridad de la oms. la administración por inhalación de vapor que se realiza actualmente en china ofrece la ventaja de acceso rápido al tracto respiratorio; sin embargo, la farmacodinamia y la farmacocinética de este modo de administración nunca se han evaluado. se sugiere no utilizar de rutina corticoides en el tratamiento de pacientes con sospecha o diagnóstico de covid- . no se puede considerar su uso profiláctico ni en pacientes con enfermedad leve sin requerimiento de oxígeno. débil en contra page en diciembre de , una serie de casos de neumonía de causa desconocida surgió en wuhan, hubei, china, con presentaciones clínicas muy parecidas a una neumonía viral; los análisis de secuenciación profunda de muestras del tracto respiratorio inferior indicaron un nuevo coronavirus, que se denominó novel coronavirus (covid- - ) ( ) . en la actualidad, en ausencia de terapia preventiva para sars-cov- , la piedra angular de atención para pacientes con covid- sigue siendo el manejo de apoyo, que va desde el tratamiento ambulatorio sintomático hasta el tratamiento intensivo completo con medidas de soporte en cuidados intensivos ( ) . dentro del manejo farmacológico se ha planteado la opción del uso de corticoides, la justificación estaría basada en la disminución de la respuesta inflamatoria del huésped a nivel pulmonar; es decir, un efecto inmunomodulador, ya que esta infección puede conducir a un síndrome de distrés respiratorio agudo (sdra); sin embargo, el beneficio puede verse superado por los efectos adversos, incluido el retraso en el aclaramiento viral y mayor riesgo de infección secundaria. a pesar de que la evidencia directa de corticoides en covid- es limitada, revisiones de los resultados en otras neumonías virales nos podrían orientar en principio en esta actual situación ( ) . teniendo en cuenta lo anterior; stockman y cols, en el . , realizaron una revisión sistemática sobre ensayos en pacientes con sars; quince ensayos examinan el uso de corticoides con diez o más pacientes en tratamiento; ensayos también recibían ribavirina; trece de estos estudios no fueron concluyentes; dos estudios describen un daño potencial con el uso de esteroides; en la literatura china estos autores encontraron catorce estudios con uso de esteroides en sars; doce fueron suspendidos por posible daño, la mayoría de estos ensayos se realizaron con muestras pequeñas de pacientes y de manera retrospectiva ( ) . arabi y cols, en noviembre de . , realizaron un estudio multicéntrico de cohorte retrospectivo en hospitales de atención terciaria de arabia saudita, donde se incluyeron pacientes; el uso de corticoides en pacientes con mers no se asoció con un cambio significativo a los días en la mortalidad y se documentó un retraso en la eliminación del arn de mers-cov ( ) . en cuanto hace referencia a la situación actual de pandemia por sars-cov- y compromiso pulmonar; wu y cols, en marzo de . realizaron un estudio retrospectivo de pacientes con covid- en china; para aquellos pacientes que desarrollaron sdra, el tratamiento con metilprednisolona estuvo asociado con una disminución del riesgo de muerte ( / [ %] con esteroides vs / [ %] sin esteroides; hr, . [ic %, . - . ]), con las limitaciones de los estudios retrospectivo, de un solo centro, con un limitado número de pacientes ( ). zha y cols, en marzo de . , describen el uso de corticosteroides en el tratamiento de pacientes con covid- ; no hallaron asociación entre la terapia con esteroides y el pronóstico de los pacientes sin sdra, siendo un estudio con una serie muy pequeña de pacientes ( ) . yang y cols, en marzo de . , en una revisión sistemática y meta-análisis que incluyó . pacientes de estudios, describen como el tratamiento con corticoides estuvo asociado con una mayor mortalidad (rr = . , ic % = . - . , p = , ), mayor estancia (wmd = . , ic % . - . , p = < , ) y una mayor tasa de infección bacteriana (rr = . , ic % . - . , p = < . ); con algunas limitaciones en este metaanálisis, la mayoría de los estudios incluidos son estudios de cohorte retrospectivos, controles históricos, con un bajo nivel de evidencia y una falta de ensayos controlados aleatorizados con buen diseño, sin un estándar uniforme para el tiempo y la dosis de los corticoides utilizado en los estudios; los efectos de los corticosteroides pueden ser influenciado también por otras opciones terapéuticas, como los medicamentos antivirales ( ) . por último, li y cols, en mayo de . , en otra revisión sistemática y metaanálisis, con respecto al uso de corticosteroides en sujetos con amci ® infecciones por sars-cov- , sars cov y mers-cov, se determinó que hubo retraso en la eliminación del virus sin mejoría en la supervivencia, reducción en la duración de la hospitalización o tasa de admisión en la uci y/o uso de ventilación mecánica; presentándose varios efectos adversos. debido a la preponderancia de los estudios observacionales en el conjunto de datos y los sesgos de selección y publicación, se concluye especialmente con respecto al sars-cov- , que se necesita mayor investigación con ensayos clínicos aleatorizados. internamente en este meta-análisis sugiere precaución al usar esteroides en pacientes con covid- ( ). horby y col en una rama del ensayo de evaluación aleatorizada de la terapia covid- (recovery), estudio aleatorizado, controlado, abierto que compara una gama de posibles tratamientos con la atención habitual en pacientes hospitalizados con covid- , compararon el uso de la dexametasona a dosis de mg día (oral o intravenosa) una vez al día por días o el alta según lo que ocurriera primero contra el manejo habitual; en pacientes aleatorizados que recibieron dexametasona se compararon con pacientes en manejo estándar; ( . %) pacientes en el grupo de dexametasona y ( . %) pacientes en el grupo control murieron a los días, con un riesgo relativo ajustado para la edad (rr . ; % ic . a . ; p < . ). la mortalidad relativa y absoluta variaron significativamente en relación al soporte ventilatorio al momento de la aleatorización; la dexametasona redujo las muertes en una tercera parte de los pacientes que recibieron ventilación mecánica invasiva ( . % vs. . %, rr . , % ic . a . ; p < . ), y una quinta parte en los pacientes que reciben oxígeno sin ventilación mecánica invasiva ( . % vs. . %, rr . , % ic . a . ; p = . ), pero sin reducir la mortalidad en paciente que no recibieron soporte respiratorio al momento de la aleatorización ( . % vs. . %, rr . , % ic . a . ]; p = . ) ( ). se recomienda no utilizar plasma convaleciente como tratamiento de rutina en paciente con sars-cov -covid- . se debe considerar su uso en el marco de un ensayo clínico y con alguno de los dos escenarios siguientes: escenario a (enfermedad severa), definida como uno o más de los siguientes: disnea, frecuencia respiratoria > /min, spo < %, pao /fio < o empeoramiento radiológico con aumento > % de los infiltrados pulmonares en - horas. escenario b (enfermedad que amenaza la vida), definida como uno o más de los siguientes: falla respiratoria, choque séptico, o disfunción multiorgánica. se recomienda no utilizar plasma convaleciente para profilaxis clínica de rutina contra la infección por sars-cov- , solo se debe considerar en el marco de un ensayo clínico. ( ) . una revisión sistemática y meta-análisis exploratorio realizado en identificó estudios de infección por coronavirus sars e influenza severa, el estudio reveló una reducción de la mortalidad, especialmente si el plasma convaleciente se emplea en la fase temprana de la enfermedad cuando se comparó con placebo o no tratamiento (or . ; ic del % . - . ); sin embargo, hay que tener presente que los estudios son de baja calidad, carecen de grupos control y puede tener riesgo moderado a alto de sesgo ( ) . se ha sugerido que el plasma convaleciente de pacientes que se han recuperado de covid- puede ser una terapia potencial, proporcionando inmunidad pasiva de los anticuerpos específicos contra sars-cov- y podría servir para prevenir y tratar la enfermedad ( ) . las personas que se han recuperado de la infección por sars-cov- pueden generar anticuerpos neutralizantes ( , ) que podrían tener aplicación en la prevención de infección en ciertos escenarios, como las personas con comorbilidades subyacentes que predisponen a enfermedad grave y aquellas con exposición de alto riesgo como los trabajadores de la salud y los expuestos a casos confirmados de covid- . existen algunos riesgos asociados con el uso de plasma convaleciente, unos conocidos y otros teóricos; los riesgos conocidos son aquellos asociados con la transfusión de hemocomponentes, incluida la transmisión de virus (ej. vih, vhb, vhc, entre otros) ( ); riesgo muy bajo, con los estándares de calidad actuales de los bancos de sangre; también se pueden presentar complicaciones no infecciosas, como las reacciones alérgicas, anafilaxia, reacción febril a la transfusión, lesión pulmonar aguda relacionada con la transfusión (trali), sobrecarga cardiaca asociada a transfusión (taco) y hemólisis si se administra plasma abo incompatible ( ) . los riesgos teóricos incluyen el empeoramiento de la infección dependiente de anticuerpos (antibody-dependent enhancement of infection -ade); el ade puede ocurrir en varias enfermedades virales e implica una respuesta inflamatoria exagerada ante la presencia de ciertos anticuerpos ( ) . otro riesgo teórico es que la administración de anticuerpos a las personas expuestas al sars-cov- puede evitar la enfermedad, pero modifica la respuesta inmune de tal manera que esos individuos monten respuestas inmunes atenuadas, lo que los haría vulnerables a la reinfección posterior, si se comprueba que este riesgo es real estos individuos podrían ser vacunados contra covid- cuando exista una vacuna disponible ( ) . durante el brote actual en china, se utilizó plasma convaleciente en algunos pacientes con covid- ( ), desde esta publicación se identificaron publicaciones relacionadas con el tema, entre todos los estudios fueron tratados con plasma convaleciente un total de pacientes( - ) (tabla ). shen y cols en marzo describieron en china el primer reporte en el cual el plasma convaleciente puede ser una opción de tratamiento en pacientes con covid- ; reportaron una serie de casos de pacientes críticamente enfermos con covid- y síndrome de dificultad respiratoria aguda (sdra), todos en ventilación mecánica, a quienes se les administró plasma convaleciente con anticuerpos neutralizantes [ a días después del inicio de la enfermedad (ddie)], todos ellos posteriormente mostraron mejoría clínica, la carga viral de los pacientes disminuyó y fueron negativas en los días posteriores a la intervención ( ) . de forma similar, duan y cols en mazo en china reportaron mejoría clínica en una serie prospectiva de casos de paciente severamente enfermos con covid- , que recibieron plasma convaleciente con un tiempo medio de . ddie ( a días) después del inicio de los síntomas, e hicieron amci ® una comparación con un grupo control histórico comparables en edad, género y severidad de la enfermedad ( ) . zhang y cols en mazo , en china reportaron una serie de casos de pacientes con covid- críticamente enfermos, en falla respiratoria con ventilación mecánica y dos de ellos con ecmo (membrana de oxigenación extracorpórea), a quienes se les dio tratamiento con plasma convaleciente en un tiempo medio . ddie, posteriormente todos tuvieron mejoría clínica ( ) . posteriormente ahn y cols en abril en corea, describieron una serie de dos casos de paciente con covid- severamente enfermos, en falla respiratoria y con ventilación mecánica, quienes además de recibir hidroxicloroquina, lopinavir/ritonavir y metilprednisolona, fueron tratados con plasma convaleciente entre - ddie, ambos pacientes se recuperaron y fueron liberados de la ventilación mecánica, uno fue dado de alta al momento del reporte ( ) . ye y cols en abril en china, describieron una serie de pacientes con covid- con anormalidades imagenológicas y deterioro clínico a pesar del tratamiento estándar y con pcr para sars -cov- persistentemente positiva, aunque no estuvieron en falla respiratoria o con ventilación mecánica, de hecho, una de las pacientes era portadora asintomática; todos recibieron plasma convaleciente entre a ddie, en todos los pacientes, excepto , hubo resolución de los cambios de vidrio esmerilado y consolidación, todos mejoraron y fueron dados de alta ( ) . zeng y cols en abril en china, reportaron una serie de casos con covid- en falla respiratoria y se compararon con controles que no recibieron plasma convaleciente por limitación en la disponibilidad y compatibilidad abo; a este grupo de paciente, se les administró plasma convaleciente en promedio . ddie, en todos los casos se negativizó la pcr para sars-cov- a los días después del tratamiento; sin embargo, contrario a los reportes de los estudios previos, en este grupo se murieron pacientes ( % vs %, p = . ), pero tuvieron mayor porcentaje de aclaramiento del virus (pcr sars-cov- negativa % vs . %, p = . ) antes de la muerte e incluso el tiempo de sobrevida fue mayor en el grupo de tratamiento (p = . ) ( ) . salazar y cols en mayo , en houston, texas, reportaron una serie de pacientes con covid- severa o amenazante para la vida, el desenlace primario fue seguridad y el secundario fue el estado clínico de los pacientes al día luego de la transfusión; al día posttransfusión, pacientes ( %) mejoraron con relación a su estado clínico basal, ( %) no tuvieron cambios y pacientes tuvieron deterioro clínico. siete de los nueve pacientes que mejoraron ( %) habían sido dados de alta; para el día post-transfusión, ( %) de los pacientes presentaron mejoría y pacientes más, habían sido dados de alta; tres pacientes permanecían sin cambios, pacientes se deterioraron y uno murió por una condición no relacionada con el plasma; el promedio de estancia hospitalaria fue de . días y la estancia hospitalaria luego de la transfusión fue en promedio días; hubo una disminución de los valores promedio de pcr desde . mg/dl el día , a , mg/dl y . mg/dl los días y respectivamente; al momento de la publicación del artículo, solo permanecían intubados pacientes; todos los pacientes que requirieron ecmo ya se habían liberado y ( %) fueron dados de alta ( ) . también en texas, estados unidos, ramachandruni y col en mayo , reportaron una serie de casos con covid- severa, falla respiratoria y en ventilación mecánica o pao /fio < , todos con comorbilidades; a los cuales les administraron metilprednisolona y posteriormente plasma convaleciente; compararon los valores basales de pao /fio y posterior a la intervención; encontrando, mejoría de la pao /fio en % luego del tratamiento con esteroides y en % luego de la administración de plasma convaleciente ( ) . finalmente, en respuesta al brote de covid- en los eeuu y las tasas de mortalidad reportadas, la fda en colaboración con la mayo clinic y la comunidad nacional de bancos de sangre desarrollaron un programa nacional de acceso ampliado para recolectar y distribuir plasma convaleciente donado por amci ® individuos que se han recuperado de covid- ; entre abril y mayo , ; fueron incluidos . pacientes con covid- severa o potencialmente mortal o con riesgo alto de progresión a covid- severa o potencialmente mortal en el programa nacional de acceso ampliado; en ese tiempo, un total de . pacientes inscritos recibieron transfusión de plasma convaleciente covid- . en una publicación reciente, joyner y cols en mayo , en estados unidos, hicieron un análisis de seguridad después de la transfusión de plasma convaleciente covid- humano con compatibilidad abo en . adultos hospitalizados con covid- grave o potencialmente mortal, % de los cuales se encontraban en uci; la incidencia de eventos adversos serios (eas) durante las horas siguientes a la transfusión de plasma convaleciente fue < %, incluyendo mortalidad ( . %); de los eas reportados, hubo incidentes reportados como eas relacionados, incluyendo muertes, eventos de taco, trali y reacciones alérgicas graves asociadas a la transfusión; sin embargo, solo (de ) eas fueron considerados definitivamente relacionados con la transfusión de plasma convaleciente por el médico tratante; en este grupo de pacientes, la tasa de mortalidad a los siete días luego de la administración del plasma convaleciente, fue del , % ( ) . a pesar que la tasa general de letalidad para la covid- parece ser aproximadamente . % ( ), la tasa de mortalidad reportada parece no ser excesiva si la comparamos con los informes de wuhan que sugieren tasas de letalidad del % para los pacientes hospitalizados ( ) y % entre los pacientes en unidad de cuidados intensivos ( ) . los nueve estudios mostraron mejoría en muchos aspectos, incluyendo el aclaramiento del virus, la disminución del suplemento de oxígeno y la ventilación mecánica, la normalización de los valores de laboratorio, y la recuperación en los hallazgos pulmonares radiológicos. todos los estudios, reportaron que no se presentaron eventos de seguridad o reacciones adversas serias relacionadas con la administración de plasma convaleciente en pacientes con covid- , excepto en casos relacionados, según criterio de los médicos tratantes, en el estudio publicado por joyner y cols ( ) . esta serie de estudios son alentadores; sin embargo, la mayoría de los reportes de casos tienen limitaciones significativas: carecen de los ajustes para factores de confusión críticos, incluidos los co-tratamientos, las características basales, la gravedad de la enfermedad y el momento de administración del plasma y deben ser seguidas de investigaciones adicionales. para establecer mejor el papel del plasma convaleciente es necesario realizar estudios dirigidos a los siguientes escenarios: . el uso como profilaxis post-exposición . evaluar si el plasma convaleciente es útil en paciente con enfermedad leve . el efecto del plasma convaleciente en pacientes con enfermedad moderada . el tratamiento de rescate con plasma convaleciente en pacientes que requieren ventilación mecánica debido a covid- . finalmente, trabajos que evalúen la seguridad y farmacocinética del plasma convaleciente en los pacientes pediátricos con alto riesgo. actualmente están en curso varios estudios para evaluar el tratamiento de pacientes infectados con sars-cov- con plasma convaleciente. una búsqueda realizada el de mayo de en clinicaltrials.gov con los términos "plasma convaleciente y covid- " mostró ensayos en curso sobre el uso de plasma convaleciente en pacientes con amci ® covid- , que nos ayudarán a resolver las inquietudes relacionadas con esta intervención ( ) . page se recomienda no utilizar de rutina las las inmunoglobulinas hiperinmunes en pacientes con infección por sars-cov- /covid- . la inmunoglobulina hiperinmune (h-igiv) se deriva de individuos con altos títulos de anticuerpos contra patógenos específicos y se ha utilizado con éxito en el tratamiento de infecciones, como el citomegalovirus y la gripe h n ( ) . se propone que la inmunoglobulina hiperinmune combinada con medicamentos antivirales puede ser efectiva en el tratamiento de pacientes con covid- , estos anticuerpos (ac) recogidos de los pacientes recuperados serán específicos contra covid- al aumentar la respuesta inmune en pacientes recién infectados ( ) . existe evidencia más sólida para el uso de h-igiv en el tratamiento de enfermedades virales. cheng y cols en enero de , realizaron una revisión retrospectiva en hong kong, que reveló que el plasma convaleciente de los sobrevivientes de sars-cov administrados a pacientes con sars-cov que tenían enfermedad progresiva resultó en tasas de alta significativamente más altas en el día y tasas de mortalidad más bajas, en comparación con los controles históricos ( ) . hung y cols en febrero de , realizaron un estudio de cohorte prospectivo sobre la efectividad del plasma convaleciente de los sobrevivientes de h n con un título de ≥ : ofrecido a pacientes de la uci con infección grave por h n ; los pacientes que rechazaron las infusiones de plasma convalecientes fueron controles; veinte de los pacientes recibieron sueros convalecientes, el tratamiento con plasma convaleciente condujo a una reducción significativa de la carga viral respiratoria, los niveles séricos de citocinas (il- , il- , tnfα) y la mortalidad ( ) . posteriormente hung y cols en agosto de , publicaron un estudio multicéntrico prospectivo, doble ciego, aleatorizado y controlado en el que compararon la efectividad de la inmunoglobulina hiperinmune (h-igiv) del plasma convaleciente de los sobrevivientes de h n versus la inmunoglobulina iv (igiv) normal, en pacientes con h n en uci con soporte respiratorio y recibiendo oseltamivir; este estudio mostró, una reducción de la carga viral y una mayor supervivencia en el grupo que recibió h-igiv dentro de los días posteriores al inicio de los síntomas, demostrando la superioridad de la inmunoglobulina hiperinmune sobre la igiv en el tratamiento de la infección grave por h n ( ). el uso de inmunoglobulina hiperinmune ha demostrado una clara efectividad en el tratamiento de la gripe y el sars-cov; sin embargo, el plasma se debe recolectar y procesar de pacientes convalecientes y verificar que tenga títulos adecuados. según la experiencia con el sars-cov, lo ideal es recolectar plasma de pacientes con un curso de enfermedad más leve ( ) . poco se sabe sobre la seguridad de la inmunoglobulina hiperinmune cuando se usa para el tratamiento de infecciones por coronavirus, los riesgos incluyen la exacerbación de la infección dependiente de anticuerpos (antibody-dependent enhancement of infection -ade)( ); el ade puede ocurrir en varias enfermedades virales, e implica una respuesta inflamatoria exagerada ante la presencia de ciertos anticuerpos; sin embargo, los estudios en sars y mers no proveen información suficiente para extrapolarse a la infección por sars-cov- . no se encontraron estudios con inmunoglobulina hiperinmune en el tratamiento de pacientes con covid- . no se puede emitir una recomendación a favor o en contra para el uso de la inmunoglobulina intravenosa como tratamiento adyuvante en pacientes con covid- severo. se debe considerar la inmunoglobulina intravenosa como tratamiento adyuvante en pacientes con covid- severo, en el contexto de estudios clínicos en los siguientes escenarios. escenario a (enfermedad severa), definida como uno o más de los siguientes: disnea, frecuencia respiratoria > /min, spo < %, pao /fio < o empeoramiento radiológico con aumento > % de los infiltrados pulmonares en - horas. escenario b (enfermedad que amenaza la vida), definida como uno o más de los siguientes: falla respiratoria, choque séptico, o disfunción multiorgánica. en las enfermedades virales, los anticuerpos ejercen su efecto por neutralización viral (bloqueo de la entrada de células virales y, por lo tanto, replicación), activación del complemento, opsonización y mediación de citotoxicidad celular dependiente de anticuerpos. la neutralización viral es específica de antígeno; otras actividades antivirales son antígeno-inespecíficas y se realizan en parte a través de interacciones fc: fc receptor. en la infección por sars-cov- , el principal antígeno objetivo asociado con la neutralización es la proteína spike, que es responsable de la unión del sars-cov- a las células epiteliales, incluidos los neumocitos; los anticuerpos en las inmunoterapias pasivas covid- son de naturaleza policlonal, con múltiples epítopos contra los paratopes de sars-cov- , incluido el dominio de unión al receptor en la proteína spike ( ) . la inmunoglobulina intravenosa (igiv) es un producto derivado del plasma de miles de donantes utilizados para el tratamiento de inmunodeficiencias primarias y secundarias, afecciones autoinmunes/inflamatorias, trastornos neuroinmunológicos y secuelas relacionadas con infecciones; la igiv proporciona protección inmune pasiva contra una amplia gama de patógenos; actualmente, la experiencia con el uso de igiv en el tratamiento de la infección por sars-cov- es muy limitada; sin embargo, la justificación del uso de ivig en la infección por sars-cov- es la modulación de la inflamación ( ) . la igiv se ha usado en el tratamiento de otros coronavirus, incluido el sars cov. stockam y cols, en septiembre de , en respuesta a una petición de "the world health organization -who", realizaron una revisión sistemática de los efectos del tratamiento en los pacientes con síndrome respiratorio agudo, incluida la igiv o el plasma convaleciente; se evaluaron cinco estudios sobre el uso de igiv o plasma convaleciente administrado además de corticosteroides y ribavirina, se consideró que estos estudios no fueron concluyentes ya que los efectos de la igiv o el plasma convaleciente no podían distinguirse de otros factores que incluían comorbilidades, estadio de la enfermedad o el efecto de otros tratamientos ( ) . wnag y cols, en mayo , hicieron un estudio prospectivo, en un solo centro, de infección por sars en taiwán, se administró igiv si el paciente tenía leucopenia amci ® o trombocitopenia, o si había progresión rápida de la enfermedad en la radiografía; un total de pacientes recibieron igiv, de los cuales tenían citopenias graves, uno de ellos tenía evidencia de síndrome hemofagocítico y paciente tuvieron progresión radiológica de la enfermedad; el estudio sugiere que la igiv condujo a una mejora significativa en el recuento de leucocitos y plaquetas, pero reconoce que no había un grupo de control para evaluar objetivamente las respuestas ( ) . lew y cols, en julio , reportaron un estudio retrospectivo de un solo centro en singapur, se encontró que los pacientes adultos con sars tratados con un régimen de pulso de metilprednisolona ( mg) e igiv ( . mg/kg) diariamente durante tres días consecutivos tuvieron una hazard ratio ajustada de . para mortalidad en comparación con el grupo no tratado, con una tendencia hacia una recuperación más temprana; sin embargo, este hallazgo no fue estadísticamente significativo (ic del %: . a . ; p = . ); además, este resultado tuvo como factor de confusión el uso concurrente de esteroides ( ) . aunque algunas de las preparaciones de igiv comercializadas actualmente (gamunex-c y flebogamma) contienen anticuerpos que reaccionan de forma cruzada contra el sars-cov- y otros antígenos de virus, in vitro( ), hasta la fecha, ningún ensayo clínico de alta calidad ha demostrado eficacia y seguridad convincentes de igiv en epidemias de coronavirus. a pesar de que los datos para el uso de igiv en la infección por sars y mers son débiles, la dosis alta de ivig puede ser útil en la infección grave por sars-cov- a través de la modulación inmune, saturando fcγr y reduciendo ade ( ) . en general, la inmunoglobulina intravenosa es bien tolerada y el perfil de seguridad es bien conocido. las reacciones adversas comunes son leves y autolimitadas, pero se sabe que en pacientes de alto riesgo se producen efectos adversos graves, como trombosis, disfunción renal y muerte. en cuanto la evidencia (tabla ); xie y cols en abril, en wuhan, china, realizaron un estudio retrospectivo, revisando casos de covid- severa (disnea, fr > /min, spo < % en reposo, pao /fio < , progresión imagenológica > % en - horas) o críticamente enfermos (falla respiratoria con ventilación mecánica, choque, disfunción orgánica múltiple) en el cual evaluaron la mortalidad a días como desenlace primario y como desenlace secundario evaluaron la mortalidad a días, días de estancia hospitalaria, de uci y la necesidad de ventilación mecánica; reportaron que el tratamiento con igiv dentro de las horas posteriores al ingreso no sólo redujo el uso de la ventilación mecánica comparado con el tratamiento luego de horas del ingreso ( . % vs . %, p = . ), sino que también redujo la duración de la estancia en el hospital ( . ± . vs . ± . días, p = . ) y la uci ( . ± . vs . ± . días, p = . ); mejorando en última instancia la mortalidad a los días (p = . ); concluyen, que el estudio demostró que el tratamiento con igiv en pacientes con covid- con neumonía grave puede mejorar los indicadores en poco tiempo y mejorar la eficiencia del tratamiento de los pacientes con alta efectividad ( ) . el tratamiento con dosis altas de igiv ( g/día durante días) al inicio del distrés respiratorio, sumado al tratamiento de soporte y en un caso combinados con antivirales (lopinavir/ritonavir) y metilprednisolona en covid- grave publicado por cao y cols en marzo, en wuhan, china ( ), demostró la elevación de los recuentos de linfocitos, disminución de los marcadores inflamatorios, recuperación de la oxigenación, resolución parcial/completa de las alteraciones radiológicas pulmonares y las pruebas de hisopos nasales y orofaríngeos negativos dentro de unos pocos días después del inicio tratamiento (< días). lanza y cols en mayo , reportaron en nápoles, italia, el caso de una mujer de años que tenía covid- severa y que venía con deterioro clínico a la cual se le venía dando tratamiento con hidroxicloroquina más azitromicina, no se le administraron amci ® esteroides por el riesgo de disminuir la depuración de la viremia, a quién se le administró igiv el día después de iniciados los síntomas con mejoría clínica rápida, normalización de los gases arteriales y disminución marcada de los infiltrados pulmonares al día y respectivamente; como evento adverso, reportaron hipotensión durante el inicio de la infusión que se mejoró al disminuir la velocidad de infusión. la paciente finalmente se recuperó, negativizó rt-pcr sars-cov- y fue dada de alta ( ) . se recomienda la tromboprofilaxis farmacológica en todos los pacientes confirmados o sospechosos de covid- severo, a menos que está contraindicada, en cuyo caso es razonable la implementación de profilaxis no farmacológica. en términos generales se reconoce que los pacientes hospitalizados con enfermedad médica aguda, incluidas infecciones como la neumonía, tienen un mayor riesgo de eventos tromboembólicos ( ) . tang y cols, en china describieron múltiples trastornos de la coagulación en pacientes con covid- , aquellos pacientes que no sobrevivieron tenían significativamente mayores niveles de dímero-d y productos de degradación de la fibrina y con tiempos de coagulación convencionales más alargados al ingreso (p < . ); . % de los no sobrevivientes y . % de los sobrevivientes cumplían criterios para coagulación intravascular diseminada( ); este mismo grupo realizó un estudio con pacientes con covid- severo, pacientes con coagulopatía asociada a covid- (cac), definida por un sic score ≥ la utilización de tromboprofilaxis redujo de manera significativa la mortalidad a días ( . % vs . %, p = . ) así como también en aquellos con un se recomienda no utilizar de rutina antiagregación en pacientes con covid- severo con el fin de prevenir desenlaces neurológicos adversos. no se encuentran estudios en la literatura para el uso de antiagregantes para el manejo específico del covid- ; el manejo de los eventos cardiovasculares en pacientes covid- no difiere de la población general sin la enfermedad. no se establecen diferencias en los estudios descritos, ni en las publicaciones hasta la fecha de la revisión ( ) . no se puede emitir una recomendación a favor o en contra sobre el uso de la anticoagulación terapéutica de rutina con heparinas de bajo peso molecular o heparina no fraccionada en pacientes con bajo riesgo de sangrado y con un curso clínico grave o crítico, que además tengan elevación del dímero d mayor a mcg/ml ( ng/ml) y/o fibrinógeno mayor a mg/dl. diferentes publicaciones describen como una estrategia de tratamiento basada en profilaxis con heparina de bajo peso molecular (hbpm) para tratar la coagulopatía grave por covid- podría no ser suficiente. especialmente porque estos pacientes tienen dentro de su coagulopatía, una predisposición mayor a la presencia de trombosis que al sangrado. además, los bajos niveles de antitrombina que se han descrito en estos pacientes, los hace más resistentes a la heparina, lo que sugiere que las dosis profilácticas ya sea de heparina no fraccionada o hbpm pueden ser inadecuadas( - ). asociaciÓn colombiana de medicina crÍtica y cuidados intensivos. amci ® no se puede emitir una recomendación a favor o en contra sobre la anticoagulación terapéutica de rutina con heparinas de bajo peso molecular en pacientes con covid- en estado crítico, que presenten elevación del dímero d mayor a mcg/ml ( ng/ml). tang y cols, en china con un estudio retrospectivo donde se incluyeron pacientes con covid- severo, se evaluó la presencia de trombosis como desenlace asociado. este estudio describe como niveles de dímero d por encima de ng/ml estaban asociados a una mayor probabilidad de muerte y en estos pacientes el tratamiento anticoagulante reduce de manera significativa la mortalidad ( . , ic % . - . ) ( , ) . no se puede emitir una recomendación a favor o en contra para la medición rutinaria de niveles de anti xa en pacientes con covid- en los que se decide hacer anticoagulación terapéutica con hbpm. se puede considerar la medición de niveles anti xa si se cuenta con la disponibilidad del recurso. harr y cols, en un estudio en donde se incluyeron pacientes con hiperfibrinogenemia relacionada a trauma, se evidenció como los niveles de fibrinógeno se correlacionaron significativamente con la consistencia del coágulo y adicionalmente como se genera una relación inversa entre los niveles de fibrinógeno y la actividad de las hbpm, lo que sugeriría una potencial resistencia a la heparina. basados en que los pacientes covid- presentan niveles de fibrinógeno en muchas ocasiones con niveles > mg/dl e incluso > mg/dl lo que hace razonable considerar que aquellos pacientes covid- que requieren dosis terapéuticas de hbpm y es posible evaluar los niveles de anti xa, hacer ajustes y monitoreo del nivel de anticoagulación sería una opción razonable ( ) . se recomienda el uso de hbpm o hnf para la anticoagulación terapéutica en pacientes con una indicación específica con diagnóstico de covid- . amci ® como previamente se describió, una revisión sistemática comparó las dosis fijas de hbpm subcutánea con dosis ajustadas de hnf intravenosa o subcutánea en personas con clínica sugestiva de tep, esta revisión demostró que la incidencia de tep recurrente fue menor con hbpm que en los participantes con hnf (or . , ic % . a , ), también se asoció con una reducción en el tamaño del trombo (or . , ic % . a . ), evidencia de baja calidad. sin embargo, no hubo diferencias en la mortalidad general entre los participantes tratados con hbpm y los tratados con ufh (or . , ic % . a . ). por otra parte, los protocolos de manejo en escenarios de coagulación intravascular diseminada (cid), proponen el uso de hnf por encima de la hbpm en pacientes en los que se indica la anticoagulación. más aún, la presencia de falla renal aguda es común en los pacientes con covid- , por lo que la opción de tratamiento con hnf tiene también escenarios en donde podrían ser de elección ( , ) . capítulo . procedimientos y covid- recomendaciÓn se recomienda realizar la preoxigenación en pacientes con sospecha o diagnóstico de covid- , cuando estén disponibles, en áreas de presión negativa con mínimo recambios de aire por hora o en instalaciones con ventilación natural o que tengan un recambio de aire de mínimo l/seg si están disponibles. se recomienda como complemento opcional durante la preoxigenación en el paciente crítico con diagnóstico o sospecha de covid- la caja de acrílico para protección durante la intubación, la caja no protege contra la generación de aerosoles fuera de esta y requiere para su uso, entrenamiento previo. si es difícil su uso retírela inmediatamente. se recomienda utilizar en la mascarilla quirúrgica sobre la mascarilla de oxigenación en el paciente crítico con sospecha o diagnóstico de covid- . se recomienda utilizar filtros hpfa entre la máscara y el dispositivo disponible para la preoxigenación en el paciente crítico con sospecha o diagnóstico de covid- . se recomienda en el paciente con sospecha o diagnóstico de covid- preoxigenación por a min, si el paciente luego de minutos no tiene incremento en la mejoría inicie la administración de medicamentos. se recomienda en caso de compromiso hemodinámico considerar ventilación a dos manos con cierre hermético de la máscara sobre la cara del paciente con frecuencias altas (> por minuto y baja presión). amci ® fundamento el manejo de la vía aérea es un procedimiento considerado generador de aerosoles, la enfermedad covid- tiene una alta tasa de transmisión y el personal de salud requiere el uso estricto del epp ( , ) , revise el enunciado para epp recomendado en esta guía. la posibilidad de permanencia del virus en algunos ambientes puede durar hasta horas, por esto una estrategia para proteger al equipo de salud y otros pacientes podrías ser estar en áreas con presión negativa, lo cual no es fácil de encontrar en nuestro contexto o que tenga un adecuado recambio de aire( ). como medidas complementarias se pueden utilizar opciones como las cajas acrílicas para intubación, esta disminuye el riesgo de contaminación por gotas, pero no elimina los aerosoles, es necesario previo entrenamiento. en caso de que la caja sea una limitante retírela inmediatamente ( ) . los pacientes covid- , clásicamente se presentan con tos, esto es un factor de riesgo para quienes manipulan la vía aérea, la utilización de mascarilla por parte del paciente debajo del dispositivo bolsa mascarilla , la cual también deberá tener un filtro de alta eficiencia para disminuir el número de partículas que pueden estar en el ambiente durante la preoxigenación y posible ventilación, ésta última la cual será evitada al máximo ( , , ) . la preoxigenación es una técnica que pretende barrer el nitrógeno y aumentar la disponibilidad de oxígeno para que cuando el paciente presente apnea por los medicamentos para la intubación o por su condición clínica, se disponga de un tiempo mayor sin desaturación crítica y riesgo de colapso cardio-respiratorio. considere que los pacientes con formas moderadas y severas de covid- , pueden tener más comprometida esta reserva respiratoria y puede no ser efectiva la preoxigenación y cuando inicia la apnea, la desaturación será más precoz. si después de minutos de preoxigenación no hay mejoría de la saturación arterial, considere fallida esta maniobra y considere mayor riesgo de hipoxemia severa con probabilidad de bradicardia extrema y paro cardiorrespiratorio. en caso de requerir ventilación por disminución rápida de la saturación de oxígeno arterial y considere necesario ventilación manual bolsa mascarilla a dos manos no debe ser vigorosa y debe utilizar filtros hpfa, estos reducirán los aerosoles en el ambiente( , ). se recomienda la utilización de cajas de acrílico para intubación del paciente con sospecha o diagnóstico de covid- como complemento durante la intubación para disminuir el riesgo de contaminación por gotas, sin embargo, no protege % la generación de aerosoles y si ésta hace más difícil la intubación retírela inmediatamente. amci ® se recomienda minimizar los intentos de intubación orotraqueal en el paciente con sospecha o diagnóstico de covid- , el primer intento debe procurar ser realizado por el más experimentado en el manejo de la vía aérea. se recomienda la intubación con videolaringoscopio en pacientes con covid- teniendo en cuenta disponibilidad y entrenamiento); esta alternativa puede ofrecer ventajas frente a la laringoscopia tradicional. se recomienda administrar medicamentos para asegurar la vía aérea en secuencia de inducción rápida, para obtener en el menor tiempo posible condiciones para la intubación ( a s). se recomienda en caso de intubación fallida por personal experto, considerar dispositivos supraglóticos como las máscaras laríngeas como medida de rescate con el riesgo de generación de aerosoles. se recomienda contar con disponibilidad de vasopresor y atropina en el sitio donde se realizará la intubación. en caso de contar con lidocaína se recomienda utilizar a dosis de mg/kg sin epinefrina. administrada minutos antes de la intubación. como se describió en el aparte de preoxigenación, se deben garantizar todas las medidas basadas en bioseguridad y protección adecuada para el personal de la salud, ubicación en áreas que cuenten con presión negativa o salas con un óptimo recambio de aire pueden ayudar a disminuir el riesgo de contagio. las medidas complementarias como la caja de taiwán o caja de intubación puede complementar de forma opcional estas medidas durante el proceso de aseguramiento definitivo de la vía aérea pero no garantiza % la eliminación de aerosoles, pudiendo ser un obstáculo para quien realiza la intubación, de ser así, se deberá retirar inmediatamente, por eso sólo es un complemento al epp, que es la verdadera protección en estos escenarios (se debe incluir máscaras n o ffp fpp ) ( , ) . el paciente críticamente enfermo puede tener comprometida de forma significativa su reserva respiratoria y hemodinámica por su cuadro clínico y puede empeorar por los medicamentos utilizados durante la intubación orotraqueal. las complicaciones en el manejo de la vía aérea se presentan cuando se realiza más de un intento dentro de los cuales están el traumatismo, desaturación e hipoxemia ( ) . es por esto por lo que se debe procurar que el primer intento sea realizado por el más experimentado en el manejo de la vía aérea e idealmente se logre la intubación en el primer intento con la menor hipoxemia secundaria ( , , ( ) ( ) ( ) . basado en el planteamiento anterior y considerando la ventaja de intubar pacientes críticamente enfermos en el primer intento dadas sus condiciones clínicas, la revisión de amci ® demandas asociadas a manejo de vía aérea en los estados unidos sigue siendo preocupante al considerar posibles causas la falta de entrenamiento y falta de aplicación de las guías y recomendaciones de manejo así como en la utilización de equipos adecuados para pacientes específicos ( ) ; se ha planteado especialmente en los pacientes covid- los cuales tienen un importante compromiso pulmonar y alto riesgo de desaturación, la posibilidad de encontrar una vía aérea difícil no predicha y dificultades en su manejo( , ). es por esto que se requiere del mejor dispositivo para manejo de vía aérea invasiva disponible, siempre y cuando se cuente con el adecuado entrenamiento previo en su uso, la ventaja de los videolaringoscopio se debe a la superioridad al compararla con la laringoscopia convencional, siendo en algunos grupos la primera opción para intubaciones electivas ( , ) . como se ha mencionado, el riesgo de una rápida desaturación en pacientes con enfermedad pulmonar, así como en pacientes con covid- , se debe utilizar la inducción de secuencia rápida la cual se utiliza para pacientes con estómago lleno en los cuales se quiere lograr condiciones de inconciencia y de intubación óptimas en el menor tiempo posible para disminuir el riesgo de broncoaspiración. en estos casos de falla respiratoria con tan mala reserva se quiere aprovechar la ventaja que ofrece esa técnica para tener en poco tiempo al paciente intubado con menor riesgo de desaturación. es así como los medicamentos en una inducción de secuencia rápida incluyen el opioide, hipnótico y relajante, estos dos últimos administrados simultáneamente y lavados con un bolo de cc. la opción del opioide en nuestro contexto suele ser fentanilo a dosis de a mcg / kg iv, dando a minutos de latencia para su efecto, luego el hipnótico que puede ser propofol entre y , mg / kg si la estabilidad hemodinámica lo permite o considera usar vasopresor simultáneo. en caso de preferir evitar la hipotensión la ketamina a dosis de a , mg / kg es una opción más estable hemodinámicamente. con relación al relajante neuromuscular la succinilcolina es la clásicamente utilizada dosis de a , mg/kg pero debido a sus efectos secundarios como hiperpotasemia, fasciculaciones, mialgias y un importante riesgo de hipertermia maligna algunos grupos no lo consideran, el rocuronio a dosis de , mg/kg ha demostrado lograr tiempos y condiciones de intubación similar a la succinilcolina sin los efectos secundarios de esta ( , ) algunos grupos han considerado no utilizarlos si la condición clínica del paciente es crítica pues éste período de latencia puede ser acompañado de una hipoxemia severa y paro cardíaco, por lo tanto sólo usan hipnótico y relajante neuromuscular. ante una vía aérea difícil no predicha en la cual no se logre la intubación, considere los dispositivos supraglóticos como las máscaras laríngeas los cuales son más fáciles de insertar en comparación con la técnica de intubación orotraqueal, y deben estar dentro del planeamiento y organización de elementos para manejo de la vía aérea invasiva. al lograr ventilar con este dispositivo se logrará una recuperación del paciente, pero se pueden generar aerosoles pues este mecanismo de cierre puede permitir escape de aire y macropartículas durante el ciclo respiratorio ya sea manual o mecánico ( , ) . el cuadro clínico de pacientes críticos y particularmente covid- , puede asociarse a inestabilidad hemodinámica y requerimiento de soporte vasopresor. la adición de medicamentos como los opioides o hipnóticos pueden asociarse a hipotensión la cual puede no responder a volumen, es necesario evitar episodios de hipotensión en especial en pacientes ancianos, con enfermedades cardiovasculares de base las cuales pueden tolerar menos estos cuadros de hipotensión, así como también pueden favorecer amci ® desbalance en la relación ventilación/perfusión a nivel pulmonar empeorando los cuadros de hipoxemia. la hipoxemia puede acompañarse de bradicardia y si no se corrige la ventilación o la bradicardia es muy probable que el paciente presente paro cardiorrespiratorio, por lo cual se recomienda utilizar una dosis de atropina para corregir la bradicardia, no mejorará la oxigenación, pero tendrá un tiempo adicional para recuperar la oxigenación y ventilación del paciente. en estos casos debe tener disponibles vasopresores y atropina desde el planeamiento de los medicamentos necesarios para el manejo de la vía aérea ( ) . se recomienda la intubación orotraqueal oportuna y no retrasar el inicio de la ventilación mecánica invasiva en los pacientes con sdra severo por covid- debido a mayor riesgo de desenlaces adversos. fundamento definir el momento de la intubación en esta población es un reto. la mayoría de los autores recomiendan el inicio "temprano" de la ventilación mecánica, sin embargo, la definición de cuando es temprano no es clara. este punto es motivo de análisis dado que a la luz de la evidencia actual la utilización de métodos no invasivos como la ventilación no invasiva y la cánula de alto flujo, para manejo inicial de pacientes con algún grado de hipoxemia es controvertido entre otras por el riesgo que supone al personal sanitario al ser un procedimiento generador de aerosoles. en el un estudio mostró que la intubación retrasada después de la falla al utilizar cánula de alto flujo o la ventilación no invasiva para pacientes con insuficiencia respiratoria moderada y grave se asoció con una mayor mortalidad. publicaciones recientes muestran que solo la quinta parte de los pacientes que murieron por covid- recibieron ventilación mecánica invasiva y soporte respiratorio más agresivo antes de la muerte, lo que indica que en muchos pacientes se habría retrasado la intubación. de los pacientes fallecidos solo el % recibieron tratamiento de oxígeno nasal o con mascarilla facial antes de su muerte. esta baja proporción puede tener varias explicaciones. primero, algunos pacientes con hipoxemia severa no tenían otros síntomas, como dificultad para respirar o disnea, es decir, desarrollaron una especie de hipoxemia silenciosa. en segundo lugar, la falta de suficientes ventiladores mecánicos invasivos es una razón importante que evitaría que los pacientes reciban intubación. tercero, el manejo de estos pacientes por un equipo de médicos no intensivistas; por lo tanto, pueden no estar seguros del momento en que un paciente requiere intubación. la serie de casos de la epidemia de covid- en wuhan mostró que la intubación tardía era común en la etapa inicial de la epidemia, mostrando que una de esas posibles razones del retraso incluía falta de ventiladores mecánicos invasivos y falta de capacitación clínica específica para el soporte respiratorio. recomendaciÓn se recomienda tomar la decisión de intubación orotraqueal en el paciente crítico con sospecha o diagnóstico de covid- utilizando una combinación de variables clínicas, gasométricas y hemodinámicas. tabla la intubación orotraqueal (iot) efectiva y segura, programada debe prevenir el colapso respiratorio y hemodinámico. siempre es necesario asegurar la escena del procedimiento de intubación con las consideraciones técnicas y de protección personal adecuadas. conocer los pasos para la realización del procedimiento de intubación orotraqueal (iot) contextualizados al paciente covid- , reduce los riesgos innecesarios. la iot es un procedimiento generador de aerosoles por lo tanto lo ideal es realizarlo en una habitación con presión negativa, sin embargo, la baja disponibilidad en el país obliga a utilizar otras alternativas de seguridad. una habitación de presión negativa es un cuarto que tiene una presión más baja que las áreas adyacentes, lo que mantiene el flujo de aire fuera de la habitación y hacía habitaciones o áreas contiguas. las puertas de la sala deben mantenerse cerradas, excepto al entrar o salir de la sala, y la entrada y la salida deben minimizarse. la intubación en el paciente crítico con covid- es de los procedimientos que mayor riesgo de aerosolización tiene, por lo tanto, se debe adoptar una posición de intervención oportuna, pero también segura, para evitar desenlaces desfavorables en el paciente y disminuir el riesgo de contaminación en el personal de salud, se recomienda individualizar cada caso mediante la combinación y análisis de los criterios clínicos, gasométricos y hemodinámicos de cada paciente. ( , ( ) ( ) ( ) se recomienda no realizar de rutina broncoscopia en los pacientes con sospecha o diagnóstico de covid- , debido al riesgo de generación de aerosoles. se puede considerar en atelectasias masivas con compromiso significativo de la oxigenación adicional a la lesión pulmonar per sé y la hemorragia alveolar para control local directo. en general la realización de broncoscopia en pacientes con sospecha o confirmación de covid- debe ser evitada y realizarse sólo con indicación de emergencia como cuerpo extraño en la vía aérea, hemoptisis masiva, obstrucción grave de la vía aérea central o atelectasia lobar o pulmonar completa( ). esto debido a la alta carga viral en la mucosa nasal y faríngea de los pacientes con infección por sars-cov- ( ) y la alta producción de aerosoles infecciosos que se generan durante este procedimiento. en caso de ser necesaria su realización, deberá ser llevada a cabo por el operador con mayor experiencia amci ® minimizando el tiempo de exploración y el personal expuesto en la sala. es mandatorio utilizar epp completo que incluya respirador fpp o fpp , bajo protocolo supervisado de donning y doffing ( ) . son de elección los broncoscopios desechables de un solo uso, pero de no ser posible se prefiere el uso de un broncoscopio flexible por encima de uno rígido por la más fácil manipulación de este. idealmente el procedimiento se llevará a cabo en el mismo cubículo del paciente que deberá contar con presión negativa y recambio de aire de a veces por hora. recomendaciÓn se recomienda no realizar de rutina la broncoscopia para la recolección de muestras para el diagnóstico de covid- en el paciente críticamente enfermo. la broncoscopia es una prueba de segunda elección para la toma de muestras respiratorias en los pacientes con sospecha o confirmación de covid- ( ) . la toma de muestras del tracto respiratorio superior por hisopado nasofaríngeo u orofaríngeo es el método primario y de elección para determinar la infección por sars-cov- . solo si resultaran dos pruebas negativas y persistiera una alta sospecha diagnóstica estaría indicado tomar muestras del tracto respiratorio inferior por broncoscopia, ya sea aspirado endotraqueal (bas) o lavado bronco alveolar (bal) ( ), prefiriendo la realización de minibal para la recolección de muestras ( ). las muestras deberán ser recogidas en un recipiente estéril, e introducidas en una bolsa con autocierre. deben manejarse con cuidado extremo evitando manipulaciones innecesarias y bajo protocolos de protección para el personal que las maneja, y trasladarse al laboratorio para su análisis. pueden almacenarse a - ºc las primeras h de su recolección; si se demorara más el análisis, precisa almacenarse a temperatura de - ºc ( ) . en una serie china la sensibilidad del bal fue de % frente a % en muestra de esputo (no recomendada) y % en hisopado nasofaríngeo ( ) por lo que el especialista que realiza el procedimiento deberá sopesar el riesgo de este procedimiento en cada caso, valorando que se beneficiarán aquellos pacientes que tengan una indicación adicional para su realización. se recomienda la realización de la traqueostomía cuando está indicada, en los pacientes covid- sospechosos y confirmados con pronóstico razonable de vida, después del o día de ventilación , previa valoración y consenso por el equipo quirúrgico y de cuidado intensivo, asegurando que las condiciones clínicas, ventilatorias y hemodinámicas se encuentran controlada. en el contexto de pacientes hospitalizados en cuidado intensivo, la traqueostomía se realiza para facilitar el destete de la ventilación mecánica, mejorar la limpieza de la vía aérea y el manejo de las secreciones, aumentar la comodidad de los pacientes y la movilización y disminuir la probabilidad de complicaciones como la estenosis traqueal; sin embargo no hay una clara disminución en la mortalidad ( ) ( ) ( ) . la infección actual por sars-cov , tiene diferentes estadios de gravedad, uno de ellos es el compromiso pulmonar el cual se caracteriza por un síndrome de dificultad respiratorio agudo (sdra). de acuerdo con el comportamiento de la covid- , entre un % a un % de los pacientes requieren ventilación mecánica ( , ) , este grupo de pacientes con manifestación grave del compromiso pulmonar requiere estrategias de protección pulmonar en la ventilación mecánica, sedación profunda y posiblemente parálisis muscular y puede tener una mortalidad entre el % y el % ( , ) . una de las principales características en éste grupo de pacientes es la mortalidad temprana, definida ésta como aquella que se produce en menos de días; de acuerdo a la experiencia en wuhan, china; leung ( ), reporta que la mortalidad se presenta en los primeros cinco días luego de la admisión hospitalaria y de acuerdo a lo referido por graselli et al ( ) , en la región de lombardía, italia, la mediana de mortalidad se presenta al día siete después del ingreso. con estas consideraciones, al principio de la pandemia y en las aproximaciones iniciales no se recomendaba realizar la traqueostomía en los primeros días posteriores a la intubación orotraqueal. sin embargo, con el conocimiento de la fisiopatología y las experiencias en otras series, como medida para facilitar la liberación de la ventilación mecánica, se ha podido realizar el procedimiento después de la primera semana de inicio de la ventilación. de acuerdo con el curso natural de la enfermedad, el paciente en promedio se intuba al día a de iniciado los síntomas, una semana posterior a la intubación para la traqueostomía, estaríamos alrededor del día de la enfermedad, donde los pacientes ya tendrán una disminución de la carga viral. esto sin embargo no evita la utilización de los epp necesarios. esta medida en específico fue discutida y consensuada entre la sociedad de medicina critica amci y la asociación colombiana de cirugía. no existe evidencia que permita evaluar el riesgo real de infección del personal asistencial de los pacientes con sospecha o diagnóstico de covid- en la realización de traqueostomías. se debe considerar en la traqueostomía y la realización de ésta como un procedimiento generador de aerosoles (organización mundial de la salud). se recomienda que los epp requeridos para la realización del procedimiento, deben incluir máscaras ffp o n , protección ocular, vestido antifluido idealmente desechable y amci ® guantes; este nivel de protección representa el mayor nivel de seguridad para realizar el procedimiento en el paciente con sospecha o diagnóstico de covid- . no existe actualmente artículos que permitan evaluar cual es el riesgo real de infección del personal asistencial en la realización de traqueostomías en pacientes con covid- ; quizás el ejemplo más cercano, es una serie de casos, reportada durante la epidemia del síndrome respiratorio agudo grave en el ( ), en el cual se realizaron traqueostomías sin ninguna infección del personal, en este reporte se aseguró un adecuado uso del equipo de protección personal (epp), el cual incluía las medidas de barrera, máscaras ffp y cuando existía la disponibilidad, respiradores con suministro de aire purificado. la traqueostomía y la realización de ésta es considerada por la organización mundial de la salud (oms) un procedimiento generador de aerosoles, bajo esta perspectiva, el epp requerido para la realización del procedimiento, debe incluir máscaras ffp o n , protección ocular, vestido antifluido idealmente desechable y guantes; este nivel de protección representa el mayor nivel de seguridad para realizar el procedimiento mencionado. es obligatorio, que todo el personal reciba el entrenamiento para la postura, el uso y el retiro de los epp, si estos pasos no se realizan de la forma adecuada, representan una fuente de contaminación( ). se recomiendan no esperar la negativización de la pcr para sars-cov para realizar la traqueostomía en el paciente con diagnóstico de covid- . fuerte en contra fundamento los estudios de zou et al y lescure et al ( , ) , muestran que la carga viral de los hisopados nasales y faríngeos es elevada en la primera fase de la enfermedad, con una disminución entre el día nueve al quince, pero esta puede permanecer detectable hasta por tres semanas ( ) . existen recomendaciones acerca de la necesidad de realizar la traqueostomía una vez la pcr para sars-cov sea negativa ( , ) ;aunque este esquema suena lógico, es importante tener presentes las siguientes consideraciones. la sensibilidad de una sola muestra para ruta-pcr puede ser sólo del %( ) y es posible que sea necesario realizar una segunda prueba para minimizar el riesgo para quien realiza el procedimiento, aunque esta aproximación no siempre puede ser viable desde el punto de vista clínico, epidemiológico y administrativo. amci ® recomendamos que la mejor estrategia es diferir la realización de la traqueostomía hasta días luego de la intubación cuando esta indicada, con el conocimiento acerca de la evolución natural de la enfermedad, en ese momento en la mayoría de los pacientes, lo más probable es que la condición ya se encuentre en la tercera semana desde el inicio de los síntomas, en cuyo caso lo más probable es que ya exista una disminución de la carga viral; este hecho no evita que se deba utilizar de la forma correcta los epp. recomendaciÓn se recomienda escoger la técnica teniendo en cuenta la experticia que tenga en el grupo tratante y la anatomía del paciente para la decisión de la técnica. se recomienda la guía ecográfica para disminuir la probabilidad de complicaciones del procedimiento si la anatomía es desfavorable para la realización de traqueostomía percutánea. se recomienda la traqueostomía quirúrgica en pacientes de riesgo elevado de complicaciones donde se requiere un control más rápido y seguro de la vía aérea. se recomienda no utilizar de forma rutinaria la utilización de broncoscopia para la realización de traqueostomía por vía percutánea. no hay evidencia directa hasta la fecha de publicación del consenso si existe superioridad entre las dos técnicas en el paciente con sospecha o diagnóstico covid- . en pacientes críticos no existen diferencia en los desenlaces cuando se evalúa el rendimiento de la técnica percutánea y la técnica quirúrgica, la elección de uno u otro método está dado por la anatomía del paciente, el entrenamiento de los profesionales y la disponibilidad de los diferentes insumos y técnicas. tampoco se ha logrado hasta la fecha evaluar durante la pandemia de covid- si existe una diferencia entre las dos técnicas y por lo tanto no es posible acercarse a una recomendación basada en la literatura. sin embargo, es importante que para la decisión de la técnica a utilizar se considere la anatomía del paciente y los siguientes aspectos:  no se recomienda la utilización de broncoscopia para la realización de traqueostomía por vía percutánea, ésta aumenta la generación de aerosoles y el número de personas expuestas a estos ( , ) . amci ®  si la anatomía es desfavorable para la realización de traqueostomía percutánea, la guía ecográfica puede disminuir la probabilidad de complicaciones del procedimiento ( ) .  la traqueostomía quirúrgica es una alternativa para la realización del procedimiento en las unidades de cuidado intensivo en momentos de sobrecarga laboral y adicionalmente puede tener un mejor y más rápido control de la vía aérea, especialmente en pacientes con riesgo elevado de complicaciones ( ) . recomendaciÓn se recomienda el uso de la terapia ecmo en sdra severo refractario por covid- (pao /fio < , posición prona, requerimiento de relajantes neuromusculares en algunos casos vasodilatadores pulmonares y maniobras de reclutamientos) sin respuesta clínica manifestado por:  pao /fio < mmhg por mas de horas  pao /fio < mmhg por mas de horas  ph < . + paco > mmhg por mas de horas además del criterio anterior, se recomienda tener en cuenta la edad, las comorbilidades y la expectativa de sobrevida del paciente con buena calidad de vida y en circunstancias donde no exista limitación de recursos. no hay estudios clínicos aleatorizados sobre el uso del ecmo en pacientes con covid- ( , ) . existe el estudio eolia ( ) , el cual fue detenido durante su realización, y de acuerdo a un análisis bayesiano posterior puede interpretarse como una disminución de la mortalidad en los pacientes en ecmo con sdra severo ( , , ( ) ( ) ( ) ( ) . de igual manera debe considerarse la racionalización de los recursos y el estado de prevalencia de la pandemia en un lugar determinado. el inicio de la terapia puede evaluarse en función de la cantidad de pacientes en falla respiratoria y la disponibilidad de personal y otros recursos; si el hospital debe comprometer todos los recursos en proveer medidas básicas de cuidado intensivo no debe utilizar el ecmo ( , ) . los pacientes jóvenes sin comorbilidades son considerados de alta prioridad al igual que los trabajadores de la salud ( , ) . amci ® se recomienda no desarrollar nuevos centros de ecmo en época de pandemia, sobre todo en situaciones con limitación de recursos. fuerte en contra fundamento actualmente se recomienda el uso del ecmo con las mismas indicaciones para sdra basado en la capacidad de las instituciones de salud para iniciar éste tipo de terapias ( ) . en épocas de crisis la capacidad de los hospitales está saturada y obliga a la reubicación y optimización de los recursos ( , ) . los centros que ofrecen la terapia en ecmo deben ser centros con resultados favorables y tiempos de soporte de pacientes relativamente cortos ( ) . cuando estamos en tiempos de capacidad hospitalaria convencional y existe disponibilidad de camas de cuidado intensivo se pueden ofrecer los servicios de ecmo vv, va, e-cpr inclusive a pacientes no covid- , cuando estamos en contingencia y capacidad nivel se debe hacer un triage respecto a pacientes jóvenes y ofrecer ecmo vv, va, escoger muy bien los casos para pacientes no covid- y no ofrecer e-cpr, cuando estamos en contingencia y capacidad nivel es porque ya se están usando sitios de expansión y están casi saturados se restringe el ecmo a todas las indicaciones y se prioriza a pacientes con indicaciones no covid- con mayor riesgo de sobrevida, el ecmo vv queda para pacientes jóvenes, con disfunción de órgano y covid- positivo, no se ofrecerá ecmo va o e-cpr y cuando estamos en capacidad de crisis es porque la capacidad total hospitalaria está sobresaturada y no es posible realizar ecmo tanto en pacientes covid- como en los no covid- ( , ) . se recomienda practicar e implementar medidas de capacitación y vigilancia continua para mejorar la higiene de manos, evaluando la adherencia a protocolos establecidos en los trabajadores de la salud mediante listas de chequeo y supervisión para evitar infecciones cruzadas en el entorno del paciente con sospecha o diagnóstico de covid- . se recomienda establecer protocolos específicos para reducir el riesgo de infecciones que se deriven de la interacción y el cuidado del paciente crítico con sospecha o diagnóstico de covid- . amci ® se recomienda implementar prácticas de cuidado para la prevención de contagio de covid- . se debe utilizar el equipo de protección personal (epp) para la prevención de enfermedades de componente infeccioso asociado a exposición con fluidos corporales derivados del paciente crítico con sospecha o diagnóstico de covid- . las infecciones relacionadas con la atención sanitaria (iras) son definidas por la organización mundial de la salud como aquellas "infecciones que se presentan en un paciente durante el proceso de atención en un hospital u otro centro sanitario que no estaban presentes o no se estaban incubando en el momento del ingreso; se incluyen las infecciones contraídas en el hospital pero que se manifiestan tras el alta hospitalaria y también las infecciones profesionales entre el personal del centro sanitario". las iras representan una importante carga de enfermedad que se asocia a un impacto negativo en la economía del paciente y del sistema sanitario. la organización mundial de la salud plantea la higiene de manos como la principal medida necesaria para reducir y prevenir las iras; por esta razón establece directrices sobre la higiene de manos en la atención sanitaria y basada en esta propone la estrategia multimodal para la mejora de higiene de manos. las estrategias mencionadas anteriormente han demostrado el incremento en el cumplimiento de higiene de manos y disminución en las infecciones relacionadas con la atención en salud. la estrategia multimodal se articula a través de cinco componentes: cambio del sistema, formación, evaluación/ retroalimentación, recordatorios en el lugar de trabajo clima institucional. a través de estos componentes, se garantiza que el centro sanitario cuenta con la infraestructura necesaria para practicar adecuadamente el lavado de manos incluyendo dentro de este el acceso a un suministro seguro continuo de agua, jabón, preparado alcohólico y toallas; a su vez se proporciona educación, evaluación y retroalimentación con regularidad a todos los profesionales sanitarios ( se recomienda realizar un plan de cuidados organizado y específico en paciente crítico con sospecha o diagnóstico de covid- , ofreciendo el uso óptimo de recursos e intervenciones. se recomienda evitar el uso de excesivo de papelería relacionada con los registros usados para gestión de insumos y atención de los pacientes. se recomienda realizar intervenciones educativas enfocadas a mejorar la adherencia y adecuado uso de los epp. se debe procurar el cuidado de los elementos de protección personal bajo un protocolo que conserve las condiciones de integridad de estos. la actual reserva de elementos de protección personal (epp) es insuficiente debido al aumento de la demanda global, por el incremento de casos de covid- y por la información errónea que ha conllevado a compras de pánico y almacenamiento. por esta razón la organización mundial de la salud a través de su guía: uso racional del equipo de protección personal para la enfermedad por covid- ha implementado las siguientes estrategias para optimizar la disponibilidad de (epp): usar los (epp) adecuadamente, minimizar la necesidad de (epp) y coordinar el suministro adecuado de (epp). ( ) la duración máxima del uso continuo de la n es de a horas, siguiendo las recomendaciones del manual de medidas básicas para control de infecciones en ips de minsalud. pero en lo cotidiano, ningún trabajador tolera a horas continuas con un respirador. por esto, su uso continuo en el sitio de trabajo dependerá de la necesidad de pausar para comer, para ir al baño, etc. en este caso, se guardará en una bolsa de papel para su nueva colocación, si tiene menos de horas, se desechará si está visiblemente contaminada o se torna húmeda. el reusó de la n dependerá de la casa del fabricante, de si contiene o no celulosa en su estructura del respirador. por ejemplo, la recomendación del consenso colombiano acin sobre la desinfección para los respiradores sin celulosa es con peróxido de hidrógeno vaporizado al % por minutos. los respiradores n de uso industrial tienen mayor contenido de celulosa que los de uso médico por lo tanto para procesos de esterilización, solo los n de uso médico podrán ser esterilizados mediante de peróxido de hidrógeno vaporizado (sterrad®)( - ). amci ® se recomienda elaborar el protocolo de pronación del paciente críticamente enfermo por covid- , garantizando el entrenamiento al personal de salud, organizando el recurso humano, dispositivos de apoyo y tiempo establecido para cambios de posición para prevenir las lesiones por presión en el paciente. fuerte a favor fundamento la estrategia de pronación es una alternativa eficiente en el manejo del síndrome de dificultad respiratoria en pacientes críticos y es fundamental la gestión del profesional de enfermería en la prevención de complicaciones y eventos adversos, lo cual aportará significativamente a la calidad del cuidado ofrecido favoreciendo las mejoras en la oxigenación. es importante optimizar los cuidados de enfermería en torno de los cuidados de piel en los pacientes en ventilación mecánica en decúbito prono, los estudios han demostrado como principal complicación las lesiones de presión con una incidencia hasta de . %, siendo las más frecuentes las grado y ( ). una lesión se puede producir si se supera una presión en el tejido capilar arterial de mmhg denominándose interfaz de presión. basándose en lo anteriormente mencionado, el cambio de posición es un componente integral de la prevención y el tratamiento de las upp, con una justificación sólida y de amplia recomendación en la práctica de enfermería ( ) . el uso de superficies especiales para el manejo de la presión (semp) a partir del estudio de defloor ( ), se determinó un antes y un después en el uso de las semp en conjunción de los cambios posturales. este realizo un importante aporte para reducir la incidencia de lesiones por presión comparado con los colchones de estándar. se recomienda promover actividades para controlar el nivel de estrés en el personal durante las jornadas de trabajo: identificar y reconocer los propios límites, buscar o proponer ayuda psicológica profesional cuando sea requerido, promover estilos de vida saludables, y organizar los turnos de trabajo asegurando periodos de descanso. se recomienda compartir las emociones con pares y superiores, analizar objetivamente las situaciones adversas, manejar fuentes de información objetivas y científicas, realizar pausas activas durante el turno y brindar espacios grupales para expresar emociones, miedos e incentivar al equipo de trabajo reconociendo su labor. amci ® se recomienda utilizar recursos de salud mental ocupacional, apoyo por enfermedad y licencia familiar, además de garantizar una adecuada dotación de personal. los estilos de superación personal y el crecimiento psicológico desempeñan un papel importante en el mantenimiento de la salud mental de las enfermeras. es razonable suponer que los niveles de ansiedad y estrés entre los profesionales de la salud son proporcionalmente más altos que los de la población general debido al contacto directo con pacientes infectados. esto puede explicar por qué las enfermeras de primera línea son excepcionalmente vulnerables a la fatiga y al agotamiento (wang, okoli, et al. ) , agotamiento mental, falta de moral del personal, control / autonomía de decisión, menor calidad de vida y baja satisfacción laboral (cheung y yip, ). ( ) . una investigación reciente realizada en china continental menciona el impacto negativo de la pandemia de covid- en los trabajadores de atención médica de primera línea, incluidos los mayores niveles de ansiedad (shanafelt, ripp y trockel, ), depresión (xiang et al. ), estrés postraumático síntomas, soledad e impotencia (xiang et al. ) ( , ) . los aspectos traumáticos y estresantes de la participación en una pandemia también ponen en riesgo el daño psicológico a los médicos ( ) . la experiencia psicológica de las enfermeras que atienden a pacientes con covid- se puede resumir en temas: primero, las emociones negativas presentes en la etapa inicial consisten en fatiga, incomodidad e impotencia que fue causado por el trabajo de alta intensidad, el miedo y la ansiedad, y la preocupación por los pacientes y sus familiares. segundo, los estilos de auto afrontamiento incluyeron ajustes psicológicos y de vida, actos altruistas, apoyo de equipo y coordinación racional. tercero, encontramos crecimiento bajo presión, que incluía un mayor afecto y agradecimiento, desarrollo de posición de responsabilidad profesional y autorreflexión. finalmente, encontraron que las emociones positivas ocurrieron simultáneamente con emociones negativas( ). se recomienda ofrecer mecanismos de apoyo para amortiguar el estrés relacionado con la pandemia por covid- . esto incluye intervenciones para pacientes y familias ofreciendo recursos de salud mental y educación al egreso, previo a este reforzar visitas virtuales. se recomienda anticipar las necesidades de salud mental de los pacientes, el personal y las familias para ofrecer una respuesta integral de salud pública. se debe incluir atención psicológica en la hospitalización para pacientes, familiares y personal afectado por covid- . se recomienda proporcionar atención de salud mental en las comunidades, mientras que se requiere distanciamiento social y los recursos del sistema de salud son limitados. amci ® se recomienda mantener una estrategia de comunicación asertiva con la familia, teniendo en cuenta la formación del personal sobre las estrategias para comunicar malas noticias. ser solidarios con el duelo de las familias y acompañar el proceso de afrontamiento aún en la distancia, identificando factores de riesgo para patología mental o duelo complicado, utilizando los recursos institucionales de salud mental para mejorar las intervenciones. la pandemia tiene el potencial de crear una crisis secundaria de angustia psicológica y desbordamiento del sistema de salud mental. los miembros de la familia pueden experimentar angustia, miedo o ansiedad por la hospitalización de un ser querido, particularmente cuando las medidas de control de infecciones restringen las visitas. la telesalud (incluida la cobertura de seguro para la telesalud), el suministro extendido de medicamentos, el aumento de la capacitación en salud mental del proveedor, el apoyo virtual de pares y los grupos virtuales de apoyo al uso de sustancias pueden ayudar a garantizar que se satisfagan las necesidades de salud mental de la comunidad ( ). el sistema de salud y los líderes de enfermería deben asegurarse de que su personal de enfermería clínica esté protegido y respaldado para que puedan proporcionar esta dimensión crucial de la atención de covid- . se recomienda crear grupos centralizados y definidos para atención de pacientes con sospecha o diagnóstico de covid- que se encarguen de elaboración, socialización e implementación de protocolos. estos deben incluir los aspectos de infraestructura, áreas delimitadas, utilización de epp, listas de chequeo, observadores, insumos y recursos que permitan atención integral. se recomienda organizar el plan de atención del paciente con sospecha o diagnóstico de covid- de enfermería con la asignación de actividades, número de personas según escalas que midan escalas de carga laboral para definir el número adecuado de los miembros del equipo de trabajo, tiempo de atención, gestión de recursos, gestión de riesgo y un líder por turno que garantice el cumplimiento fuerte a favor fundamento la implementación de estrategias de gestión en contingencias genera un trabajo organizado, enfocado en la prevención y tratamiento centralizado, elaboración y socialización de protocolos claros, áreas específicas, delimitadas y asignadas, con un uso racional del recurso humano que se despliega en fases, desde el inicio de la emergencia considerado como detección temprana hasta la atención directa de pacientes con sospecha amci ® o confirmación de sars-cov- . dentro de las fases tempranas, se busca la gestión de los recursos necesarios para la atención de estos pacientes, con una asignación de zonas o servicios y unas condiciones particulares, tratamientos específicos y actividades de atención especiales para las cuales se discriminan medicamentos, dispositivos e insumos necesarios para el cuidado de enfermería. los grupos de atención deben contar con capacitación, gestión y supervisión, apoyo logístico, apoyo psicológico y retroalimentación ( , ) . el plan de atención de enfermería debe tener presente la minimización de exposición, la prevención de infecciones en el personal y cuidados especiales derivados de la condición clínica de los pacientes con esta infección, altamente contagiosa y con síntomas o necesidades que rompe el modo operacional convencional y que requiere implementación basada en la práctica clínica. por lo tanto, el plan debe ser centralizado oportuno, ordenado, seguro y eficiente e incluye: relación enfermería/paciente de acuerdo a criticidad, capacitaciones y entrenamiento al personal de enfermería de línea de frente en el área crítica de aislamiento mediante videos, infografías y procesos prácticos (el contenido de capacitación incluye el uso de elementos de protección personal, higiene de manos, desinfección de áreas, manejo de residuos y esterilización de dispositivos de atención al paciente y manejo de exposición ocupacional), asignación de actividades clínicas (atención directa) y administrativas (supervisión, observador, líderes, gestión de recurso humano y medicamentos), soporte y contratación de personal adicional ante la contingencia con preparación académica o inducción, asignación de turnos razonables con períodos de descanso (alimentación, eliminación), coordinación con otros departamentos y optimización de flujos de trabajo, estrategias de control de infecciones y trabajo en equipo ( , ) . se recomienda que las muestras clínicas tomadas para el diagnóstico de covid- deben conservarse a temperatura entre - a °c, y luego de las horas deben permanecer congeladas a una temperatura de - °c. se recomienda que se realice el envío al laboratorio de salud pública de referencia dentro de las horas posteriores a la toma de la muestra del paciente. se recomienda que el transporte de las muestras debe realizarse con geles o pilas congeladas. se recomienda considerar que las muestras del tracto respiratorio bajo presentan la mejor certeza diagnóstica en pacientes con neumonía para adultos intubados y ventilados mecánicamente con sospecha de covid- . se recomienda contar con elementos de protección personal de acuerdo con las precauciones establecidas para el paciente con sospecha o diagnóstico por covid- para evitar la transmisión a profesionales de la salud. se debe evitar perder el circuito cerrado en los pacientes ventilados mecánicamente y valorar el riesgo de las acciones en pacientes con peep alta. se recomienda realizar la toma de muestra post mortem no invasiva por hisopado nasofaríngeo dentro de las primeras seis ( ) horas posteriores al fallecimiento, para que esta sea útil para su análisis. las muestras clínicas tomadas para el diagnóstico de coronavirus deben conservarse a temperatura entre - a °c, y luego de las horas deben permanecer congeladas a una temperatura de - °c. sin embargo, la muestra puede conservarse en un tiempo máximo de refrigeración por horas. no obstante, se sugiere que se realice el envío al laboratorio de salud pública de referencia dentro de las horas posteriores a la toma. si no se conserva la cadena de frío adecuada, la muestra puede ser inviable. el transporte de las muestras debe realizarse con geles o pilas congeladas ( , , ) .se debe tener en cuenta que no conservar la cadena de frío durante el transporte de la muestra, degradan la partícula viral, obteniéndose falsos negativos ( ) . las muestras del tracto respiratorio bajo presentan la mejor certeza diagnóstica en pacientes con neumonía. para adultos intubados y ventilados mecánicamente con sospecha de covid- en comparación al tracto respiratorio superior (nasofaríngeo u orofaríngeo). en el caso de aspirado traqueal, es importante considerar que para la obtención de las muestras para el diagnóstico de covid- se deben contar con elementos de protección personal de acuerdo a las precauciones estándar para evitar la transmisión a profesionales de la salud, circuito cerrado y valorar su realización en aquellos pacientes con peep alta ( ) . la toma de muestra post mortem no invasiva por hisopado nasofaríngeo se debe hacer antes de seis ( ) horas post mortem, para que esta sea útil para su análisis( , - ). se recomienda en los pacientes con diagnóstico covid- , monitorizar continuamente la oxigenación mediante saturación arterial de oxígeno con pulso oxímetro y la aparición temprana de signos clínicos de dificultad respiratoria durante la monitorización (aleteo nasal, cianosis, tirajes intercostales). se recomienda no suministrar de forma rutinaria suministrar oxígeno si la saturación de oxígeno (spo ) está por encima de %, y no se evidencian signos clínicos de dificultad respiratoria durante la monitorización continua del patrón respiratorio. se recomienda como parámetro importante para evaluar la oxigenación y guiar el suministro de oxígeno mediante los diferentes dispositivos la transferencia de oxígeno, medida por la pao / fio o sao /fio . se propone iniciar la oxigenoterapia por cánulas de bajo flujo y ajustar el flujo (máximo l) hasta alcanzar la spo objetivo ≥ %; si el paciente se encuentra en estado crítico iniciar con mascarilla con bolsa de reserva (a - l / min). una vez que el paciente esté estable, el objetivo de oxigenación es mantener niveles de spo entre y % en pacientes no embarazadas y entre - % en pacientes embarazadas. se recomienda no utilizar de forma rutinaria el uso de dispositivos que generan aerosoles durante la administración de oxígeno (dispositivos venturi o nebulizador de alto flujo o jet) en pacientes con sospecha o diagnóstico covid- . en las diferentes guías publicadas para manejo de pacientes positivos para covid- las metas de oxigenación durante la terapia de oxígeno en adultos recomiendan iniciar la oxigenoterapia a l / min y ajustar el flujo hasta alcanzar la spo objetivo ≥ % durante la reanimación; o use mascarilla con bolsa de reserva (a - l / min) si el paciente está en estado crítico. una vez que el paciente esté estable, el objetivo de oxigenación es > % de spo en pacientes, no embarazadas y ≥ - % en pacientes embarazadas( ). los dispositivos para la oxigenoterapia se pueden dividir en dos grupos, dependiendo de si cubren la totalidad o una parte de los requerimientos respiratorios del paciente. unos son de bajo flujo o para esfuerzos mínimos del paciente, estos dispositivos completan su ventilación con aire ambiente y los sistemas de alto flujo cubren la totalidad de los requerimientos inspiratorios del paciente. escalones terapéuticos: oxigenoterapia convencional a diferentes concentraciones de bajo flujo (son las cánulas nasales, las mascarillas simples y las mascarillas con reservorio), es el primer escalón terapéutico ante cualquier paciente que presente una situación de hipoxemia (spo ) < % respirando aire ambiente. el objetivo debe ser ajustar la fio (hasta . ) para mantener un nivel de oxigenación adecuado, considerado este como una spo > %. la administración de oxígeno se considera un procedimiento generador de aerosoles de riesgo bajo y por lo tanto es adecuado para pacientes covid- positivos( ). b. en adultos con signos de emergencia (respiración obstruida o ausente, dificultad respiratoria severa, cianosis central, shock, coma y / o convulsiones) deben recibir vía aérea amci ® de emergencia manejo y oxigenoterapia durante la reanimación para apuntar a spo ≥ %. una vez el paciente está estable, objetivo> % de spo en adultos no embarazadas y ≥ - % en mujeres embarazadas. c. para el manejo del paciente con covid- la máscara de no re inhalación se considera como la opción de preferencia para escalar el paciente antes de la intubación y considerar la transferencia a uci; esto se debe a que puede proporcionar altas fracciones inspiradas de oxígeno ( ) . d. los dispositivos que generan aerosoles durante la administración de oxígeno (dispositivos venturi o nebulizador de alto flujo o jet), no están indicados para manejo de covid- ( ). se recomienda aplicar las estrategias de retiro de la ventilación mecánica habituales para pacientes adultos críticos en general, hasta el momento no se ha construido una evidencia contundente para el destete en covid- . se recomienda en el paciente críticamente enfermo por covid- un descenso de la presión de soporte (psv) según tolerancia clínica, de esta forma el paciente podrá ser sometido a la realización de prueba de respiración espontánea con una presión de soporte de entre - cm h o. se recomienda que el destete automatizado puede ser considerado como una herramienta útil según disponibilidad de equipos para realizarlo. se recomienda no utilizar las maniobras que incrementan la aerosolización como la prueba de respiración espontánea en pieza en t o el cuff-leak test en el momento de realizar la medición de los predictores de éxito en el destete. fuerte en contra fundamento la realización de las pruebas de respiración espontánea sigue siendo un factor predictor importante en el éxito en el retiro del soporte ventilatorio mecánico y la indicación de tiempo de duración sigue siendo de a minutos debido a que las intubaciones realizadas en el mismo periodo de tiempo no han tenido diferencias significativas en el éxito del destete ( , ) . en los pacientes que han sido ventilados por más de horas y que el motivo por el cual fueron llevados a ventilación mecánica ya ha sido superado se debe establecer un protocolo de destete que debe incluir una prueba diaria de respiración espontánea y la minimización o retiro de la sedación (si no existe alguna contraindicación)( , ). la movilización temprana como factor coadyuvante en el éxito de la liberación mecánica ya se ha documentado en otros escenarios similares, razón por la cual la implementación amci ® temprana de este tipo de estrategias será un punto de vital importancia para recuperar la funcionalidad de los pacientes con covid- ( ) . se recomienda que la extubación de los pacientes críticamente enfermos por covid- se debe realizar con los elementos de protección personal requeridos para el riesgo de aerosoles. se recomienda no estimular la tos y el esputo inducido en los pacientes con sospecha o diagnóstico de covid- posterior a la extubación inmediata. se recomienda no utilizar de forma rutinaria la vmni en la falla respiratoria post extubación en pacientes críticos que no tengan una enfermedad concomitante que sea respondedora a la vmni como el epoc o edema pulmonar de origen cardiogénico en pacientes con sospecha o diagnóstico de covid- . fuerte en contra se recomienda mantener un umbral bajo para decidir intubación en caso de sospecha de fallo en la extubación en el paciente con sospecha o diagnóstico de covid- . la estricta monitoria y manejo del paciente posterior a la extubación surgen como un reto insoslayable para el personal de cuidado intensivo, enfocando todos sus esfuerzos en evitar la re-intubación, lo que se traducirá en un descenso significativo de la morbilidad y la mortalidad que supone una re-intubación ( ), la cual se puede definir como el no requerimiento de re intubación en las primeras horas post extubación ( , ) . en los últimos años la cánula nasal de alto flujo (caf) se ha convertido en una herramienta útil en el soporte de oxigenoterapia en los pacientes extubados que presenten riesgo de reintubación ( , ), y a la vez no presenten hipercapnia ( ) . la utilización de ventilación mecánica no invasiva de manera profiláctica en la falla respiratoria post extubación no ha demostrado tener éxito evitando la re-intubación en las primeras horas ( , ) excepto en las situaciones donde el paciente presente una enfermedad pulmonar o alteración cardiaca concomitante que sea respondedora a el manejo con vmni como lo son la enfermedad pulmonar obstructiva crónica (epoc) y el edema pulmonar de origen cardiogénico ( , ) . los pacientes extubados en los que se halla documentado epoc, se sugiere posterior a la extubación la implementación de una estrategia de niv de manera protocolaria ( , ), con una intensidad de hora cada horas durante un período mínimo de horas ( ). se recomienda utilizar en los pacientes con extubación reciente que no expresen predictores de riesgo de fracaso, sistemas de oxigenoterapia convencionales de bajo flujo que generen menos riesgo de aerosolización, fuerte a favor se podría considerar cánulas de alto flujo de oxígeno y/o la ventilación mecánica no invasiva (con una máscara facial adecuadamente ajustada y ramas inspiratorias y espiratorias separadas) como terapia de puente después de la extubación, pero se deben brindar las condiciones estructurales necesarias (habitaciones de presión negativa o habitaciones aisladas de puertas cerradas) y con epp estrictos para el personal sanitario. fuerte a favor fundamento las pautas de anzics establecen que la caf y/o la ventilación no invasiva (con una máscara facial bien ajustada y ramas inspiratorias y espiratorias separadas) pueden considerarse como terapia de puente después de la extubación, pero deben proporcionarse epp estricto en el aire. la terapia cpap o bipap (con alta presión espiratoria final) podría ser útil para prevenir la eliminación del reclutamiento en estos pacientes. en el momento de la extubación, los pacientes a menudo han estado enfermos durante más de una semana. es probable que su carga viral disminuya en ese punto, por lo que el riesgo de transmisión del virus puede ser menor (en comparación con la intubación inicial) ( ) . de no contar con predictores de que nos indiquen que podría fracasar la extubación se deben utilizar entonces sistemas de oxigenoterapia convencionales de bajo flujo que generen menos riesgo de aerosolización( ). se recomienda limpiar y desinfectar con frecuencia el área de retiro de epp, incluso después de que se haya completado cada procedimiento de eliminación. se debe limpiar esta zona, pasando de las áreas más limpias a las más sucias, antes de ingresar a la habitación del paciente y realizar el manejo y disposición final de residuos. se recomienda realizar la limpieza de superficies con un desinfectante adecuado o con una solución de hipoclorito sódico que contenga ppm de cloro activo (por ejemplo, un producto con hipoclorito en una concentración de - gr/litro, se hará una dilución : en el momento de su utilización). amci ® se recomienda que los recipientes que contengan los residuos deberán quedar en el lugar designado a tal efecto, que permanecerá cerrado hasta que, según el procedimiento de gestión de residuos de la institución sean retirados. los circuitos, filtros, succión cerrada y tot deben ser dispuestos en bolsas de color rojo las cuales deben ser de polietileno de alta densidad de . milésimas de pulgada y deben contar con un rótulo donde se indiquen: el nombre del generador, las palabras residuos biolÓgicos (covid- ) . una vez dispuesto, apretar y asegurar con nudo la bolsa de residuos y remover la bolsa de residuos del recipiente de residuos. posteriormente, desinfectar el exterior de la bolsa con solución desinfectante. luego colocar la bolsa de residuos en otra bolsa adicional de residuos y apretar y asegurar con nudo la bolsa de residuo. finalmente desinfectar la exterior bolsa de residuos con solución desinfectante. una vez terminada la disposición de los residuos de extubación, desinfectar los guantes con que manipuló los residuos con solución desinfectante y ubicar la bolsa de residuos dentro del vehículo de recolección interna de residuos. finalmente desinfectar el exterior de la bolsa de residuos con solución desinfectante. una vez terminada la disposición de los residuos de extubación, desinfectar los guantes con que manipuló los residuos con solución desinfectante y ubicar la bolsa dentro del vehículo de recolección interna. acogerse a la ruta sanitaria que asegure el menor riesgo de contaminación en el traslado interno de los residuos en la habitación del paciente (zona limpia) y zona sucia, se debe garantizar la ubicación de recipiente plástico de color rojo, liviano, resistente a los golpes, en material rígido impermeable, de fácil limpieza, y resistentes a la corrosión. los recipientes deberán ser lavados y desinfectados de acuerdo con los procedimientos establecidos por el prestador de servicios de salud( - ). se recomienda utilizar un ajuste de peep del paciente crítico por covid- , basado adicional a la tabla de peep, en las condiciones clínicas del paciente, en los índices de oxigenación, en la mecánica respiratoria del paciente y en los métodos de monitoreo disponibles. se recomienda titular la peep más alta que mantenga o mejore la relación safi y permita una presión plateau ≤ cmh o. se recomienda utilizar otras estrategias de titulación de peep probadas y con las cuales el equipo de trabajo esté familiarizado, dependiendo de la disponibilidad del recurso: ensayo peep decremental precedido por una maniobra de reclutamiento; titulación mediante la amci ® estimación de la presión transpulmonar con catéter esofágico o tomografía de impedancia eléctrica. fuerte a favor fundamento la titulación de la peep debe hacerse en función de la distensibilidad, oxigenación, espacio muerto y estado hemodinámico. puede titularse la peep mediante la estimación de la presión transpulmonar con catéter esofágico o tomografía de impedancia eléctrica. podría también titularse a partir de la fórmula (dp=plateau-peep) teniendo en cuenta que sea lógico el acoplamiento matemático fisiológico (lo que resultaría en una peep de cmh o si la presión plateau es de cmh o). la titulación de la peep requiere consideración de los beneficios (reducción de atelectrauma y mejora del reclutamiento alveolar) frente a los riesgos (sobre distensión inspiratoria final que conduce a lesión pulmonar y mayor resistencia vascular pulmonar)( , , ). se recomienda aplicar los protocolos de rehabilitación física como estrategia beneficiosa en el tratamiento respiratorio y físico de pacientes críticamente enfermos por covid- . se recomienda realizar la movilización precoz del paciente críticamente enfermo por covid- durante el curso de la enfermedad siempre que sea posible hacerlo de forma segura, asegurando la protección personal del personal sanitario. derivado del tratamiento médico intensivo para algunos pacientes con covid- , incluida la ventilación pulmonar protectora prolongada, la sedación y el uso de agentes bloqueantes neuromusculares, los pacientes con covid- que ingresan en la uci pueden presentar un elevado riesgo de desarrollar debilidad adquirida en la uci empeorando su morbilidad y mortalidad. por lo tanto, es esencial la rehabilitación temprana después de la fase aguda del síndrome de distrés respiratorio agudo (sdra) para limitar la gravedad de la debilidad adquirida en uci y promover la recuperación funcional. según la guía de la oms y la ops, enfatizan extremar el uso de los elementos de protección personal (epp) durante las intervenciones de rehabilitación física. la rehabilitación física proporciona intervenciones a través de movilizaciones, ejercicio terapéutico y programas individualizados a las personas que superan la enfermedad crítica asociada con covid- durante la ventilación mecánica y luego de esta, con el fin de permitir un retorno al hogar con funcionalidad. la prescripción de la movilización y ejercicio terapéutico debe de ser considera cuidadosamente en función del estado del paciente teniendo en cuenta, la estabilidad hemodinámica y clínica de la función respiratoria. cuando las movilizaciones, ejercicio terapéutico o programas de rehabilitación están indicados, debe realizarse una correcta planeación teniendo en cuenta amci ® la identificación/uso del personal mínimo necesario para realizar la actividad de manera segura. y el aseguramiento de todo el material que requerido esté a la mano y funcione correctamente y esté perfectamente limpio y desinfectado. si el material/equipo tiene que ser compartido con otros pacientes, límpielo y desinféctelo después de cada uso, entre paciente y paciente. se requiere personal entrenado específicamente para la limpieza y desinfección de los equipos, en una habitación aislada. y siempre que sea posible, evitar el traslado del material entre las áreas infectadas y no infectadas del hospital, manteniendo el equipamiento en las zonas aisladas ( ) ( ) ( ) ( ) ( ) . ( basados en un estudio preliminar aún sin publicar, se podría sugerir el uso de dexametasona a dosis de mg (oral o venosos) por días o hasta el alta si ocurre primero en pacientes hospitalizados con sospecha o diagnóstico de covid- que requieren suplencia de oxígeno, incluyendo aquellos con ventilación mecánica, que sean menores de años y con más de días de síntomas. amci ® actualmente no existe una terapia dirigida que se a efectiva para el manejo del virus; un número alto de estudios han surgido en los últimos dos meses, la mayoría sin el rigor metodológico suficiente para tomar decisiones adecuadas con respecto al manejo del paciente con infección por sars-cov- . el conocimiento en la estructura del virus y el mejor entendimiento en la fisiopatología de la enfermedad genera un sinnúmero de potenciales fármacos que han sido ensayados para el manejo de la enfermedad. en tiempos de pandemia, con una patología catastrófica en términos de vidas humanas y costos hospitalarios; es importante encontrar soluciones a desenlaces importantes como mortalidad, días de estancia en uci y en el hospital, aumento en los días libres del ventilador, disminución de complicaciones mayores debido a la enfermedad entre otros. hasta el momento no se ha documentado ninguna terapia específica que pueda impactar sobre estos desenlaces; pero la calidad de los trabajos, tampoco dejan claro sin él no usar ningún tratamiento específico mejora los desenlaces al menos al disminuir el número de complicaciones. este nuevo beta-coronavirus es similar al coronavirus del síndrome respiratorio agudo severa (sars-cov) y del síndrome respiratorio del medio este (mers-cov); por lo tanto, varias moléculas que habían sido evaluadas en este tipo de enfermedad rápidamente se abrieron paso a ensayos clínicos en paciente con covid- . estos ensayos principalmente observacionales, aleatorios pero abiertos con un número pequeño de pacientes no han permitido sacar adecuadas conclusiones y es frecuente como ver las diferentes guías de las principales sociedades del mundo cambiar de forma frecuente sus recomendaciones; no existes evidencia de estudios clínicos aleatorios y controlados que midan desenlaces fuertes, la premura de un tratamiento efectivo ha sacrificado el rigor metodológico que una investigación requiere. una estructura viral y replicación conocidas generan posibles dianas para que diferentes fármacos puedan ser investigados, antivirales tipo arbidol el cual inhibe la fusión de la membrana en la envoltura viral a algunos receptores; antimaláricos como la hidroxicloroquina y la cloroquina, las cuales inhiben la entrada viral y endocitosis por múltiples mecanismos, así como los efectos inmunomoduladores demostrados en el huésped; antivirales que impiden la replicación como el lopinavir o darunavir inhibiendo las proteasas o la ribavirina, el remdesivir o el favipiravir que actúan como análogos de nucleótidos o fármacos que actúan modulando la respuesta específica del huésped como el tocilizumab el cual se une al receptor de la il- inhibiendo el punto de acción de esta; los corticosteroides con múltiples efectos en la modulación del sistema inmunológico del paciente o los fármacos para evitar la respuesta secundaria a esta cascada inflamatoria como son los anticoagulantes. por último, se han buscado estrategias con el fin de mejorar la inmunización pasiva del huésped en el uso del plasma de pacientes convalecientes o el uso de inmunoglobulinas enriquecidas entre otros tratamientos propuestos para esta enfermedad. amci ® a los diferentes medicamentos que han sido usados en la pandemia del sars-cov- /covid- . de manera reciente en datos preliminares aún sin publicar horby y col en una rama del ensayo de evaluación aleatorizada de la terapia covid- (recovery), estudio aleatorizado, controlado, abierto que compara una gama de posibles tratamientos con la atención habitual en pacientes hospitalizados con covid- , compararon el uso de la dexametasona a dosis de mg día (oral o intravenosa) una vez al día por días o el alta según lo que ocurriera primero contra el manejo habitual; en pacientes aleatorizados que recibieron dexametasona se compararon con pacientes en manejo estándar; ( . %) pacientes en el grupo de dexametasona y ( . %) pacientes en el grupo control murieron a los días, con un riesgo relativo ajustado para la edad (rr . ; % ic . a . ; p < . ). la mortalidad relativa y absoluta variaron significativamente en relación al soporte ventilatorio al momento de la aleatorización; la dexametasona redujo las muertes en una tercera parte de los pacientes que recibieron ventilación mecánica invasiva ( . % vs. . %, rr . , % ic . a . ; p < . ), y una quinta parte en los pacientes que reciben oxígeno sin ventilación mecánica invasiva ( . % vs. . %, rr . , % ic . a . ; p = . ), pero sin reducir la mortalidad en paciente que no recibieron soporte respiratorio al momento de la aleatorización ( . % vs. . %, rr . , % ic . a . ]; p = . ) ( ) . no se emite recomendación a favor ni en contra para el inhibidor de la janus quinasa (baricitinib) en los pacientes con sospecha clínica o diagnóstico de covid- severo. uno de los reguladores conocidos de la endocitosis es la proteína quinasa asociada a ap (aak ); la interrupción de aak podría, a su vez, interrumpir el paso del virus a las células y también el ensamblaje intracelular de partículas del virus. uno de los seis fármacos de unión a aak de alta afinidad es el inhibidor de la janus quinasa (jak y jak ), llamado baricitinib, que también se une a la quinasa asociada a la ciclina g, otro regulador de la endocitosis ( ) . el baricitinib alcanza concentraciones plasmáticas suficientes para inhibir aak con mg o mg una vez al día; por su baja unión a proteínas plasmáticas y a su mínima interacción con las enzimas cyp, permite combinarlo con los antivirales. sin embargo, algunos piensan que el bloqueo de la señal jak-stat por baricitinib puede producir un deterioro de la respuesta antiviral mediada por interferón, con un posible efecto facilitador sobre la evolución de la infección por sars-cov- ; otras limitantes son la linfopenia (no dar si < cel./ mm ) y el aumento de la cpk. ( , ) . cantini y cols, en abril , en italia, administraron baricitinib a mg/día vía oral por semanas a pacientes con covid- moderado y los compararon con un grupo control; la terapia mejoró significativamente los parámetros clínicos, respiratorios y de laboratorio (pcr); ninguno de los pacientes requirió uci vs % del grupo control, sin eventos adversos. se amci ® trata de un estudio piloto de seguridad e impacto clínico en pacientes que no estaban en uci ( ) . ¿en pacientes hospitalizados con sospecha o diagnóstico de covid- el uso de n-acetil cisteína modifica el curso clínico de la enfermedad o genera beneficios en desenlaces clínicos de interés? basados en evidencia indirecta para el manejo del sdra y resultados observaciones en covid- , se podría utilizar el uso de n-acetil cisteina a dosis de mg/kg/día durante los primeros cinco días del sdra, aunque no se ha demostrado impacto en la mortalidad, su utilización parece relacionarse con una disminución significativa en la estancia en la unidad de cuidados intensivos y con disminución de los marcadores inflamatorios en pacientes con covid- . la severidad de la infección en covid- , en gran parte depende de la respuesta inmunológica de cada persona, sin embargo, se encuentran mecanismos fisiopatológicos de relevancia. sobreproducción de moco en vía aérea superior e inferior, que en parte explica la dificultad en la mecánica ventilatoria y los retos de ventilación en estos pacientes, la descarga desmedida de citoquinas proinflamatorias que se asocian a la falla multiorgánica y la coagulopatía asociada a la disfunción endotelial. esto mecanismos fisiopatológicos son comunes en el sdra, incluido los casos asociados a covid- ( ). amci ® enfermo con falla respiratoria aguda, la cual engloba falla respiratoria hipóxica (tipo ), falla respiratoria hipercápnica aguda (tipo ), sdra y lesión pulmonar aguda, se revisaron ensayos clínicos, más de pacientes. el análisis del grupo de n acetilcisteína intravenoso mostró una reducción de estancia en uci, de . días, con una heterogeneidad muy baja del %, con valoración de la evidencia calificada como de alta calidad y baja probabilidad de sesgo ( ) . en covid- , fue utilizada con recuperación completa en un caso severo de un paciente con déficit de glucosa fosfato deshidrogenasa (g pd), con control de la hemolisis y resolución del compromiso pulmonar. en pacientes sin déficit de g pd, también ha sido asociada a mejoría clínica y disminución significativa de los niveles de pcr y ferritina ( ) . en una revisión de costo efectividad nacional, se identificaron referencias, de ellos era revisiones sistemáticas de la literatura, dos de las cuales incluían metaanálisis (lu y zhang ), y fueron incluidos en la evaluación. estos estudios incluyeron información de ensayos clínicos que comparaban la aplicación de nac intravenosa frente a placebo o cuidado usual en pacientes con sdra. los tres estudios reportan como resultado de mortalidad rr de . con ic al % de . a . (lu ), rr de . con ic al % de . a . (lewis ) y rr de . con ic al % de . a . (zhang ) . para el tiempo de estancia en uci solo las revisiones con metaanálisis reportaron resultados, encontrando una diferencia de promedio de días de estancia de - . días con ic al % de - . a - . (lu ) y de - . días con ic al % de - . a - . (zhang ). una de las revisiones reportó que en ninguno de los estudios analizados se presentaron eventos adversos. no se encontraron resultados para los desenlaces de infección, sobreinfección, ni uso y tipo de antibiótico utilizado. en el análisis se encontró una reducción estadísticamente significativa de los días de estancia en uci de los pacientes que recibieron tratamiento con n-acetilcisteína intravenosa con dosis entre y mg/kg/día durante los primeros cinco días del sdra, en comparación con los pacientes que recibieron placebo o manejo usual. no se reportó diferencia estadísticamente significativa en la reducción de la mortalidad de los pacientes que recibieron nac. ( ) calidad de vida . ¿cómo podemos medir la calidad de vida, en los pacientes con covid- que egresan de la uci? se recomienda utilizar los marcadores disponibles de severidad y del riesgo de mortalidad por covid- en los pacientes internados en la uci. amci ® las secuelas inmediatas en los pacientes víctimas del devastador ataque sistémico del covid- durante su estancia en la uci son valorables, pero no se dispone de herramientas que permita medir el grado de afectación de la calidad de vida de estos pacientes posterior al egreso de la uci o de alta hospitalaria, por lo tanto, se sugiere realizar estudios de creación, validación y utilización de instrumentos de valoración de la calidad de vida en pacientes con covid- posteriores al alta hospitalaria. parte importante de los pacientes con diagnóstico de covid- que ingresan a la uci, evolucionan tórpidamente presentando deterioro progresivo de los diferentes órganos llegando en pocos días a una falla multiorgánica ( ) , estos pacientes presentan características clínicas y de laboratorio que se relacionan de manera significativa con mayor severidad y riesgo de mortalidad ( , ) . a pesar de conocer con alguna precisión el riesgo de severidad y mortalidad de los pacientes que ingresan a la uci, no disponemos de un score que nos permita evaluar y predecir el grado de afectación en la calidad de vida de los pacientes que logran sobrevivir. aproximadamente un , % del total de pacientes con enfermedad por covid- ingresan a uci, y de estos , % sometidos a ventilación mecánica ( ); lamentablemente los pacientes con enfermedad severa que logran sobrevivir y recuperarse han sido sometidos a una larga estancia en la uci y a ventilación mecánica invasiva con una intubación prolongada, que puede producir disfunción en la deglución impidiendo a la persona alimentarse de forma correcta y segura. es importante diagnosticar esta disfagia en los pacientes que se están recuperando del covid- y tratarla correctamente desde el principio para evitar complicaciones importantes como la malnutrición y la deshidratación, así como el riesgo de neumonía aspirativa. además de la disfagia, la fibrosis pulmonar y el riesgo de trombos son los problemas más frecuentes, pero no los únicos. una de las características de la enfermedad severa por covid- es que el virus provoca una enfermedad multiorgánica, con un amplio y heterogéneo abanico de secuelas cuyo alcance todavía se desconoce y aunque el órgano más afectado es el pulmón, puede afectar también otros órganos o sistemas incluido el snc, que en los casos más graves puede presentar encefalitis, delirios, desorientación y confusión, síntomas que pueden persistir tras el alta de la uci. otra secuela frecuente son las polineuropatías, esta afectación suele comenzar con una sensación de hormigueo en las extremidades y en los pacientes con covid- se presenta además con un cuadro de miositis que provoca debilidad y cansancio al caminar, a veces incluso en reposo; en algunos pacientes se presenta tal debilidad que dificulta llevar el alimento a la boca e incluso deglutirlo. sin embargo, la primera y más frecuente de las manifestaciones neurológicas del covid- es la pérdida del olfato, que a veces perdura como secuela tiempo después del alta. un estudio en pacientes ingresados en el hospital clínico san carlos de madrid revela que el % había sufrido anosmia en mayor o menor grado. la importancia de este síntoma radica en que las fosas nasales pueden ser la vía de acceso del virus al sistema nervioso central. amci ® otras posibles secuelas neurológicas asociadas a la infección por covid- son la ageusia, la cefalea y amnesia a corto y mediano plazo. también son importantes las secuelas que afectan al sistema cardiovascular. un estudio publicado en la journal o the american medical association advierte que un % de los pacientes presenta una elevación de las enzimas que indican daño en el miocardio. la inflamación que provoca el virus puede provocar directamente ese daño y también puede agravar el estado de pacientes que ya tengan una patología cardiovascular de base, muchas miocarditis son reversibles, pero hay una parte importante que deja como secuela una pérdida de la función contráctil. todavía se desconoce el alcance y es difícil medir el impacto de la enfermedad sobre el corazón porque en algunos casos, los síntomas de insuficiencia cardíaca se confunden con los de la neumonía. otra de las complicaciones más frecuentes, y potencialmente más grave, afecta al mecanismo de coagulación de la sangre. durante el ingreso hospitalario se han visto numerosos casos de ictus. la secuela más importante es el riesgo de que se formen trombos, que pueden ir al pulmón o al cerebro, y si se producen en las arterias, pueden dar lugar a un infarto, aunque este efecto es mucho menos frecuente. eso explica que algunos pacientes de covid- dados de alta hayan tenido que volver a ingresar por trombosis. finalmente es importante tener presente que a las afectaciones que haya podido producir el virus, hay que sumar las secuelas neurológicas propias de una estancia prolongada en una unidad de cuidados intensivos que también pueden ser graves y a veces no se distinguen bien unas de otras. debilidad muscular, desorientación, depresión y problemas psicológicos son secuelas muy habituales entre los pacientes que salen de la uci por enfermedades diferentes. por la anterior razón es difícil, por ahora, saber qué es efecto directo del virus y que puede derivarse del proceso de hospitalización. aún es difícil decir si los daños a largo plazo dependen del propio virus o de los efectos adversos del proceso tratante. sin embargo, este análisis de las posibles secuelas del covid- en el cuerpo, se presenta con más dudas que certezas. como es habitual en medicina, las causas pueden ser múltiples y muchas veces reflejan la participación de varias complicaciones que se han podido dar durante el proceso infeccioso directo o por la hospitalización, la información sobre los mecanismos de invasión del sars-cov- en todos los órganos sigue siendo, por ahora, escasa. y también lo es nuestro conocimiento sobre los efectos adversos de los medicamentos, muchos de ellos experimentales, que se han utilizado durante esta crisis. gran parte de lo que conocemos actualmente sobre los efectos de este virus proviene de la experiencia clínica de otros colegas y de las historias de pacientes que han sufrido la enfermedad, quedando todavía mucho por descubrir. dado el actual panorama, diferentes hospitales e instituciones de salud se preparan en torno a la rehabilitación, habilitando ya unidades multidisciplinares poscovid- para el seguimiento de estos pacientes y algunos centros están contactando con los pacientes dados de alta para evaluar su estado y hacer un seguimiento a su salud. también están en marcha varios estudios multicéntricos para evaluar el alcance de las secuelas, prácticamente todos los centros sanitarios deberán tener pautas de seguimiento y control para los pacientes ya dados de alta, creándose necesario la utilización simultánea de amci ® instrumentos de medición de calidad de vida, que en nuestro país colombia, ya se han utilizado previamente con este fin ( , ) . con este instrumento de medición de la calidad de vida se realizaron algunos estudios piloto tanto en pacientes crónicos como en la población general con el fin de determinar la comprensión del instrumento y factibilidad de aplicación del mismo en cuanto a la consistencia interna, la revisión realizada por vilagut y cols demostró que la aplicación de la escala arrojó en diversos estudios un alfa de cronbach igual o superior a . en todas las escalas excepto en función social ( ) . y aunque un número cada vez mayor de estudios mide los resultados físicos, cognitivos, de salud mental y de calidad de vida relacionada con la salud (cvrs) en los sobrevivientes adultos de la uci, los datos sobre las propiedades de medición de tales instrumentos son escasos y, en general, de calidad deficiente a justa. se necesitan análisis empíricos que evalúen el rendimiento de los instrumentos en adultos sobrevivientes de la uci para avanzar en la investigación en este campo ( ) . finalmente, el conocimiento de las secuelas y complicaciones dejadas por la infección del covid- , permitirá identificar importantes variables clínicas que acompañan a esta enfermedad y que afectan de manera importante la calidad de vida de los pacientes que padecen la enfermedad severa en la unidad de cuidados intensivos. en la actualidad no existen estudios para evaluar el riesgo de malnutrición aguda en pacientes hospitalizados por sars-cov- . experiencias con otras infecciones virales por influenza, se han identificado como factores asociados con mortalidad, la presencia de malnutrición, la adquisición de infección intrahospitalarias, la falla respiratoria y la presencia de infiltrados en la radiografía de tórax ( ) . las guías espen recomiendan utilizar el must o el nrs- ( ), para la tamización del riesgo nutricional, estos puntajes de tamización previamente han sido validados en múltiples patologías y contextos clínicos; sin embargo, existen otros puntajes útiles desde la perspectiva clínica como la valoración global subjetiva, mini-nutritional asessment( ), puntaje nutric ( ) y la global leadership initiative on malnutrition (glim) ( ) . el proceso de diagnóstico nutricional debe involucrar, dos componentes: la identificación del riesgo con la utilización de alguno de los puntajes previamente validados en otros contextos y posteriormente el diagnóstico de los pacientes con malnutrición y la valoración de la gravedad de ésta; en este último paso es importante la valoración del índice de masa corporal, los hábitos de consumo calórico y proteico, la presencia de inflamación, los trastornos gastrointestinales, las enfermedades de base y siempre que sea factible el cálculo de la masa muscular. tabla . en vista del riesgo de infección al personal de salud, no siempre será necesario la visita nutricional al paciente, ésta podría ayudarse con entrevista al familiar, interrogatorio vía amci ® telefónica y sólo en caso necesario el examen del paciente para lo cual se requiere el uso de equipo de protección personal completo. esta estrategia de interrogatorio al familiar o al paciente por vía remota o telefónica puede ayudar a identificar los patrones de consumo y los hábitos nutricionales de riesgo y en caso de ser necesario la valoración nutricional disminuye el tiempo de exposición a un ambiente contaminado. para la atención presencial de pacientes en el ámbito de cuidado intensivo, es necesario definir cuál es el riesgo que existe de infección para el personal de salud, para aclarar esta pregunta se debe definir si hay un riesgo de generación de aerosoles( ). aunque no existen pautas específicas para la nutrición en pacientes con covid- , las diferentes sociedades científicas han desarrollado guías de pauta clínica para la nutrición de pacientes con esta enfermedad ( , ) . idealmente la nutrición debe ser iniciada de forma temprana, esto se refiere al inicio en las primeras a horas del ingreso a cuidado intensivo o en las primeras horas luego de la intubación y el inicio de la ventilación mecánica( ) y se prefiere la vía enteral. aunque no existen estudios para evaluar el momento del inicio de la nutrición en pacientes con infección por sars-cov , el inicio temprano de la nutrición ha mostrado beneficios en términos de mortalidad y reducción de infecciones con dicha estrategia ( , ) . adicionalmente es importante, evaluar el riesgo de morbilidad y mortalidad asociado a la malnutrición aguda en el ámbito del paciente crítico, en los pacientes que no se alcance la meta de aporte calórico y proteico por vía enteral o que exista contraindicación para ésta, se debe considerar el inicio de nutrición por vía parenteral, especialmente cuando su riesgo nutricional agudo sea elevado (puntaje nutric ≥ , nrs ≥ ) ( ) ( ) . el choque no es una contraindicación para la utilización de nutrición enteral ( ) y no es una indicación para el uso de nutrición parenteral, quizás la mejor estrategia, es vigilar la presencia de disfunción gastrointestinal, en combinación con la presencia de intolerancia a la nutrición enteral, especialmente en pacientes con acidosis láctica en progreso y cuando sea necesario escalar la dosis de vasopresores o exista incapacidad para la reducción de éstos. no es necesario medir el residuo gástrico de rutina, es preferible iniciar procinéticos de forma rutinaria. la sonda debe colocarse con cuidado de evitar riesgo de contaminación, preferiblemente al entubar al paciente. algunos pacientes pueden presentar diarrea, ya que se ha descubierto la presencia de la proteína ace (receptor del virus sars-cov- ) en células del esófago, estómago, duodeno y recto. no existe evidencia que indique que la nutrición enteral durante la posición prono aumente el riesgo de complicaciones. sugerimos no suspender nutrición enteral al durante la pronación, se debe iniciar con dosis trófica de ml/h. amci ® se recomienda una estrecha monitorización de la tolerancia a la nutrición enteral para pacientes en posición prono. se recomienda para aumentar la tolerancia de la ne a los pacientes en posición prona, una elevación del tórax entre - º (posición de trendelenburg inversa) no realizar endoscopias digestivas para ubicación de sondas avanzadas recomendaciones de nutrición parenteral los pacientes con covid- pueden requerir niveles significativos de sedación y bloqueo neuromuscular, lo que puede aumentar la incidencia de intolerancia gastrointestinal. la nutrición parenteral (np) debe utilizarse donde la alimentación enteral no está disponible o no logra completar el % de los requerimientos. si existen limitaciones para la ruta enteral, se podría recomendar nutrición parenteral periférica (npp) en la población que no alcanza el objetivo proteico energético por nutrición oral o enteral. la np temprana debe 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has garnered global attention. the aim of this article is to overview available evidence on the use of widely used antirheumatic drugs hydroxychloroquine, methotrexate and colchicine for additional indications. hydroxychloroquine has endothelial stabilizing and anti-thrombotic effects. its use has been explored as an adjunctive therapy in refractory thrombosis in antiphospholipid syndrome. it may also prevent recurrent pregnancy losses in the absence of antiphospholipid antibodies. hydroxychloroquine favourably modulates atherogenic lipid and glycaemic profiles. methotrexate has been tried for modulation of cardiovascular events in non-rheumatic clinical conditions, although a large clinical trial failed to demonstrate a benefit. colchicine has been shown to successfully reduce the risk of recurrent cardiovascular events in a large multicentric trial. potential antifibrotic effects of colchicine require further exploration. hydroxychloroquine, methotrexate and colchicine are also being tried at different stages of the ongoing coronavirus disease (covid- ) pandemic for prophylaxis and treatment. while the use of these agents is being diversified, their adverse effects should be timely diagnosed and prevented. hydroxychloroquine can cause retinopathy and rarely cardiac and auditory toxicity, retinopathy being dose and time dependent. methotrexate can cause transaminitis, cytopenias and renal failure, particularly in acute overdoses. colchicine can rarely cause myopathies, cardiomyopathy, cytopenias and transaminitis. strong evidence is warranted to keep balance between benefits of repurposing these old antirheumatic drugs and risk of their adverse effects. rheumatologists are at the forefront of research and practice of using various anti-inflammatory and immunomodulatory drugs. their decades-long clinical experience can be valuable at the time of drug repurposing and attempting to use widely tested antirheumatic drugs for newer indications [ ] . numerous immunomodulatory drugs have been employed to induce remission of autoimmune disease and prevent adverse effects of concomitant high-dose corticosteroid and other synthetic disease-modifying anti-rheumatic drug (dmard) therapies, particularly in elderly patients and those with comorbidities [ ] . although precise mechanisms of action of most old antirheumatic drugs remain unknown, these are exemplified for their relative safety and utility, allowing corticosteroid dose tapering and long-term disease control. a scopus search conducted on th august of the number of publications mentioning these drugs to this day revealed an ongoing interest in these drugs in the published literature. overall, , documents are retrievable for methotrexate, , for colchicine, and , for hydroxychloroquine (fig. ) . the repurposing of traditionally used dmards has garnered significant attention recently with the advent of the coronavirus disease pandemic [ ] . this opinion article aims to sensitize readers towards the potential utility of these drugs in cardiovascular disease and fibrosis (both of which have been reported as sequelae of [ ] [ ] [ ] as well as serve as a reminder of their adverse effect profile. such potential adverse effects need to be kept in mind by specialists who do not routinely use these drugs when they are explored for other indications. in this article we overview some of the effects of hydroxychloroquine, methotrexate and colchicine and reflect on their positive and negative sides to guide repurposing of drug therapies. the choice of articles for review reflects the authors' understanding and viewpoints regarding the potential therapeutic roles of these drugs. hydroxychloroquine (hcq), a relatively safe derivative of chloroquine, is effectively used for the treatment of systemic lupus erythematosus and other inflammatory rheumatic diseases. it acts by accumulating in lysosomes, where its basic ph modifies the normally acidic milieu. this interferes with the loading and presentation of antigens by class ii major histocompatibility complex (mhc) protein. also, it interferes with the activation of toll-like receptors (tlr) by deoxyribonucleic acid (dna) and ribonucleic acid (rna) moieties [ ] . the use of hcq reduces the risk of thrombosis and diminishes vascular inflammation and endothelial dysfunction in a murine model of antiphospholipid syndrome (aps) [ ] . in patients with antiphospholipid antibodies (apl) fig. annual publication activity in scopus as of august , with the keywords "methotrexate", "colchicine" and "hydroxychloroquine" for articles published up to . graph might appear to dip for some entries as the year is still ongoing. the graph seems to suggest that there is still ongoing interest in these older disease-modifying antirheumatic drugs treated with hcq, levels of soluble tissue factor reduced after three months of hcq therapy. however, other markers of thrombogenic potential, such as annexin activity and anti-domain immunoglobulin g activity, and complement activation did not differ [ ] . in patients with systemic lupus erythematosus (sle) from italy ( positive for apl) followed up over a median of years, there was a markedly reduced risk of vascular events in those treated with hcq for five years (hazard ratio [hr] . , % confidence interval [ci] . - . ) [ ] . another retrospective study analysed insurance databases in taiwan ( patients with sle on hcq during first year compared with a group of sle patients not on hcq during the same period) [ ] . over a mean period of . years, hcq therapy was associated with a small but non-significant reduction in the risk of vascular events (hr . , % ci . - . ). the data was limited due to the lack of data about apl for these patients [ ] . a multicentric randomized controlled trial (rct) reported patients with apl positivity in the absence of any systemic autoimmune disease who received either hcq or placebo. over a mean of . years of follow-up, neither group developed thrombotic events, resulting in premature termination of the trial and a subsequent inability to draw meaningful conclusions [ ] . another recent open-label study in greece with primary aps treated with standard anticoagulation regimen (with or without antiplatelets) randomized patients to receive either hcq in addition to standard care or standard care only. over mean . years follow-up, those on hcq had a lower risk of incident thrombosis, which did not remain significant (hr . , % ci . - . ) after adjusting for confounders. possibly, a larger sample size might have attained results with statistical significance [ ] . the european league against rheumatism (eular) recommends hcq therapy (based on expert opinion and anecdotal evidence) for patients with aps and recurrent pregnancy loss despite optimal doses of anticoagulation and antiplatelet agents during pregnancy [ ] . various ongoing clinical trials are evaluating the role of add-on hcq in aps. the hibiscus multicentre multinational trial is evaluating the effect of hcq ( mg daily for the duration of pregnancy) compared to placebo, in addition to standard therapy (i.e. preventative dose of low molecular weight heparin with aspirin), on live births in women with primary aps. a related study, the hibiscus-t trial, is evaluating the preventative role for recurrent thrombosis in patients with thrombotic aps treated with hcq or placebo in addition to oral anticoagulation with vitamin k antagonists for months [ ] . the hypatia trial is another multicentric trial spanning multiple european nations assessing the effect of hcq in a placebo-controlled rct involving patients with either aps or persistent apl positivity. this trial intends to assess the impact of hcq, started before pregnancy and continued for months, on pregnancy morbidity associated with apl, i.e. a composite of early pregnancy losses, pre-term delivery (< weeks) or placental insufficiency [ ] . another french multicentric trial, the hydrosapl trial, is also assessing the effect of hcq on pregnancy outcomes in primary aps [ ] . apart from aps, hcq is also being explored for its potential utility in patients with recurrent pregnancy loss. in this french rct (the bbq trial), patients with recurrent pregnancy loss shall be treated with hcq mg daily or placebo starting pre-conception until the th week of pregnancy, with the objective of assessing whether treatment improves pregnancy outcomes in such patients [ ] . other cardioprotective effects of hcq, apart from reduction of thrombotic events, have also been proposed. in a model of induced myocardial infarction in rats, the administration of hcq was associated with reduced apoptosis in the infarcted myocardial tissue after weeks [ ] . in patients with rheumatoid arthritis (ra) and sjogren's syndrome, the use of hcq has been associated with a reduced risk of developing diabetes mellitus (table ) [ ] [ ] [ ] [ ] . in animal models of insulin resistance induced by a high fat diet, the addition of hcq was associated with better survival of the insulin-producing islet of langerhans, as well as decreased release of inflammatory adipokines and markers of endothelial stress. thus, a beneficial effect of hcq on mechanisms driving insulin resistance has been postulated [ ] . in a small proof-of-concept study of patients with type diabetes mellitus (n = ) inadequately controlled with a combination of metformin and sulfonylurea, the addition of hcq mg daily was compared with pioglitazone mg daily. although pioglitazone was more effective in reducing levels of glycated haemoglobin and achieving glycaemic control, nearly two-thirds of the patients on hcq could attain less than . % level of glycated haemoglobin [ ] . another small clinical trial of patients with pre-diabetes to receive hcq (n = ) or placebo (n = ) demonstrated improvements in glycaemic control over months hcq therapy [ ] . these studies suggest the potential for hcq as an add-on agent for diabetes mellitus. however, until the utility of hcq is proven for the indications of diabetes mellitus, dyslipidemia or cardiovascular disease, its indiscriminate use for these indications should be avoided [ ] . considering the evidences for anti-platelet and anti-thrombotic actions of hcq, as well as favourable lipid profile in patients treated with hcq, it is possible that this drug might have an adjuvant role in the prevention of incident or recurrent cardiovascular events. this might need exploration in future clinical trials [ ] . methotrexate is the first-line dmard for the management of inflammatory arthritides such as rheumatoid arthritis and psoriatic arthritis. the drug's anti-folate mechanism of action is historically implicated in the treatment of neoplastic (lymphoblastic) diseases. the immunosuppressive action of low-dose methotrexate therapy (up to mg/week) primarily relies on the inhibition of the enzyme -aminoimidazole- -carboxamide ribonucleoside (aicar) transformylase (atic), resulting in higher levels of aicar that inhibits adenosine monophosphate deaminase and adenosine deaminase. the latter results in higher extracellular levels of adenine, further converted to adenosine, which acts via adenosine receptors to exert antiinflammatory actions. further, inhibition of the enzyme dihydrofolate reductase (dhfr) also results in uncoupling of nitric oxide (no) synthetase, by increasing levels of dihydrobiopterin and tetrahydrobiopterin, resulting in greater release of no as opposed to reactive oxygen species. methotrexate also exerts an effect to decrease downstream inflammatory signalling operating via nuclear factor kappa b (nfκb). while supplementation with folic acid antagonizes the anti-folate action of methotrexate (responsible for most of its side effects), it does little to inhibit other anti-inflammatory pathways [ ] . recently accumulated evidence suggests a favourable modulation of cardiovascular risk in patients with rheumatic diseases treated with methotrexate [ , ] . a pilot study evaluated the safety of methotrexate therapy ( mg/week) in patients with coronary artery disease undergoing percutaneous coronary intervention (pci), started weeks prior to the procedure and continued for weeks after the procedure. no major safety signals were observed during this period [ ] . a rct, the tethys trial, evaluated the role of methotrexate in patients with st segment elevation myocardial infarction (stemi). methotrexate administered at a dose of . mg/ kg, repeated h after pci in patients, was compared to patients receiving placebo. this trial aimed to analyse area under the curve for serial measurements of creatine kinase and troponin i over the first h after stemi as a surrogate for area of infarct. no significant differences in the area of infarct measured either using creatine kinase or troponin i could be demonstrated between the two groups. a matter of concern was that the left ventricular ejection fraction (lvef) was significantly lesser in patients treated with methotrexate compared to those receiving placebo, three months after completion of the treatment [ ] . another large multicentric rct, the cirt trial, evaluated the role of methotrexate in the secondary prevention of cardiovascular events. in this trial, patients who had a prior myocardial infarction or had triple vessel coronary artery disease on a background of diabetes mellitus or metabolic syndrome were randomized to receive methotrexate - mg/week ( patients) or placebo ( patients). the primary endpoint was a composite of first occurrence of a major adverse cardiovascular event (non-fatal myocardial infarction, stroke, or death due to cardiovascular cause), later modified to include hospitalization for unstable angina resulting in a revascularization procedure. the results of this trial did not favour the use of methotrexate, with a small but insignificant reduction in the risk of developing the primary end point with methotrexate (hr . , % ci . - . ). apart from a higher incidence of non-basal cell carcinoma in the group receiving methotrexate, no other significant safety signals were observed [ ] . these studies do not seem to suggest a role for methotrexate in the prevention of cardiovascular events. an ongoing study (nct ) is evaluating the targeting of left ventricular remodelling following stemi with a nano-emulsion containing methotrexate. it remains to be seen whether such a strategy is useful to favourably modulate cardiovascular events. colchicine has been used for the management of gout, familial mediterranean fever (fmf) and behcet's disease for decades [ ] . it acts predominantly on neutrophils, monocytes and macrophages, decreasing their chemotaxis and release of various inflammatory cytokines. it interferes with the activation of the nlrp- inflammasome complex, thereby reducing the secretion of downstream cytokines such as il- ß and il- [ , ] . knocking out the expression of nlrp- has been shown to reduce atherosclerosis as well as dampen the severity of myocardial injury following myocardial infarction in animal models [ ] . in a murine model, the administration of colchicine improved survival and residual left ventricular function following myocardial infarction [ ] . in a model of atherosclerosis induced by high fat diet in rabbits, the administration of colchicine reduced the uptake of -fluodeoxyglucose in atherosclerotic plaques in the abdominal aorta. moreover, in those rabbits with an increased cholesterol level in the peripheral blood, the burden of atherosclerotic plaques was significantly lesser along with greater circulating high-density lipoprotein (hdl) cholesterol [ ] . in platelet-rich plasma from healthy volunteers, in-vitro treatment with colchicine significantly reduced platelet aggregation [ ] . refractoriness to colchicine when used in the longer term in disease states such as fmf has been associated with oxidative stress [ ] . several clinical studies have also assessed the modulation of cardiovascular risk by colchicine. in a parallelgroup observational study, patients with coronary artery disease (cad) were treated with standard care and compared with another patients also treated with colchicine . mg daily in addition to standard care. multivariable analysis revealed a significant effect of colchicine on the reduction of the volume of atherosclerotic plaques [ ] . in a double-blind placebo-controlled trial of patients with cad short-term colchicine therapy significantly reduced systemic inflammation and improved endothelial function [ ] . in another study, patients with acute coronary syndromes (acs) planned for pci were randomized to treatment with add-on colchicine (n = ) or just standard treatment (n = ); those on colchicine had lower levels of chemokines, suggesting a reduction in local inflammatory activity at the site of pathology [ ] . in the low dose colchicine for myocardial infarction (lodoco-mi) study, patients with acute myocardial infarction were randomized to receive colchicine or placebo in addition to standard care, and c-reactive protein (crp) levels were compared at month. there was no significant difference in the proportions of patients attaining crp levels lesser than mg/l amongst patients treated with colchicine or placebo [ ] . the ongoing lodoco trial is evaluating clinical cardiovascular outcomes in , patients with stable cad treated with add-on colchicine or placebo [ ] . the colin trial, evaluated treatment with add-on colchicine (n = ) compared to placebo (n = ) in patients with stemi. neither the primary outcome of difference in peak serum crp during hospital admission between the two groups, nor secondary outcomes for differences in other biochemical, clinical or imaging outcomes (assessed by echocardiography or magnetic resonance imaging) at month were successfully met [ ] . a large multicentric trial evaluated the role of add-on colchicine (n = ) compared to placebo (n = ) in patients following myocardial infarction. the trial assessed the composite end-point of cardiovascular death, successful resuscitation after cardiac arrest, stroke, recurrent myocardial infarction, or angina requiring an intervention for revascularization. followed up for a median . months, treatment with colchicine was associated with a reduced risk of the composite outcome compared to placebo (hr . , % ci . - . ). gastrointestinal side-effects were similar, however, a greater incidence of pneumonia was observed in patients receiving colchicine ( . % vs . % on placebo) [ ] . a recent systematic review also confirmed an atheroprotective effect of colchicine, with a significant reduction in future cerebrovascular ischemic events with colchicine (odds ratio . , % ci . - . ) as observed in six clinical trials [ ] . colchicine may also exert anti-fibrotic effects. clinical trials have demonstrated the potential role of colchicine in reducing the occurrence of atrial fibrillation following catheter ablation and cardiac surgeries [ ] . it has been postulated that the modulation of atrial fibrosis by colchicine is mediated by its effects on interleukin- and renin-related pathways [ ] . in a trial of patients undergoing coronary artery bypass grafting, perioperative treatment with colchicine was associated with reduction in post-surgical constrictive pericarditis [ ] . in a rat model of hypertension induced by nephrectomy, treatment with colchicine ameliorated both glomerular and interstitial fibrosis in the remaining kidney [ ] . in another animal model of renal injury induced by ureteric obstruction in rats, treatment with colchicine reduced both cortical fibrosis and tubulointerstitial injury [ ] . anecdotal reports also exist of the regression of retroperitoneal fibrosis in patients treated with colchicine [ ] . in ten patients with keloids of the ear, treatment with colchicine for a month prior to keloid excision (along with pressure therapy and intralesional corticosteroid therapy in some) was associated with lack of recurrence of the keloids, although long-term follow-up data were unavailable for a majority of patients [ ] . a potential role of colchicine in retarding the progression of oral submucous fibrosis has also been proposed [ ] . treatment with topical colchicine was also effective in reducing induced spinal epidural fibrosis in an experimental model in rats [ ] . in a clinical trial involving patients with liver cirrhosis of different aetiologies, patients were treated with standard treatment alone and another with add-on colchicine. followed up over a mean of . years, those treated with colchicine had a greater proportion of survival. biochemical tests revealed that colchicine therapy was associated with lower serum levels of pro-collagen iii peptide, a marker of fibrosis [ ] . another report described the combination of colchicine with ursodeoxycholic acid (udca) in patients with primary biliary cirrhosis who did not respond to udca alone. after a followup of years, two-thirds of patients continued to survive when treated with this combination treatment regimen [ ] . it is possible that the anti-fibrotic effects of colchicine are mediated by its inhibitory actions on cytotoxic t lymphocytes as well as stimulation of endogenous mechanisms to regress fibrosis such as collagenases [ ] . the potential antifibrotic effects of colchicine merit systematic evaluation by clinical trials in patients with systemic fibrosing diseases such as systemic sclerosis, or major organ fibrosis such as interstitial lung diseases. the covid- pandemic has swept the world. based on preliminary evidence, abnormal activation of the immune system and cytokine storm appear to be associated with disease severity. in this context, commonly used antirheumatic drugs are being explored in covid- [ ] . while hydroxychloroquine has been recommended for prophylaxis of high-risk contacts of covid- , there is little evidence at present to support this [ ] . occasional reports have even described patients on long term hydroxychloroquine therapy for rheumatic diseases who eventually developed covid- [ ] . overall, the level of enthusiasm regarding the use of hydroxychloroquine in covid- has dampened due to mixed, often negative preliminary results of trials [ ] . it has been hypothesized that the timing (early initiation) of hydroxychloroquine might play a role in protection against severe covid- , and the results of trials on chemoprophylaxis of covid- with hydroxychloroquine might help answer this question [ ] . in this context, it is necessary to mention that hydroxychloroquine (and its analogue, chloroquine) have been previously tried in other viral infections such as zika virus and ebola virus infections. while in-vitro efficacy had been observed, this did not translate into clinical efficacy, possibly due to the fact that the drugs were unable to attain a concentration in the endosome necessary for their anti-viral properties to take effect [ , ] . anecdotal reports have suggested reduced severity of covid- in patients on colchicine for other indications [ ] . another retrospective review of records of patients from an european centre compared patients treated with standard of care (varying combinations of hydroxychloroquine, lopinavir/ritonavir and corticosteroids) with patients treated with colchicine at . - mg/day in addition to standard of care. treatment with colchicine was associated with % reduction in hazard of death (hazard ratio . , % confidence intervals . - . ) [ ] . however, in another recent study, six out of seven children with paediatric autoinflammatory syndromes who developed covid- were on colchicine [ ] . ongoing clinical trials identified on a search on clinicaltrials.gov on august are summarized in table . the results of these trials shall help better understand the role of antirheumatic drugs in covid- in the coming times. it is increasingly being understood that recovery from covid- is prolonged. nearly one-half of patients with covid- from italy had persistence of symptoms, including fatigue and joint pains, after months of onset of clinical disease [ ] . neuro-cognitive disorders are also being described in covid- survivors, at least in part driven by the endothelial injury due to the infection [ ] . a significant proportion of patients with pneumonitis due to covid- are likely to develop residual pulmonary fibrosis [ , ] . covid- causes endothelial injury, and this might be responsible for long-term cardiovascular consequences of the disease, including a predisposition towards future cardiovascular events [ , ] . therefore, age-old rheumatic drugs should be explored in such select recovered populations. particularly, it can be hypothesized that the anti-fibrotic effects of colchicine might help in post-covid- fibrosis, including lung fibrosis. table summarizes side effects of the examined drugs. hcq entails the risk of toxicities in the long term [ ] . ocular toxicity occurs in the form of retinal damage. up to in patients might develop retinopathy after five years of using hydroxychloroquine, and this is often silent in the earlier stages [ ] . in the later stages, macular vision may be affected, resulting in maculopathy and vision loss. fundoscopy and perimetry are insensitive for picking up earlier changes of hcq retinopathy. recent recommendations suggest limiting the dose of hcq to mg/kg/day, since higher doses increase the risk of retinopathy after years [ ] . before initiation of hcq, baseline fundus evaluation with the macula assessment is recommended. if this is abnormal, more sensitive techniques such as spectral domain optical coherence tomography may be employed and repeated annually [ ] . other adverse effects hcq-related skin pigmentation due to sun exposure, myopathy, and conduction blocks [ ] . cardiac toxicity may be potentiated by other drugs resulting in prolongation of the qt interval such as azithromycin [ ] . rare instances of sensorineural hearing loss have also been noted [ ] . in the context of the ongoing covid- pandemic, potential cardiac toxicity of hcq has received widespread attention. however, speaking from personal experience as well as the published literature in rheumatic diseases [ ] , cardiac toxicity with hcq (including qt prolongation) is very rare. pharmacological interaction with azithromycin can potentially prolong qt interval when these two drugs are taken together, as has been tried in the management of covid- . hence, due caution and pre-treatment with follow-up ecgs to seek any developing cardiac arrhythmias might be suggested if and when hcq is combined with azithromycin or any other drug that can cause qt prolongation [ ] . the adverse effect profile of methotrexate is mainly due to its antifolate actions [ ] . methotrexate toxicity can result in mucositis with oral ulcers, crusting of lips and diarrhoea, skin rashes, pancytopenia, transaminitis, and acute kidney injury [ ] . the risk of toxicities is greater in the presence of renal failure. in a series of patients with rheumatoid arthritis with concomitant renal impairment treated with methotrexate, nearly % developed features of methotrexate toxicity such as leucopenia, transaminitis and renal failure. interestingly, a protective effect towards methotrexate toxicity in the presence of renal failure was observed in those individuals also taking hydroxychloroquine [ ] . acute methotrexate toxicity requires discontinuation of the drug and administration of folinic acid. varying dose regimens of folinic acid are used, although often mg hourly for doses is administered. supportive treatment in the form of granulocyte colony stimulating factor, blood component transfusions, treatment of coexistent infections and nutritional care are also essential. untreated methotrexate toxicity is associated with significant risk of mortality [ , ] . rarely, methotrexate may result in acute interstitial pneumonitis [ ] . colchicine therapy can result in diarrhoea and other gastrointestinal adverse effects. long-term colchicine therapy is rarely associated with myopathy, polyneuropathy, transaminitis, and rhabdomyolysis [ ] . the risk of toxicity is increased in the presence of renal failure. also, coadministration with drugs inhibiting the enzyme cytochrome table adverse effect profile a risk of retinopathy generally occurs after years of use, and is related to cumulative doses. those with renal failure or on concomitant tamoxifen therapy are at greater risk b per se, cardiac toxicity is very rare when hydroxychloroquine is used in the context of rheumatic diseases c generally seen with methotrexate toxicity, such as in overdose (daily rather than weekly administration), or with concomitant underlying renal impairment p oxidase a (cyp a , such as clarithromycin) or the drug efflux protein p-glycoprotein portends a greater risk of adverse effects [ ] . occasional reports exist regarding the use of intravenous colchicine for prolonged periods for difficult to treat conditions such as familial mediterranean fever. despite intravenous use for longer term, little toxicity was observed in most patients, other than gastrointestinal side effects [ , ] . drug withdrawal is recommended if such toxicities develop [ , ] . rarely, these drugs can have cardiovascular side effects also, as holds true for most anti-rheumatic drugs: methotrexate has been associated with pericardial and myocardial injury, colchicine rarely causes myocardial injury, and hcq therapy occasionally results in conduction system abnormalities [ ] . clinicians should be aware for the risk of cardiac adverse events when these drugs are considered for repurposing and combined therapies in different patient cohorts. keeping in mind the diverse impacts on different body systems seen with these drugs, it is imperative to generate high-quality evidence for the use of hydroxychloroquine, methotrexate and colchicine for other indications. they are already being tried in patients with cardiovascular disease for secondary prevention. clinical trials might also attempt to decipher the potential utility of hydroxychloroquine, methotrexate or colchicine for primary prevention of cardiovascular events in a high risk population, such as those with metabolic syndrome and in individuals with inflammatory arthritides which predispose to greater cardiovascular risk. furthermore, the potential anti-fibrotic effects of colchicine need to be systematically studied in local fibrosing diseases (such as idiopathic interstitial lung diseases) or systemic fibrosing diseases (such as systemic sclerosis) by suitably powered clinical trials. although recent promising developments in vaccines for covid- hold promise for community level prevention, these are still preliminary and it is likely that millions of individuals shall be infected before such vaccines come into regular clinical use [ ] . therefore, the role of drugs with anti-fibrotic potential such as colchicine in preventing or managing long-term sequelae of covid- should also be explored. repurposing of old antirheumatic drugs is being attempted for numerous indications today. hcq, methotrexate and colchicine are primarily used for favourable modulation of cardiovascular risk. clinicians must be aware of the propensity of these drugs to cause side effects, such as cytopenias with methotrexate, retinopathy with hcq, and gastrointestinal adverse effects with colchicine. in view of the risk of adverse events, it is essential to carefully consider the risk-benefit ratio before prescribing these drugs for newer, unlicensed indications, taking particular care to identify high-risk populations such as those with underlying renal impairment and avoid drug interactions. the role of these drugs in covid- shall be clarified in ongoing trials. it may also require evaluation whether these drugs have a role in managing sequelae of covid- . drug 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with regard to jurisdictional claims in published maps and institutional affiliations key: cord- - pcnuqgy authors: morrisette, taylor; lodise, thomas p.; scheetz, marc h.; goswami, srijib; pogue, jason m.; rybak, michael j. title: the pharmacokinetic and pharmacodynamic properties of hydroxychloroquine and dose selection for covid- : putting the cart before the horse date: - - journal: infect dis ther doi: . /s - - - sha: doc_id: cord_uid: pcnuqgy coronavirus disease (covid- ), caused by the novel severe acute respiratory syndrome coronavirus (sars-cov- ), is currently responsible for a global pandemic. to date, only remdesivir and dexamethasone have demonstrated a positive response in a prospective, randomized trial for the treatment of patients with covid- . hydroxychloroquine (hcq) is an agent available in an oral formulation with in vitro activity against sars-cov- that has been suggested as a potential agent. unfortunately, results of randomized trials evaluating hcq as treatment against a control group are lacking, and little is known about its pharmacokinetic/pharmacodynamic (pk/pd) profile against sars-cov- . the objective of this review was to describe the current understanding of the pk/pd and dose selection of hcq against sars-cov- , discuss knowledge gaps, and identify future studies that are needed to optimize the efficacy and safety of treatments against covid- . the novel severe acute respiratory syndrome coronavirus (sars-cov- ), which is responsible for coronavirus disease (covid- ) , was first identified in a group of patients that had contracted pneumonia-like illness of unknown etiology in wuhan, china [ ] . despite variations in symptom severity, a subset of patients have been shown to progress to acute respiratory distress syndrome, often leading to mechanical ventilation and death [ ] . despite an impressive response by the medical community to identify therapies, the clinical efficacy of most potential antivirals remains uncertain [ ] . to date, remdesivir has shown a moderate response effect (faster time to recovery) for patients with covid- in a double-blind, randomized, placebo-controlled clinical trial (effect size greatest in those hospitalized patients requiring any supplemental oxygen); however, remdesivir is currently only available in limited rations and as an intravenous formulation, which may pose challenges [ ] . furthermore, dexamethasone (preliminary, unpublished data) has been shown to reduce -day mortality compared to usual care in hospitalized covid- patients receiving oxygen or invasive mechanical ventilation. theoretically, use of interleukin (il- ) inhibitors as immunomodulators and other immunomodulatory agents may be promising in severe cases, but supportive care currently remains one of the cornerstones of therapy for covid- [ ] . hydroxychloroquine (hcq) is an antimalarial agent with in vitro antiviral activity, anti-inflammatory effects, and immunomodulatory actions [ ] [ ] [ ] . given its wide availability and in vitro activity against sars-cov- , hcq became one of the earliest and most studied therapies for the treatment of and prophylaxis against covid- . unfortunately, little is known about the appropriate dosage regimen(s) of hcq to optimize efficacy and safety against covid- , as current knowledge is primarily derived from hcq use in healthy volunteers and other non-covid- disease states (e.g., malaria, rheumatoid arthritis, etc.) [ ] [ ] [ ] [ ] [ ] . this is further highlighted by the fact that current enrolling clinical trials are utilizing different hcq dosing regimens. furthermore, there is considerable heterogeneity among studies regarding the pharmacokinetics (pk) sampling matrix (i.e., important because of varying concentrations depending on the matrix [blood versus plasma/serum]) and patient populations that have attempted to characterize hcq's pk/ pharmacodynamic (pk/pd) properties against sars-cov- . although studies completed thus far show variable results, arshad and colleagues performed a large multicenter, retrospective, observational analysis that evaluated patients hospitalized because of a covid- -related admission receiving hcq mg twice daily on day , followed by hcq mg twice daily on days to [ ] [ ] [ ] [ ] . when comparing hcq monotherapy (n = ) versus no treatment (n = ), in-hospital mortality was significantly lower in the hcq monotherapy group ( . % vs. . %). importantly, there was also significantly more steroid usage in the hcq monoptherapy group compared to the no treatment group ( . % vs. . %), which may have contributed to this mortality difference [ ] . furthermore, the world health organization discontinued the hcq arm in the solidarity trial because it showed ''little or no reduction in the mortality of hospitalized covid- patients when compared to standard of care'' (hcq dosed mg twice daily on day , followed by hcq mg twice daily for a total of days), and the food and drug administration revoked the emergency use authorization to utilize hcq for the treatment of covid- [ ] [ ] [ ] . given these critical gaps and inconsistencies in the literature, the objective of this review was to describe the current understanding of the pk/ pd of hcq for the treatment of covid- as it relates to the efficacy and safety of dosage regimens. the focus of this review is on initial therapy of hcq based on the most common treatment durations for covid- (when available). given the scarcity of literature on hcq and similarities in mechanisms of action and chemical structure, data describing chloroquine in vitro activity will also be reviewed. we also discuss gaps in the literature and considerations going forward regarding in vitro and in vivo analyses of hcq and other covid- treatment options and identify future pk/pd studies that are needed to optimize the efficacy, if possible, and safety against covid- . it is important to note that the authors' primary goal was to summarize and evaluate the pk/pd of hcq to help clinicians in this challenging time. the summaries described are not meant to advocate for or endorse the widespread use of hcq for the treatment of covid- . this article is based on previously conducted studies and does not contain any studies with human participants or animals performed by any of the authors. hydroxychloroquine is currently only available as an orally administered tablet, available in mg as the sulfate salt form (equivalent to mg base) [ ] . there are numerous pk studies on the bioavailability of the oral tablet. however, most are in healthy volunteers or in patients with disease states other than covid- [ ] [ ] [ ] [ ] [ ] . most studies have concluded that hcq peak concentrations are estimated to be observed within - h [ , , ] . in healthy males who received a single hcq mg oral dose, a mean peak blood hcq concentration of . mcg/ml was achieved in . h, while a peak plasma hcq concentration of . mcg/ ml was achieved in . h. tett and colleagues performed a randomized, crossover study in which the hcq mg oral tablet was compared to an intravenous infusion of racemic hcq mg to evaluate the absolute bioavailability of the commercially available hcq tablet. these authors concluded that the mean (± sd) fraction of the oral dose absorbed (estimated from urine and blood) was . (± . ), while a wide range of absorption was calculated from plasma data [ , ] . despite the lack of data and recommendations, current centers around the world have described crushing the hcq tablets into suspensions and administering them via feeding tubes in patients otherwise unable to take oral medications, despite the institute for safe medication practices (ismp) listing hcq filmcoated tablets on the ''do not crush'' medication list [ [ ] , direct communications]. although this approach is commonplace in some inpatient centers among covid- patients, there are no data on the impact of crushing hcq tablets, administration via feeding tubes, and overall bioavailability or the timing of absorption. given the uncertainty in pk with this approach, this further emphasizes the importance of understanding and optimizing pk and pd of hcq against sars-cov- . most studies have shown that the binding of hcq to protein is moderate (* %) [ , ] . albumin and alpha -acid glycoprotein have been the two proteins associated with the majority of hcq binding. hcq exists as (r)-and (s)-enantiomers, and stereoselective protein binding has been documented [ ] . it has been shown that hcq exhibits linear pk [ ] . due to hcq's sequestration in deep tissues, the volume of distribution (vd) that hcq displays is extremely large. tett and colleagues reported a blood and plasma vd of l and , l, respectively, following intravenous hcq infusion in healthy volunteers [ ] . hcq and chloroquine, which show similar patterns of tissue distribution, have been shown to concentrate quite highly in the lungs, kidney, liver, and spleen in animal models [ ] . maisonnasse and colleagues found that hcq concentrations in the lung were higher than in plasma, with lung:plasma ratios ranging from to in macaques [ ] . notably, these data may very well be quite different in humans, and lung:plasma ratios could be lower because of differences in the metabolic composition and lower drug recovery rates. cyp enzymes catalyze the dealkylation of hcq to pharmacologically active metabolites, and hcq/chloroquine has been documented to be metabolized through cyp a, d , and c systems. the metabolism of hcq leads to the three active metabolites, desethylhydroxychloroquine, desethylchloroquine, and bisdesethylhydroxychloroquine, although they have been shown to increase more significantly following chronic administration. it is anticipated that there are lower levels of these active metabolites present in the initial days of therapy for covid- patients, and it is unclear how the various concentrations of active compounds translate to overall activity against sars-cov- . limited research has been conducted on investigating the association between genetic polymorphisms in cyp a, d , and c and hcq drug concentration levels [ ] [ ] [ ] [ ] [ ] . little is known about the role of membrane transporters on hcq pk/pd. there is literature that suggests that hcq inhibits uptake activity of human organic anion transporting polypeptide a [ ] . also, hcq/chloroquine has been shown to be an inhibitor of p-glycoprotein [ ] . reports have described a median of * % of hcq being excreted renally as unchanged drug in humans [ , ] . urinary elimination of hcq as metabolites and unchanged drug has been reported to be between % and % [ , , ] . lim and colleagues reported hcq clearance to be . l/h (two-compartment model best described these data). most reports have described a terminal elimination half-life of - days (in contrast to a terminal blood halflife reported by carmichael and colleagues of . h) [ , ] . using plasma data following administration of the oral hcq tablet, tett and colleagues calculated a mean (± sd) terminal elimination half-life of (± ) days [ ] . hydroxychloroquine/chloroquine mechanism, concentration, and in vitro inhibitory activity against sars-cov- although the mechanism of action of hydroxychloroquine and chloroquine against sars-cov- has not been fully elucidated, it is thought that these agents may prevent terminal glycosylation of its functional entry receptor (angiotensin-converting enzyme receptor), thus inhibiting viral entry [ , ] . furthermore, it has been shown that these agents can alkalinize intracellular compartments through incorporation of lysosomes and endosomes, leading to inhibition of viral replication and infection [ ] . the majority of in vitro analyses of hcq and chloroquine have utilized vero cell lines (table ) yao and colleagues performed an in vitro evaluation of hcq and chloroquine against sars-cov- -infected vero cells at a moi of . for h, followed by treatment concentrations for or h. the authors concluded that hcq was more potent than chloroquine, with ec values of . lm and . lm, respectively, at h and ec values of . lm and . lm, respectively, at h. furthermore, hcq was shown to exhibit a superior antiviral effect compared to chloroquine when cells were pretreated prior to viral challenge [ ] . an evaluation from maisonnasse and colleagues analyzed the in vitro activity of hcq against sars-cov- in veroe cells (moi: . ) and a model of reconstituted human airway epithelium (moi: . ). in veroe cells at and h, the half maximal inhibitory concentration (ic ) values were . lm and . lm, respectively. however, hcq at lm or lm was not shown to significantly reduce viral titers in the reconstituted human airway epithelium at h compared to untreated control [ ] . despite these in vitro evaluations, it is important to note that very little is known regarding the relevance of in vitro ec values in optimizing the pk/pd of hcq in humans. furthermore, depending on testing conditions, the reported studies have reported a * -fold range in ec values. finally, it is currently unknown which cell line is optimal for showing activity, and the discordant results between different cell lines introduce additional uncertainty in the relevance of these values. these limitations further emphasize the need to define the ''optimal target'' and how to correlate this target to efficacious hcq exposures. to date, limited pk/pd profiling studies have been performed to identify optimal dosage regimens against covid- in humans, and the exposures targeted in these assessments are of questionable relevance. in a study conducted by garcia-cremades and colleagues, translational pk/pd modeling was used to propose optimized hcq dosage regimens for initial treatment to ensure the highest likelihood of success (predict viral decline and risk of qtc prolongation). a published two-compartment population pk model for hcq was used to predict plasma concentrations for different dosing regimens of hcq ( mg, mg, mg, and mg twice daily for , , and days, with and without various loading doses, and all simulations included virtual patients that were simulated times). evaluating the same ec range as previously published ( . - . lm), the authors of this study performed simulations by fixing one of the reported ec values, with each hcq regimen simulated with each value times, to determine hcq's effect on viral replication. the authors found that the extrapolated hcq plasma concentration for the % viral inhibition value following simulations was . lm, and the authors' targets were hcq concentrations ''close or above the clinical ec values and below . lm'' for each simulation. importantly, however, the authors do not provide sufficient support that these exposures would lead to the proposed results, and the data do not support [ . lm as a toxic target. a mechanistic pk/virologic/qtc model for hcq was developed, and a simulation study was performed to predict viral decline and qtc prolongation. the authors concluded that hcq regimens [ mg twice daily for at least days were predicted to rapidly decrease viral loads, while regimens [ mg twice daily were predicted to prolong qtc intervals (because of levels [ . lm) [ ] . yao and colleagues used a physiologically based pk (pbpk) model for hcq to simulate lung fluid hcq concentrations under five hcq dosing regimens to evaluate the most effective and safe regimen. quantitative markers were ratios of estimated free lung tissue trough concentration to ec (r ltec ) based on human pk data and rat lung penetration data, which were compared to the ''efficacious'' chloroquine mg twice daily that was reported to improve outcomes by gao and colleagues [ , ] . importantly, whether or not chloroquine mg twice daily is an efficacious regimen is unclear as these data remain unpublished, and a recent report suggested high-dose chloroquine may be associated with increased toxicity and mortality [ ] . the targeted hcq r ltec values were based on r ltec values of . , . , and . on days , , and , respectively, simulated from the chloroquine mg twice-daily regimen. all hcq regimens produced r ltec values higher than target r ltec values for each day evaluated; however, the authors recommended hcq mg twice daily on day , followed by mg twice daily for days while ''considering efficacy, safety, and patient compliance.'' furthermore, the r ltec values of hcq were found to be higher than those of chloroquine on days , , , and . it is important to note that the hcq ec values were * . less than those of chloroquine, which plays a role into hcq's higher r ltec values, and also that this enhanced potency is contrary to other in vitro analyses performed [ , ] . perinel and colleagues performed a prospective analysis to characterize the pk of hcq in patients admitted to the intensive care unit with laboratory-confirmed sars-cov- infection. patients included in this analysis received initial hcq regimens of mg orally three times per day [ ] . based on previously published literature, hcq trough values [ mcg/ ml and \ mcg/ml were considered to ''optimize efficacy and safety,'' respectively [ , ] . it is important to note that there is no evidence in support of these values for optimal treatment against covid- . thirteen patients were included in this analysis ( of which were mechanically ventilated, % male), with a median age of years ( - years), body weight of . kg ( . - . kg), and renal function of . ml/min ( . - . ml/min) as estimated via the ckd-epi formula. based on the hcq initial regimen ( mg orally three times/day), only % of patients achieved ''therapeutic'' levels ([ mcg/ml) at a mean of . days ( . - . days), while % of patients achieved what the authors classified as ''toxic'' levels ([ mcg/ml). to more clearly understand the variability of hcq pk parameters and assess an optimal dosing regimen, a simulation study was performed across various dosing regimens. the authors proposed that a hcq mg loading dose on day followed by mg twice daily for days provided ''optimal'' exposures (targeting levels [ mcg/ml and \ mcg/ml) compared to the other hcq regimens simulated [ ] . al-kofahi and colleagues performed simulations to model and identify possible pre-and postexposure hcq dosage regimens that would achieve target exposure over the sars-cov- ec and help guide clinicians in dose selection for covid- . of note, the population pk parameters were derived from hcq plasma concentrations from healthy volunteers and malaria patients, not patients with diagnosed covid- . the authors of this study chose to evaluate the percentage of patients with trough concentrations above the sars-cov- ec values of . lm and . lm (to account for plasma protein binding). these ec values were chosen as targets as lower ec values are typically associated with lower mois, and the authors presumed that post-exposure prophylaxis may be associated with lower viral loads compared to acute infection with sars-cov- ; however, there is no evidence to support this claim [ ] . regimens simulated included post-exposure prophylaxis one: no loading dose, followed by mg daily for days; post-exposure prophylaxis two: mg loading dose, followed by mg daily for days; post-exposure prophylaxis three: no loading dose, followed by mg three times daily for days; post-exposure prophylaxis four: mg loading dose, followed by mg daily for days; post-exposure prophylaxis five: mg loading dose, followed by mg daily for days; and post-exposure prophylaxis six: mg loading dose, followed by mg daily for days. with the exception of post-exposure prophylaxis regimen one, all other regimens had between and % troughs above target on day , with a median time above target being - h. post-exposure prophylaxis regimens three, five, and six maintained the optimal pd targets compared to the other regimens at day [ ] . despite these results, it is currently unclear if the target chosen by the authors is, in fact, an appropriate target. importantly, boulware and colleagues conducted a randomized, double-blind, placebo-controlled trial assessing the effect of hcq for post-exposure prophylaxis and found no significant difference in the incidence of new covid- illness [ ] . overall, there are notable and important limitations across the highlighted studies. the primary limitation is that a proper exposure-response analysis was not performed in any of the human simulation studies. targeted exposures were all related to published ec values to predict pd response, despite an absence of data to support this strategy. it is currently unknown if ec values are meaningful values and what exposures relative to these values would be associated with an antiviral effect. furthermore, if ec values prove to be an effective target, it would be of importance to determine what conditions and cell line are associated with a reproducible and meaningful value. it warrants mention that the only true pd study currently available (maisonnasse et al. [ ] macaques study described above) failed to demonstrate an antiviral effect with hcq despite having serum and lung concentrations above the ec . furthermore, if a target is identified, there is likely to be significant variation in pk in patients infected with covid- that is not properly accounted for in the simulation studies published. when considering pk, it is also likely important to consider hcq intracellular concentrations, as hcq has been shown to accumulate in cells through ''lysosomal trapping'' [ ] . overall, there is a clear need for clinical data that links hcq dose, pk, response, and safety in patients with covid- . although the most common adverse effects of hcq are gastrointestinal in nature, ocular and cardiac toxicities are arguably the most concerning [ ] [ ] [ ] [ ] . ocular adverse effects associated with hcq include retinopathy, corneal changes, and decreased visual acuity; however, these are most commonly associated with longterm use and would likely not be of principal concern with the short durations of hcq utilized for covid- [ ] . nevertheless, a recent report associated both maximum (p = . ) and mean (p = . ) hcq levels to predict hcq-induced retinopathy. in this report of patients, the overall frequency of retinopathy was . %. hcq-induced retinopathy was found in . %, . %, and . % of patients with a maximum hcq blood concentrations of . - . mcg/ml, . - . mcg/ml, and . - . mcg/ml, respectively, and found in . %, . %, and . % of patients with mean hcq blood concentrations of . - . mcg/ml, . - . mcg/ml, and . - . mcg/ml, respectively. notably, risk of retinopathy increased with hcq duration, with only % of patients experiencing this adverse effect within the first years of hcq use [ ] . the most immediate life-threatening adverse effect of hcq is ventricular arrythmias arising from qtc prolongation. unfortunately, to our knowledge, there has been no evaluation linking hcq exposure to cardiac toxicity. evaluating a dose-response relationship with cardiac toxicities, including qtc prolongation, cardiomyopathy, torsades de pointes, and ventricular arrhythmia, is of utmost importance in future studies. it is also important to note that, although cardiac issues can arise with hcq monotherapy, most cardiotoxicity is likely in combination with other drugs (e.g., azithromycin) or those with baseline pre-disposing conditions, such as baseline prolonged qtc and/or electrolyte abnormalities (although a dose-dependent effect with hcq monotherapy is possible). in the evaluation conducted by perinel and colleagues, qtc prolongation occurred in two patients receiving hcq mg orally three times/day, with qtc values elevating from to ms and to ms with hcq levels of . mcg/ml and . mcg/ml, respectively. given these large variations in hcq levels, baseline comorbidities or concomitant qtc-prolonging medications could have contributed to qtc prolongation; however, these potential confounding variables were not reported by the authors. a limitation of this study was that there were limited hcq exposures that were correlated to toxicities. this evaluation utilized a ''toxic'' level of [ mcg/ ml, which was derived as ''…a number of arguments suggest that a concentration of mcg/ml should not be exceeded to avoid ocular toxicity'' [ ] . despite this basis, hcq-induced ocular toxicity is most commonly associated with prolonged exposures and will likely be of less concern with the short durations utilized for covid- ; cardiotoxicity is of much more concern during acute ingestion of hcq [ , ] . indeed, mercuro and colleagues conducted a retrospective, observational study evaluating hospitalized covid- patients and found that those receiving hcq monotherapy had a median (iqr) change in qt interval of . [- . to . ] ms [ ] . also important to note, given the large vd and high tissue sequestration of hcq, it is possible that patients may remain at risk for qtc prolongation after hcq discontinuation. baseline and periodic qtc monitoring should be used to help guide the safe use of hcq in patients receiving this medication for covid- . despite the work completed to date, many data need to be considered for the future testing of hcq and other covid- therapies to determine optimal dose(s) of these therapies. antiviral ''activity'' in vitro and in vivo needs to be adequately defined (ec , ec , etc.) against sars-cov- in the most pertinent cell line. the previously cited in vitro studies display a broad range of ec values that were determined utilizing different mois and applying different time periods utilized for their samplings, despite the uncertainty of whether or not ec values are meaningful targets [ ] [ ] [ ] . further studies need to assess exposures in animal models in both blood and lung that are related to covid- pd response (e.g., viral load reductions, resolution of symptoms, survival, etc.). studies also need to further examine the penetration of hcq into relevant tissues, taking into account intracellular concentrations (macrophages, neutrophils, etc.) due to ''lysosomal trapping'' associated with hcq. when studying intracellular concentrations of hcq, macrophage concentrations in humans need to be determined as concentrations will need to be corrected for if serum to macrophage concentrations differ between species. following the optimal determination of the pd ''target,'' serum exposures in blood/plasma/serum should be correlated with lung concentrations throughout in vivo evaluations and then which dose(s) this translates to in humans needs to be determined. following all of these considerations, these dosage regimens should be evaluated for efficacy and safety in well-designed, randomized clinical trials. also, the current ongoing clinical trials will need to be evaluated for possible efficacy and safety signals associated with different dosage regimens. current dosing protocols and guidelines for hcq are a result of extrapolating data and findings from other disease states and/or pharmacology simulation studies performed. given the ''cart before the horse'' approach with hcq in response to covid- , it will be important to examine the results of ongoing clinical trials to determine whether dose response(s) were observed (if any). it will be important to analyze possible differences in outcomes in those with mild versus moderate versus severe disease and whether combination therapy may be an effect modifier. covid- is likely to be a clinical issue for the foreseeable future. if hcq is shown to fail in clinical trials, this emphasizes the problem with relying on in vitro activity of antimicrobials to translate to improved patient outcomes. while the initial pandemic may not have afforded the opportunity for robust preclinical work to inform agent selection and dosing, this should not be the case moving forward. we have been able to work towards improving outcomes in patients with covid- with non-pharmacologic approaches and (possibly) some potential treatment options in the interim; however, we as clinicians should learn from the mistakes made and attempt to world health organization ( ) who timeline-covid- clinical characteristics of coronavirus disease in china pharmacologic treatments for coronavirus disease (covid- ) remdesivir for the treatment of covid- -preliminary report the cytokine release syndrome (crs) of severe covid- and interleukin- receptor (il- r) antagonist tocilizumab may be the key to reduce the mortality chloroquine is a potent inhibitor of sars coronavirus infection and spread structural and molecular modeling studies reveal a new mechanism of action of chloroquine and hydroxychloroquine against sars-cov- infection chloroquine analogues in drug discovery: new directions of uses, mechanisms of 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issue? life-threatening severe qtc prolongation in patient with systemic lupus erythematosus due to hydroxychloroquine hydroxychloroquine retinopathy-implications for research advances for rheumatology care risk of qt interval prolongation associated with use of hydroxychloroquine with or without concomitant azithromycin among hospitalized patients testing positive for coronavirus disease funding. no funding or sponsorship was received for this study or publication of this article. place the horse before the cart as we proceed with future therapies.authorship. all named authors meet the international committee of medical journal editors (icmje) criteria for authorship for this article, take responsibility for the integrity of the work as a whole, and have given their approval for this version to be published. all authors have provided writing and editing assistance.disclosures. taylor morrisette, thomas p. lodise, marc h. scheetz, srijib goswami, and jason m. pogue have no relevant conflicts of interest to disclose. michael j. rybak is the editor-in-chief of this journal.compliance with ethics guidelines. this article is based on previously conducted studies and does not contain any studies with human participants or animals performed by any of the authors.open access. this article is licensed under a creative commons attribution-noncommercial . international license, which permits any non-commercial use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/bync/ . /. key: cord- -p j caf authors: monzani, alice; genoni, giulia; scopinaro, alice; pistis, gianfranco; kozel, daniela; secco, gioel gabrio title: qtc evaluation in covid‐ patients treated with chloroquine/hydroxychloroquine date: - - journal: eur j clin invest doi: . /eci. sha: doc_id: cord_uid: p j caf in late december , a cluster of pneumonia cases caused by a novel coronavirus occurred in wuhan, china and has spread rapidly initially throughout europe and later usa ( ). the pathogen was originally called novel coronavirus ( -ncov) and later named severe acute respiratory syndrome coronavirus (sars-ncov- ) by the world health organization (who). in late december , a cluster of pneumonia cases caused by a novel coronavirus occurred in wuhan, china, and has spread rapidly initially throughout europe and later in the united states. the pathogen was originally called novel coronavirus ( -ncov) and later named severe acute respiratory syndrome coronavirus (sars-ncov- ) by the world health organization (who). although most human infection linked by coronavirus are mild, the severe acute respiratory syndrome coronavirus (sars-cov) and the middle east respiratory syndrome coronavirus (mers-cov) were responsible for and infections with and deaths, with a mortality rate of % and %, respectively. , , , sars-cov- shares many similar clinical symptoms with sars-cov and mers-cov, including fever, dry cough, fatigue and worsening dyspnoea with interstitial pneumonia that in up to %- % might unfortunately evolve in a severe acute respiratory distress syndrome (ards). , by april , confirmed cases have been reported globally, with deaths. the infection is now spread in countries, with a median mortality rate up to %. the rapid worldwide diffusion of sars-cov- prompted the who to proclaim the state of pandemic on march . while social-distancing measures were rapidly implemented, a safe and effective vaccine is likely to take many months or years. up to now, since no effective sars-cov- therapy is available, patients are treated with several nonspecific drugs. despite poor real clinical evidence of unequivocal beneficial effect of chloroquine/hydroxychloroquine (cq/hcq), the absence of an effective covid- treatment and the social pressure raised the demand of these drugs for its compassionate use, both in hospital and outpatient settings. high-dosed hcq showed promising potential effect in reducing sars-cov- viral load in covid- patients with enhanced effects in combination with azithromycin or several antiviral drugs. cq/hcq are two old anti-malarial drugs also used in long-term treatment of connective tissue disease making both drugs widely available and economically sustainable. nevertheless, cardiac toxicity may occur following qt prolongation and sodium-channel inhibition, resulting in ventricular arrhythmias, conduction blockade and cardiovascular collapse figure . ecg qt interval prolongation could reflect prolongation of ventricular repolarization and thus the effective refractory period. many factors affect the duration of ventricular repolarization, and the qt interval varies from beat-to-beat, day-to-day and daytime to night-time. it is affected by several factors such as age, sex, electrolyte concentrations, autonomic tone, myocardial ischaemia and importantly by several different classes of drugs. this pharmacological property is used to prevent ventricular arrhythmias, but it could also cause them predisposing to intraventricular circuits of depolarization that manifest as potentially lethal polymorphic malignant ventricular tachyarrhythmias (torsades de pointes). chloroquine/ hydroxychloroquine provoke sodium and calcium channel blockades, which lead to membrane-stabilizing effects that might result in conduction disturbances with atrioventricular block, and qrs interval widening and a dangerous qt prolongation figure . this side effect might be even enhanced in the setting of covid- setting because of drug-to-drug interaction (azithromycin, antivirals, etc) and the clinical setting of acute infection that usually sees fever, dehydration and electrolyte abnormalities. finally, sars-cov- is primarily responsible for an acute respiratory infection that might present a trigger for acute cardiovascular events like myocarditis or acute coronary syndrome. , qtc prolongation and cardiotoxicity closely correlate with cq/hcq dose and duration, and a recently published review of complications affecting patients treated with a long time high cumulative dose of cq showed that cardiac conduction disorders were the main side effects with an incidence reported up to % of all complications. considering this, it could be argued that an incautious use of cq/hcq might be responsible of a certain amount of undiagnosed arrhythmic deaths improperly attributed to covid- itself. in so forth, we believe that is probably time to have prospective clinical trials that will evaluate the safety and efficacy of hcq during treatment of covid-infected patients. in the meantime, if pneumologist, internal doctor or specialist in infective disease will recommend hcq, especially in combinations with azithromycin or antivirals we suggest an easy step-by-step flow chart for hospitalized covid- patients that could offer an easy way to control the risk of hcq cardiotoxicity figure . firstly, the concomitant use of other drugs that could result in qt prolongation and sodium-channel inhibition should be highly discouraged. moreover, a careful checklist that will exclude the presence of long-qt genetic disease (both for the patient and for the family) and familiar unexplained sudden cardiac death should be the first anamnestic step. if yes, the patient should be referred to a complete cardiological evaluation. if not, we recommend having a baseline -lead ecg and, if possible, a blood sample with evaluation of electrolytes followed by our flow chart for qtc monitoring during hcq administration. in case of outpatients setting, we recommend an ecg every days. in case of qtc > ms, please consider the need of hospital admission and continuous ecg monitoring. nonetheless, the mandatory requirement of a basal ecg would be a considerably limiting requirement for the prescription of hcq in outpatients, especially in consideration of the limited availability of healthcare professionals and personal protective equipment to protect them in a pandemic situation. a pneumonia outbreak associated with a new coronavirus of probable bat origin summary of probable sars cases with onset of illness from middle east respiratory syndrome coronavirus (mers-cov) sars: prognosis, outcome and sequelae prevalence of comorbidities in the middle east respiratory syndrome coronavirus (mers-cov): a systematic review and meta-analysis clinical characteristics of 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hydroxychloroquine dosing for patients with covid- : an integrative modeling approach for effective drug repurposing stay at home is an important message able to contain spreading of the virus, and awaiting the widely availability of telemedicine ecg-telemetry, the use of smartphone-based pocket ecg reliable devices should be highly encouraged and can potentially save healthcare resources in the current situation. key: cord- -s u g authors: isaksen, j. l.; holst, a. g.; pietersen, a.; nielsen, j. b.; kanters, j. k. title: chloroquine, but not hydroxychlorquine, prolongs the qt interval in a primary care population date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: s u g background: chloroquine (cq) and hydroxychloroquine (hcq) have recently been suggested as treatment for the current corona virus disease (covid- ) pandemic. however, despite their long-term use and only few case reports on adverse effects, cq and hcq are listed as a known risk of the lethal ventricular arrhythmia torsade de pointes and their cardiac safety profile is being questioned. thus, we aimed to investigate the electrocardiographic and mortality effects of cq and hcq in a primary care population. methods: we used danish health care registers and electrocardiograms (ecgs) from primary care to define three studies. ) a paired study of subjects with ecgs before and during use of cq/hcq, ) a matched ecg study of subjects taking cq/hcq compared to controls, and ) a mortality study on people taking hcq matched to control. in both matched studies, we adjusted for connective tissue diseases, use of qt-prolonging drugs, and cardiac disease. we used the qtc interval as the marker for electrocardiographic safety. in the mortality study, cases were followed from first claimed prescription until days after estimated completion of the last prescription. % confidence intervals follow estimates in parenthesis. results: use of cq was associated with a . ( . ; ) ms increase in qtc in the paired study (n= ). in the matched study (n= , controls= ), qtc was insignificantly increased in subjects taking cq by . (- . ; ) ms. with a {delta}qtc of . (- . ; . ), use of hcq was not associated with an increased qtc in the paired study (n= ). in the matched study (n= , controls= , ), qtc also was not different between groups (p= . ). in the mortality study (n= , ), use of hcq was associated with a hazard ratio of . ( . ; . ). conclusions: in subjects free of covid- , we found a small increase in qtc associated with use of chloroquine, but not hydroxychloroquine. we found no increased mortality associated with use of hydroxychloroquine. the corona virus disease pandemic has led to investigation and use of chloroquine (cq) and hydroxychloroquine (hcq) in the treatment of covid- . cq and hcq are -aminoquinilines approved for medical use in and , respectively, and both drugs interfere with important physiological mechanisms that may improve the clinical course of patients with covid- . cq and hcq are weak bases and accumulate in lysosomes increasing ph which interferes with virus entry and fusion( ). cq and hcq also inhibit viral gene expression and post translational modification necessary for viral replication( ). furthermore, cq and hcq have direct anti-inflammatory actions by inhibition of b-and t-cell receptors and especially by decreasing tnf-α and cytokine production (interleukin- and interleukin- )( ). the drawback is that cq and hcq have known cardiotoxic effects( ) including vasodilatation, hypotension, hypokalemia, negative inotropy, and arrhythmias. the risk of arrhythmias may partly be mediated through the other cardiotoxic effect and partly due to ion channel blockade. both cq and hcq are known herg-blockers, but also block a variety of other ion channels including sodium, calcium, i k potassium and pacemaker funny channels( ). since cq and hcq are old drugs, the approvals were granted long before thorough qt studies were required and only limited ecg safety data exists. despite the known herg block, there is a paucity of documented torsades de pointes ventricular tachycardia (tdp) during treatment,( ) which may be attributed to the concomitant calcium block protecting against tdp.( ) only three cases of tdp have been published( - ), all in patients with concomitant morbidities as cirrhosis, heart failure, or preexisting qt-prolongation. using the sl algorithm version (ge healthcare, wauwatosa, wi), we obtained heart rate, qt interval, qrs duration, jt interval, pr interval, and presence of right bundle branch block (rbbb) or left bundle branch block (lbbb). the qt interval was corrected with bazett (qtcb) and framingham (qtcfrh) formulae. based on the jt interval, jtc was obtained by linear correction for heart rate (jtc = jt - . *(rr- ), whereby rr is the r-to-r interval in seconds and jt is measured in ms). from the primary study population, we defined three subsets, each with a cq and a hcq population: ) paired study, ) matched study, and ) mortality study. as an additional analysis, we conducted a fourth study on connective tissue diseases with three populations independent of cq/hcq prescriptions. the paired study is comprised of people with an ecg taken during a period of cq or hcq treatment, respectively, and with a prior baseline ecg without hcq/cq treatment. thus, in this study, the subjects served as their own controls. . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / classification of diseases, th revision (icd- ). diabetes mellitus, hypertension, and congestive heart failure were all defined using a combination of icd- and atc codes at baseline as done previously. ( ) for subjects in the paired population, subjects served as their own controls and the results were thus inherently adjusted for comorbidity, age, and sex. we adjusted for a change in use of qt-prolonging drugs where appropriate. we used a random-effects regression model for continuous variables. for subjects in the matched population, we compared continuous electrocardiographic variables using linear regression crudely as well as with adjustment for ra, sle, ss, use of qt-prolonging drugs, hypertension, ischemic heart disease congestive heart failure, and diabetes mellitus, as appropriate. categorical variables were tested using a two-sample test of proportions. for subjects in the mortality population, we used cox regression with full adjustment as with the matched population, as appropriate. subjects were censored upon emigration . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . https://doi.org/ . / individually or days after the last claimed prescription for each case/control pair. we used time-on-study as underlying time variable in the cox model. the danish data protection agency approved the use of de-identified data ( - - ) on the conditions that the exact number of subjects in groups of < subjects not be disclosed and that no calculations (including p-values) be reported on groups of < subjects. all estimates were reported along with a % confidence interval indicated as (lower to upper). data management and statistical computations were performed in stata (version ), and a p-value < . was considered significant. . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) we identified ten subjects who had an ecg taken during a treatment of cq and who had a prior ecg without treatment of cq ( compared to their baseline ecg, subjects taking cq had a significantly longer qtcfrh by . ms ( . to , p= . ). we found no evidence of an increased qtcfrh after use of hcq ( . ms, - . to . ms, p= . ). we identified and subjects with an ecg taken during a period of treatment with cq or hcq, respectively, and matched them : with controls. subjects on cq (table ) , compared to controls, were more likely to suffer from ischemic heart disease, but not hypertension. with full adjustment for ra, sle, ss, qt-prolonging drugs, hypertension, diabetes, ischemic heart disease, and congestive heart failure, qtcfrh was . ms longer in the cq group compared to control, but with a wide confidence interval (- . to ms, p= . ). subjects on hcq (table ) more often than controls had ra, sle or ss, as well as hypertension, ischemic heart disease, congestive heart failure, and diabetes. in the unadjusted analysis, we found an increased qtcfrh interval of ms (p= . ). however, . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . after adjustment for ra, sle, ss and qt-prolonging drugs, there was no significant difference in qtcfrh between cases and controls ( . ms, - . to . ms, p= . ) and after additional adjustment for ischemic heart disease, hypertension, diabetes, and congestive heart failure, the difference in qtcfrh was reduced further to . ms (- . to . , p= . ). qrs duration followed a similar pattern in the adjusted and unadjusted analyses, whereas the jt interval also in the unadjusted analysis was comparable between the groups. we identified , subjects with a prescription for hcq irrespective of an ecg (table ) . subjects on hcq were more likely to have ra, sle, ss, hypertension, ischemic heart disease, and congestive heart failure. in the most crude analysis, adjusted only for sex and age, mortality was not different between subjects on hcq and controls (hr= . , . to . , p= . ). with adjustment for ra, sle, ss, and use of other qt-prolonging drugs, we still found no significant difference between hydroxychloroquine and matched controls (hr= . , . to . , p= . ). further adjustment for ischemic heart disease, diabetes, hypertension, and congestive heart failure did not change the association materially. kaplan-meier survival curves are shown in figure . . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . in the ra/sle/ss study we defined three populations of cases and controls with and without ra, sle, and ss respectively. we found no difference in crude or adjusted qtcfrh compared to control in any of the populations (supplementary tables - ) . heart rate was significantly higher in subjects with ra and sle, but lower in subjects with ss. crude pr interval was increased in subjects with ss, but not with adjustment for diabetes, ischemic heart disease, hypertension, and congestive heart failure. in the fully adjusted adjusted for sex, age, ss, sle, qt-prolonging drugs, ischemic heart disease, diabetes, hypertension, and congestive heart failure, patients with ra had a shorter qrs duration compared to controls. in this real-world study, we found a small, but significant, heart rate-corrected qt-prolongation of ms with cq, but no qt prolongation with hcq. we did not find increased mortality in subjects taking cq or hcq. cq and hcq are both listed as known causes of tdp on crediblemeds.org,( ) and cq is recognized by the world health organization (who) as prolonging the qt/qtc interval. ( ) however, who states that cq is associated with a low risk of cardiotoxicity based on pk/pd modelling.( ) in the present study, we found an increase in qtcfrh of . ms in the . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / paired cq population, and no difference the paired hcq population. another study ( ) showed that a , mg dose of cq yielded an increase in qtc of - ms. matched on sex and age, subjects receiving hcq showed a ms increase in qtcfrh. however, the increased qtc disappeared upon adjustment for use of qt-prolonging medication and connective tissue diseases. hence, the increased qtcfrh was likely caused by rheumatic disease and not hcq. thus, in both the matched and the paired analyses, we were unable to demonstrate any statistically significant increase in qtc associated with use of hcq. we did not find that use of hcq was associated with an increased mortality. this is not surprising, since the toxicology profile of hcq is better than that of cq.( ) who reports that the few deaths associated with use of hcq were caused by overdosing and chronic indications for hcq. ( ) only three case studies have demonstrated arrhythmic adverse events in association with use of cq/hcq. one study( ) involved a syncope in a patient with sle and end stage renal disease and qt prolongation before hcq initiation. another study( ) documents tdp in a patient with sle, with a history of cirrhosis, hbv-related hepatoma, prior myocardial infarction, and ventricular septal defect. the third study( ) involved a patient with sle, who also suffered from congestive heart failure, chronic kidney disease stage , and hypertension. all three case studies thus features patients with severe qt-prolonging comorbidity and chronic rather than episodic use of hydroxychloroquine. collectively, . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint evidence from the present and other studies found little risk of death or arrhythmic events associated with episodic use of hcq. malaria was and still remains an indication for hcq in denmark, although it is no longer recommended. ( ) hcq is commonly prescribed for connective tissue diseases such as rheumatoid arthritis, systemic lupus erythematous, and sjögren's syndrome. we were only able to identify the reason for the hcq treatment in forty to sixty percent of the cases, likely because milder connective tissue cases were treated in primary care and therefore never obtained a hospital icd- diagnosis detectable in our registries. furthermore, some subjects may have been prescribed hcq as malaria prophylaxis. cq/hcq has emerged as a possible treatment for covid- .( ) naturally, given the known risk of increased qtc, concerns about the safety of the drugs have been raised.( , ) we have found different electrocardiographic safety profiles for cq and hcq. with cq, we have demonstrated an increased qtcfrh of . ms. with hcq, we found no increase in qtcfrh. for use of cq and hcq, respectively, we found no increased mortality. cq and hcq have been used as malaria prophylaxis for decades without raising red flags and the use of hcq in higher doses to treat auto-immune diseases also has not been . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint associated with an increased mortality. the few case studies that exist were associated with chronic use of hcq beyond the duration corresponding to a covid- treatment and with severe qt-prolonging comorbidity. arrhythmias are commonly seen in patients with covid- (one study ( ) . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / using matched and paired studies of subjects free of covid- receiving chloroquine or hydroxychloroquine, we found that use of chloroquine but not hydroxychloroquine was associated with a small increase in qtc. we were unable to show increased mortality associated with hydroxychloroquine. . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint h y d r o x y c h l o r o q u i n e i n t h e t r e a t m e n t o f c o v i d - i n f e c t i o n -a s y s t e m a t i c l i t e r a t u r e r e v i e w [ p r e p r i n t s e r v e r ] . . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . a l e h m , g a b r i e l s j , c h a n g d , k i m b s , m a n s o o r a , m a h m o o d e , e t a l . t h e e f f e c t o f c h l o r o q u i n e , h y d r o x y c h l o r o q u i n e a n d a z i t h r o m y c i n o n t h e c o r r e c t e d q t i n t e r v a l i n p a t i e n t s w i t h s a r s -c o v - i n f e c t i o n . c i r c a r r h y t h m e l e c t r o p h y s i o l . . . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint dp: not disclosed due to data protection restrictions (groups of < ). na: not applicable. . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . , diabetes, ischemic heart disease, hypertension, and congestive heart failure. + excluding chloroquine and hydroxychloroquine dp: not disclosed due to data protection restrictions (groups of < ). . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / supplemental material for chloroquine, but not hydroxychlorquine prolongs the qt interval in a primary care population. jonas l. isaksen, anders g. holst, adrian pietersen, jonas b. nielsen, claus graff, jørgen k. kanters use of chloroquine was defined when a prescription for chloroquine (atc code: p ba ) was claimed. use of hydroxycloroquine was defined in the same way using atc code p ba . systemic lupus erythematous was defined as any of the icd- codes dm , dg a, dg c, di b, di c, dj c, or dn a, or either icd- code . or . . rheumatoid arthritis was defined as any of the icd- codes dm or dm , or any of the icd- codes . , . , or . . ischemic heart disease was defined as any of the icd- codes i , i , i , i , i or the icd- code . diabetes mellitus was defined as any of the icd- codes e , e , e , e or e , or use of insulin (atc: a a) or oral antidiabetics (atc: a b), or the icd- code . . hypertension was present if any of the icd- codes i or i , or the icd- code was found, or if we found concurrent use of two of these classes of medication: alpha blockers (atc: c a, c b, c c), non-loop diuretics (atc: c l, c a, c b, c d, c e, c x, c x, c c, c d, c g, c da, c ba, c da, c xa ), vasodilators (atc: c db, c dd, c dg, c , c ), beta blockers (atc: c ), calcium blockers (atc: c , c bb, c db), or angiotensin converting enzyme inhibitors (atc: c ). heart failure was present if the icd- codes i , i , or j , or the icd- code . was found, or if a prescription of loop diuretics (atc: c c) was found. the following drugs and atc codes were considered qt-prolonging in the present study. the list was taken from crediblemeds.org on april , (revision march , , and is comprised of those drugs categorized as having a known risk of torsade de pointes (tdp), with the omissions of chloroquine and hydroxychloroquine. . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint rbbb ( . %) ( . %) too few cases too few cases lbbb < ( . %) too few cases too few cases # except chloroquine and hydroxychloroquine. *adjusted for sex, age, rheumatoid arthritis, sjögren's syndrome, and qt-prolonging drugs # . § adjusted for sex, age, rheumatoid arthritis, sjögren's syndrome, qt-prolonging drugs # , diabetes, ischemic heart disease, hypertension, and congestive heart failure. dp: not disclosed due to data protection restrictions (groups of < ). . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . https://doi.org/ . / ( %) , ( %) ischemic heart disease key: cord- -mjdg ny authors: cohen, i. v.; makunts, t.; moumedjian, t.; issa, m.; abagyan, r. title: determinants of cardiac adverse events of chloroquine and hydroxychloroquine in years of drug safety surveillance reports date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: mjdg ny chloroquine (cq) and hydroxychloroquine (hcq) are on the world health organization's list of essential medications for treating non-resistant malaria, rheumatoid arthritis (ra) and systemic lupus erythematosus (sle). in addition, both drugs are currently used off-label in hospitals worldwide and in numerous clinical trials for the treatment of sars-cov- infection. however, cq and hcq use has been associated with cardiac side effects, which is of concern due to the higher risk of covid- complications in patients with heart related disorders, and increased mortality associated with covid- cardiac complications. in this study we analyzed over thirteen million adverse event reports form the united states food and drug administration adverse event reporting system to confirm and quantify the association of cardiac side effects of cq and hcq. additionally, we identified several confounding factors, including male sex, nsaid coadministration, advanced age, and prior diagnoses contributing to the risk of drug related cardiotoxicity. these findings may help guide therapeutic decision making and ethical trial design for covid- treatment. chloroquine (cq) and hydroxychloroquine (hcq) were initially used as antimalarial agents with broad-spectrum antiviral effects . the use of cq worldwide dates back to a -year mark with a well-established safety profile as a first line drug for the treatment . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) and prophylaxis of malaria . hcq, closely structurally related to cq, has been used at higher doses and for longer duration in some autoimmune diseases such as rheumatoid arthritis (ra) to systemic lupus erythematosus (sle) , . additionally, these agents have been observed and investigated for their antiviral properties . most common adverse events of (aes) of these agents include eye disorders, proximal myopathy, neuropathy, neuropsychiatric events, hypoglycemia, worsening of psoriasis, and particularly, cardiac including cardiomyopathy and qt prolongation , . recently, cq and hcq were reported as potentially beneficial treatments for severe acute respiratory syndrome coronavirus (sars-cov- ) infection, also known as covid- , and several clinical studies are currently investigating these drugs for potential therapeutic efficacy , . on march th, the fda issued an emergency use authorization of hcq and cq products "to be distributed and prescribed by doctors to hospitalized teen and adult patients with covid- , as appropriate, when a clinical trial is not available or feasible" . the use of hcq as an antiviral agent against sars-cov- was identified in an in vitro study which was a source of encouraging results . recent trials in china and france reported the potential efficacy of these agents against sars-cov- , . specifically, in an early non-randomized controlled trial conducted in france, % of the included patients treated with combination hcq and azithromycin tested negative for sars-cov- on day post-treatment compared with . % of the controls . similarly, in a narrative letter, china reported testing cq and hcq in patients with covid- -induced pneumonia in over hospitals across the nation. the authors had claimed that they have demonstrated the efficacy and safety of cq phosphate in "more than patients" . currently, several international ongoing clinical trials are investigating hcq and cq for treatment efficacy and prevention (as examples: nct , nct , nct , nct , nct , nct , nct , and nct . despite limited efficacy data, hcq and cq are currently being administered as part of first-line treatment for sars-cov- in various hospitals across the world , . as previously mentioned, cardiac complications attributed to hcq and cq have been described in various reports. a systematic review of the literature published in june examined reports including individual cases and short series from an online database search. the report identified patients with cardiac events attributed to hcq and cq administered in the context of various inflammatory disorders with a median daily dose of mg for cq and mg for hcq . recent disproportionality analysis study observed an elevated prevalence of torsade de pointes and qt prolongation reports in cq and hcq . the cardiac aes of these therapeutics are of increased concern since a subset of patients infected with covid- present with cardiac injury, suggesting a relevant cardiovascular involvement in the pathophysiology . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) of the disease . in a single-centered cohort study from wuhan, china, shi et al. examined the incidence of cardiac injury in hospitalized patients with covid- . among those, of the patients demonstrated cardiac injury, and higher in-hospital mortality rates were seen in patients with cardiac issues ( . %) compared to those without ( . %) . guo et al. described similar findings in a retrospective single-center case series analysis in which out of hospitalized patients suffered from myocardial injury. the mortality rate in those patients was . % compared to . % in those without cardiac injury . in support of these findings, recent studies have shown that major cardiac outcomes associated with cardiomyopathy ( %) and cardiac injury ( %) are common in critically-ill patients , . similar lines of evidence are also followed by an italian case report of a -year-old woman with lab-confirmed covid- who was admitted to the hospital for severe lv dysfunction and acute myopericarditis. this case highlights that sars-cov- can impact the cardiovascular system even in the absence of major respiratory tract involvement . other studies have observed covid- cardiac complications such as fulminant myocarditis, ventricular tachycardia , , . the goal of this study is to reanalyze the extensive clinical data of cq and hcq cardiac aes collected during the last years to derive the strength of the associations and, more importantly, contributing risk factors. the findings may improve the safety of these therapeutics for covid- treatment in patients that are already at higher risk of cardiac complications. the study used over thirteen million ae reports available from the united states food and drug administration adverse event reporting system (faers) and its older version, adverse event reporting system (aers) data sets. at the time of the study the faers/aers set contained reports from years - , all available online at: https://www.fda.gov/drugs/questions-and-answers-fdas-adverse-event-reportingsystem-faers/fda-adverse-event-reporting-system-faers-latest-quarterly-data-files data preparation faers/aers reports are collected through voluntary reporting to the fda through medwatch and stored in quarterly format data subsets with their respective parameters (age, sex, drug, ae etc.), and common case identifiers. faers data format has had changes historically, requiring each quarterly set to be individually downloaded and modified into consistent data tables , , . since the faers/aers set has reports . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may , . . from all over the world with their respective drug brand names, unique terms were recognized and translated into single generic cq and hcq names. the final data set contained , , ae reports. sle, ra, and malaria were considered for possible sources of reports, however due to low sample size of malaria treatment with hcq or cq in faers, only sle and ra were included in the analysis. cohort selection and data cleaning , reports were obtained from the fda faers database to form three cohorts for analysis by logistic regression: cq cohort, hcq cohort and control cohort. the control cohort for both drugs was defined by reports with ra and sle patients where hcq and cq were not used (n= , ). the cq and hcq cohorts were defined by reports with ra and sle indication with cq (n= , ) and hsq (n= , ) was used in addition to other therapeutics. rstudio (version . . ) and r (version . . ) were employed for data cleaning and logistic regression modeling. faers/aers data sets historically include a small fraction of duplicate reports. the set was scanned for these entries with the r package "dplyr" "distinct" function and were removed as appropriate. a summary of the records demographic factors is made available in table . in order to define the list of possible cardiac aes in this database a table was generated and manually checked for errors by the investigators. for a copy of this table of all aes considered cardiac related see supplementary table s . similarly, a list of nonsteroidal anti-inflammatory drugs (nsaids) was generated for use in our analysis and is made available in supplementary table s . note that the number of nsaids did not include aspirin, as this was to be modeled separately due to expected divergent effects such as higher cardiovascular risk in the patient demographics. during the data cleaning stage, age was limited to a range of to years. for the purpose of our analysis, only values of "f" or "m" from fears/aers were analyzed. the r package "dplyr" function "mutate" and "str_detect" were employed for counting the number of nsaids and cardiac aes observed in each report. a subsample of the original database that included reports with non-empty values for age and sex was also prepared. for a summary of the sample size of the subgroups see supplementary table s . the primary outcome of interest was incidence of any cardiac ae as defined by one of one of terms listed in supplementary table s . the r package "glm" was employed for logistic regression modeling via the "binomial" family function. cardiac aes were the outcome of interest in logistic regression modeling and were coded as a . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) binary (" " if occurred in the report or " " if not). covariates explored in the modeling included age (as a continuous variable expressed in years), sex (as a binary), disease state (coded as a binary of either sle or ra), nsaid usage (as a integer variable equal to the number of nsaids observed in the record), and aspirin usage (as a binary). aic (akaike information criterion) and n number of records included are reported for each of the eight models presented. note that due to absence of some values in the raw data from the fda, models including age and sex have lower sample size than those without these covariates (supplementary table s ). subject weight was not used for model building due to extreme paucity of the data. models a, b, a, and b are built using the control and cq cohorts (described above and presented in tables and , and supplementary table s ). similarly, models a, b, a, and b are built using the control and hcq cohorts (described above and presented in table and , and supplementary table s ).f coefficient estimates, standard error, adjusted odds ratio (adj. or), % confidence intervals ( %ci), and p-values are reported (tables and ). p-values that meet the significance threshold of less than . are marked in tables with an asterisk (*). the adjusted odds ratio is defined as an odds ratio that controls multiple predictor variables in a model and allows for quantification of individual contributions of different variables to a single outcome , in this case cardiac adrs. the adjusted or is calculated by the following equation: , and is intended to account for biases in association between variables from the sample data. demographics of the three cohorts to be analyzed for cardiac side effects by logistic regression is presented in table . all three cohorts were comprised of patients who were treated for either sle or ra. the control cohort (n = , ) was compiled from sle or ra faers/aers reports with no report of cq or hcq use. the cq cohort (n = , ) was composed of records of sle or ra patients with reports of cq use but not hcq. similarly, the hcq cohort (n = , ) was composed of records of sle or ra patients with reports of hcq use but not cq. as shown in table the mean patient was in their mid-fifties and about kg. the patients were predominantly female, with only . % of the controls listed as male. additionally, the patients were predominantly treated for ra. the cq cohort had the most sle patients at . %. many of the patients in all three cohorts received various nsaids and experienced cardiac adverse events (for a listing of the aes considered cardiac related see supplementary table s ). these numbers of records are large, in particular for hcq, they cover a range of demographic parameters, and are sufficient to evaluate the contributors to the cardiac . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . aes with logistic regression. the last row of table indicates an elevated number of cardiac side effect-containing reports for both cq and hcq, from . % to . % and . % respectively. interestingly, the most common individual adverse effects (not grouped by the category) for each cohort were calculated and they did not include cardiac aes (see supplementary fig. s ). table . demographics and distribution of seven selected variables of the three cohorts analyzed by logistic regression. sd is given in parentheses and stands for standard deviation. several logistic regression analyses were performed in order to evaluate whether cardiac aes were related to cq. binary logistic regression was employed to determine the confounding variables contributing to the apparent effects of the drugs on occurrence of cardiac side effects. in the first, simplest analysis explored, model a ( is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . generated in order to explore effects of nsaid and aspirin use on the coefficients explored in model a. similar to model b, both aspirin use and nsaids were shown to both independently increase risk. model b improved upon model a's aic by , indicating the significant contribution of the added variables. moreover, cq's deleterious effects on cardiac ae risk in the more complex model b were still profound, with an adjusted odds ratio of . (p < x - ). (table ) . . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . table . logistic regression analysis of hydroxychloroquine aes. * = coefficients with significant p-values . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint in our study we analyzed , fda adverse event reports divided into cq, hcq and control cohorts to determine their association with cardiac aes when taking into account other factors with increased cardiac risk. although both drugs were significantly associated with increased cardiac ae risk, it was observed that this effect was substantially larger for cq than hcq (fig. ) . additionally, age, sex, concurrent nsaid use, and disease state were identified to contribute to the risk of cardiotoxicity with both therapeutics. after controlling for these factors, it was observed that the deleterious effects of cq and hcq on cardiac ae risk remained significant (tables and ) . we expanded the current safety surveillance evidence by using a more comprehensive list of cardiac aes (supplementary table s ), to avoid diluting the safety signal with many individual terms. additionally, we performed multivariate analysis and identified several at-risk populations. the results of the multivariate binary logistic regression were validated by prior knowledge from literature: ) sex, when compared females, males are more often diagnosed with myocardial infarction, fatal coronary heart disease, among other cardiac diseases ; ) age, cardiovascular disease (cvd) has been shown to increase with age across multiple populations ; ) nsaids, especially preferential and selective cyclooxygenase- inhibitors, increase the risk of cvd , .two other essential factors were shown to be necessary to correct for, due to their association with cardiac side effects in the patient population used for our analysis and the current utilization of cq and hcq: ) sle: sle patients have been shown to have a greater risk of cardiac aes than patients with ra. this makes sense, as it is well known that cvd is one of the major complications and is one of the leading factors in mortality in patients living with sle . additionally, the host inflammatory response seen in sle may be similar to that in covid , . ) aspirin: it may be expected for aspirin to present a protective effect, however the opposite effect was observed. use of aspirin in itself does not increase cvd risk, in fact it is mainly used for preventing cardiac events . aspirin use in our study population is likely heavily associated prior cardiac related medical history, that is not listed in fears, and could not be otherwise accounted for in the model. aspirin increases the robustness of the model by correcting for prior treatment of cardiac disease or prevention in high risk patients. the regression model was instrumental to exclude this aspirin association from the quantification of the direct cardiac side effects of cq and hcq, which remained significant after adjustment ( this analysis revealed increased cardiac risk factors associated with cq and hcq that likely apply to a wide range of patients. although our study was not performed on . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . reports from covid- patients, due to absence of such reports at the time of the study, the results may still be of value because of our mode of evaluation. additionally, the cardiac complications and related mortality of sars-cov- patients , are attributed to the inflammatory nature of the infection , , which is also seen in sle pathophysiology , . in fact, several case reports describe the heart related mortality being associated with inflammation of the myocardium , . cq and hcq use in sle and ra cohorts for the study was a beneficial coincidence, since they are also inflammatory conditions affecting the cardiovascular system that are also treated with cq and hcq , . in this study we observed increased risk of cardiac aes in faers/aers reports of cq and hcq with respect to other therapeutics used for ra and sle. the association remained significant when demographic parameters and concurrent medications were accounted for in the analysis. it may be beneficial to closely monitor patients for cardiac complications. hcq may be safer for use than cq in patients at higher risk of cardiac complications. although, when compared to cq, hcq use was associated with a lower risk of these events, the risk was still statistically significant. if and when available, alternative therapeutics may be safer to use for sars-cov- patients who are already at higher risk of cardiovascular complications due to age, pre-existing cardiovascular issues, concomitant medications and the sars-cov- infection itself. due to the voluntary nature of the faers/aers reports, actual population incidences of the adverse events cannot be derived. medwatch reporting may also be biased by newsworthiness and legal variables. the safety surveillance data misses comprehensive medical records and medication history limiting the scope of the analysis. as with any association study, causality may not be derived from association, since the cases were not uniformly evaluated for causality by clinical specialists. however, the postmarketing surveillance data analysis of over , reports provides population scale evidence which can be used to identify safety signals that might go unnoticed in small scale studies. while our approach with multivariate logistic regression controls for important biases in the data, such as sex, age, and concomitant drug use, some unaccounted-for factors may remain. . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid- ? chloroquine for the novel coronavirus sars-cov- targeting endosomal acidification by chloroquine analogs as a promising strategy for the treatment of emerging viral diseases hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies hhs accepts donations of medicine to strategic national stockpile as possible treatments for covid- patients remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro nct ) hydroxychloroquine treatment for severe covid- pulmonary infection (hydra trial) (hydra) clinicaltrials.gov. 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covid- rheumatoid arthritis and cardiovascular disease international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity we thank members of the abagyan lab for support during this project. we also thank da shi for the contribution to processing the faers/aers data sets. . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint key: cord- - yvjbr q authors: hashem, anwar m.; alghamdi, badrah s.; algaissi, abdullah a.; alshehri, fahad s.; bukhari, abdullah; alfaleh, mohamed a.; memish, ziad a. title: therapeutic use of chloroquine and hydroxychloroquine in covid- and other viral infections: a narrative review date: - - journal: travel med infect dis doi: . /j.tmaid. . sha: doc_id: cord_uid: yvjbr q the rapidly spreading coronavirus disease (covid- ) pandemic, caused by the severe acute respiratory syndrome coronavirus (sars-cov- ), represents an unprecedented serious challenge to the global public health community. the extremely rapid international spread of the disease with significant morbidity and mortality made finding possible therapeutic interventions a global priority. while approved specific antiviral drugs against sars-cov- are still lacking, a large number of existing drugs are being explored as a possible treatment for covid- infected patients. recent publications have re-examined the use of chloroquine (cq) and/or hydroxychloroquine (hcq) as a potential therapeutic option for these patients. in an attempt to explore the evidence that supports their use in covid- patients, we comprehensively reviewed the previous studies which used cq or hcq as an antiviral treatment. both cq and hcq demonstrated promising in vitro results, however, such data have not yet been translated into meaningful in vivo studies. while few clinical trials have suggested some beneficial effects of cq and hcq in covid- patients, most of the reported data are still preliminary. given the current uncertainty, it is worth being mindful of the potential risks and strictly rational the use of these drugs in covid- patients until further high quality randomized clinical trials are available to clarify their role in the treatment or prevention of covid- . a receptor for cell entry, suggesting a possible similar effect of cq on sars-cov- at this step of virus replication [ ] . cq can also affect early stage of virus replication by inhibiting virus-endosome fusion, likely via increasing endosomal ph [ ] . covs such as sars-cov were shown to be able to enter target cells via ph-dependent mechanism in which the acidic ph of the lysosome triggers fusion of the viral and endosomal membranes resulting in viral particle uncoating and subsequent release of viral nucleic acid into the cytoplasm [ ] . cq can also impair posttranslational modifications of viral proteins through interfering with proteolytic processes [ ] and inhibition of glycosylation via specific interactions with sugarmodifying enzymes or glycosyltransferases [ ] . cq can also hamper lysosomal protein degradation and lysosomal fusion with autophagosomes [ ] [ ] [ ] . moreover, it has been suggested that cq has the ability to affect the cytotoxic mechanisms and works as antiautophagy agent in vitro [ ] . cq works as anti-inflammatory agent through reducing tumor necrosis factor (tnfα) release and suppressing tnf receptors on monocytes [ , ] . on the other hand, hcq has a similar effect to cq in interfering with the glycosylation of ace , blocking virus/cell fusion and inhibiting lysosomal activity by increasing ph [ ] . hcq can also impede major histocompatibility complex (mch) class ii expression which inhibits t cell activation, expression of cd and cytokines release [ ] [ ] [ ] . furthermore, hcq has been shown to impair toll-like receptors (tlrs) signaling through increasing endosomal ph and interfering with tlr and tlr binding to their dna/rna ligands thereby inhibiting transcription of pro-inflammatory genes [ ] [ ] [ ] . the aforementioned immunomodulatory properties of cq and hcq have raised the interest in using these drugs in covid- patients at risk of cytokines release syndrome (crs) [ ] . the fact that both cq and hcq are considered for the management of covid- patients clearly highlights the need to better understand their pharmacokinetics (pk) parameters. however, a full understanding of these parameters has been challenging despite the numerous reported studies. generally, pk parameters for cq and hcq are comparable (table ) [ , ] . following oral administration of cq and hcq, their bioavailability can reach up to % with plasma peak time around - hours [ ] [ ] [ ] . thus, parenteral administration, if available, might be a better route especially that oral administration has shown huge interpatient variability [ , , ] . the long half-life of both cq and hcq which could range from to days is likely attributed to their large volume of distribution ( to l/kg) and extensive tissue uptake [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . cq and hcq are metabolized via cyp- enzymes to other active compounds, which are responsible for the extended pharmacological actions and increased toxicity [ , ] . up to % of cq and hcq is primarily excreted renally as unchanged or metabolized forms, and the remaining ( %) is usually cleared through the liver, feces and skin or stored in other lean body tissues [ , [ ] [ ] [ ] [ ] [ ] [ ] . it's important to note that cq and hcq have a chiral center, which produces two enantiomers r(−) or s(+) forms or isomers [ ] , in which little is known about the differences in their pharmacological activity and their corresponding metabolites. most clinically used cq and hcq exist as a racemic mixture ( : ) of both isomers which complicates the understanding of their pk and associated toxicity as they could behave differently inside the body [ , [ ] [ ] [ ] . the most common cq and hcq adverse effects are gastrointestinal symptoms such as nausea, vomiting and abdominal discomfort [ ] , and uncommonly worrisome fulminant hepatic failure [ ] , toxic epidermal necrolysis (ten) [ ] and cardiotoxicity that could manifest with qt abnormality [ ] [ ] [ ] . nevertheless, over the years cq and hcq have maintained a good safety profile when used in several chronic diseases such as ra and sle. despite some animal experiments suggesting that hcq is probably less toxic than cq, there is a lack of high quality evidence from clinical trials supporting this claim [ , [ ] [ ] [ ] [ ] . these toxicities could be related to the very long half-life and the large volume of distribution of both drugs. one of the significant toxic effects of cq and hcq is the possible ocular pigmentation due to their binding to melanin, which could lead to damage in different parts of the eye including the cornea, ciliary body and retina [ ] . notably, the incidence of such ocular toxicity is usually rare. for instance, it was shown that only . % out of ~ patients treated with hcq (≤ . mg/kg/day) for years due to ra or sle had developed ocular related complications [ ] . most studies have shown that such complications might only occur with long term treatment of chronic diseases which extends for more than years with doses above or equal to . mg/kg/day [ , ] . however, ocular toxicity and changes could still occur with shorter treatments. other complications such as development of proximal myopathy associated with respiratory failure have also been reported in patients treated with either cq or hcq [ ] [ ] [ ] [ ] . nonetheless, most of these complications were seen in elderly patients with an average age of years suffering from chronic ra or autoimmune diseases. both cq and hcq were also shown to be associated with rare but life-threatening cardiomyopathy [ ] [ ] [ ] . other less reported cq and hcq toxicities include urticaria [ ] , ototoxicity [ , ] and some neurological effects [ , ] . the antiviral effects of cq were suggested at least years ago [ , ] . since then, several studies have tested the ability of cq and hcq to inhibit the replication of a wide range of covs and non-cov viruses in vitro as shown in tables and , respectively. the majority of these studies have revealed a substantial ability of cq and hcq as well as some of their derivatives to inhibit viral replication with no to low toxicity. specifically, cq has been shown to inhibit the replication of different covs including sars-cov, mers-cov and sars-cov- among others in several studies (table ) [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . only two studies showed no significant inhibitory effects of cq on mers-cov and mouse hepatitis virus (mhv ) [ , ] . other cq derivatives such as amodiaquine (amd), ferroquine (fq), hydroxy ferroquine (hfq) have been also shown to exerts some antiviral activity [ , ] . interestingly enough, while hcq does not seem to have a significant effect in reducing sars-cov and feline cov replication [ ] , it was recently shown to have a potent in vitro inhibitory effect against sars-cov- replication [ , ] . similarly, these compounds have shown excellent in vitro antiviral activity against several non cov (mostly rna viruses) with low toxicity in most cases (table ) . for instance, hiv was shown to be inhibited by cq alone or in combination with hcq, hydroxyurea (hu ), didanosine (ddi), zidovudine (zdv), indinavir (idv), saquinavir (sqv) or ritonavir (rtv) [ , [ ] [ ] [ ] [ ] [ ] . while other derivatives such as hcq and hfq have been also shown to inhibit hiv replication [ , , ] , one study showed no effect of hcq and fq on hiv [ ] . similarly, it was found that cq could enhance epstein-barr virus replication [ ] . furthermore, another study has suggested possible enhanced hiv replication with cq treatment through protection of tat protein from proteolytic degradation [ ] . influenza a and b viruses have also been shown to be inhibited by cq [ , [ ] [ ] [ ] [ ] [ ] although contradicting results have been seen for some subtypes and strains such as avian h n strains (a/mallard/it/ / and a/ty/it/ / ) [ , ] . several other studies have also reported in vitro inhibitory effect of cq on multiple viruses such as chikungunya virus (chikv) [ , , ] , zika virus (zikv) [ ] [ ] [ ] , ebola virus (ebov) [ ] [ ] [ ] , dengue viruses (denv) in mammalian cells [ , , ] but not insect cells [ ] as well as several others [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . nonetheless, some reports failed to observe antiviral activity of cq, hcq and fq on several other viruses including polio virus, reovirus, respiratory syncytial virus (rsv), herpes simplex viruses, coxsackie virus, vesicular stomatitis virus (vsv), vaccinia virus, sindbis virus, parainfluenza- virus and punta toro virus [ , ] . there are limited studies established to investigate the possible antiviral effect of cq or hcq in animal models (table ). in general, studies showed no significant effect of cq on covs including sars-cov and feline infectious peritonitis virus (fipv) replication or clinical scores in mice and cats, respectively [ , ] . however, it has been found that cq significantly reduced hcov-oc dissemination and replication in mice central nervous system (cns) after cq administration [ ] and increased the survival rate of hcov-oc infected newborn mice when their mothers treated by cq most probably through placental and maternal milk transfer [ ] . on the other hand, cq administration has shown contradicting outcomes when used against non-covs rna viruses in different animal models. some studies have demonstrated antiviral efficacy of cq in influenza a virus h n , zikv and ebov infected mice [ , , ] . interestingly, cq was effective against zikv in both wild type and ifnar deficient mice, and protected infected suckling pups from infection and microcephaly when given to their mothers [ , , ] . however, several other studies showed no significant antiviral effect of cq against influenza a h n and h n viruses in mice and ferrets, respectively [ ] . similarly, cq was ineffective against ebov in guinea pigs, mice and hamsters [ , ] , nipah virus (niv) in ferrets and hamsters [ , ] , hendra virus (hev) in hamsters [ ] , chikv in cynomolgus macaques [ ] , lassa virus (lasv) in mice [ ] and semliki forest virus (siv) in mice [ ] . importantly, most of these previous in vivo studies showed toxicity in animals [ , , , , ] . furthermore, it was shown that cq could lead to disease exacerbation correlating with increased type i ifn response and delayed immune responses in chikv infected macaques [ ] , increased mortality rate of sfv-infected mice [ ] and niv or hev infected hamsters [ ] . there are very limited published clinical trials that studied the possible antiviral effect of cq or hcq in cov and non-cov infected patients (table ). these published clinical trials have clearly shown no significant benefit of using cq in the prevention or treatment against influenza, denv or chikv infections in patients [ , [ ] [ ] [ ] [ ] . in fact, in one study, patients treated with cq were more likely to develop adverse effects such as arthralgia at day post-treatment [ ] . on the other hand, few studies have reported that hcq could decease hiv- viremia, stabilize cd t cell count and reduce il- and igg levels in infected patients [ ] , although others showed contradicting finding of increased hiv rnaemia in hcq treated patients [ , ] . interestingly, while few clinical studies have suggested that the use of hcq alone or with azithromycin (azt) could be beneficial for covid- patients as it reduces viral shedding and time to clinical recovery [ ] [ ] [ ] , others have reported no effect in infected patients [ , ] . however, it is important to note that most of these studies have several limitations in study designs with small sample sizes. nonetheless, around clinical trials are ongoing in different countries to asses and evaluate the therapeutic and prophylactic effects of both cq and/or hcq in covid- patients (table ). the covid- pandemic has spread out of control and has caused considerable morbidity and mortality in several countries. in this unprecedented situation, clinicians have tried all kinds of treatments in an effort to stem the progression of this disease. one treatment that has received huge attention was the empirical use of anti-malarial cq/hcq. while there is no strong and enough scientific and clinical data to support their use, several countries have already included cq/hcq in covid- treatment protocols [ , ] , not only as a treatment option for severely ill patients but also as a prophylactic measure. in this comprehensive review of the antiviral effects of cq and hcq on sars-cov- as well as other viruses, we show a broad variation in the research outcomes. both cq and hcq demonstrated promising in vitro results, however, such data have not yet been translated into meaningful in vivo studies. while few clinical trials have suggested some beneficial effects of cq and hcq in covid- patients, most of the reported data are still preliminary [ , ] , [ ] . furthermore, at least of the ongoing trials were canceled or stopped and it is not yet clear if this was due to possible adverse effects, ineffectiveness or other reasons. there are several toxicities associated with these drugs [ ] [ ] [ ] , the one that is foremost concerning is the possibility of qt prolongation and the risk of torsades de pointes, which is a potentially life-threatening arrhythmia [ ] [ ] [ ] . nevertheless, while our literature review showed that this is quite rare, it is not yet evident whether there would be any additive or possible synergistic risk when these drugs are combined with other medications such as azt [ ] . in fact, it is challenging to base a treatment decision in the absence of a complete research cycle and a clear vision of drug efficacy and safety. given the current uncertainty, it is worth being mindful of the potential risks and strictly rational the use of these drugs in covid- patients until further high quality randomized clinical trials are available to clarify their role in the treatment or prevention of covid- . this work was funded by king abdulaziz city for science and technology (kacst) grant number - , which is a part of the targeted research program (trp). azt: azithromycin; vit c: vitamin c; vit d: vitamin d; lst: losartan; rdv: remdesivir; ifß- a: interferon ß- a; nivo: nivolumab; tcz: tocilizumab; lev: levamisole; bud: budesonide; form: formoterol; sof: sofosbuvir; ldv: ledipasvir; otv: oseltamivir; atv: atazanavir; cobi: cobicistat; tdf: tenofovir disoproxil fumarate hrct: pulmonary inflammation resolution time, imv: invasive mechanical ventilation; hcw: healthcare workers; ; ars: 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two patients with acquired immunodeficiency syndrome and active inflammatory arthritis hydroxychloroquine treatment of patients with human immunodeficiency virus type the effect of chloroquine on immune activation and interferon signatures associated with hiv- key: cord- -jg twk authors: oscanoa, teodoro j.; vidal, xavier; kanters, jørgen k.; romero-ortuno, roman title: frequency of long qt in patients with sars-cov- infection treated with hydroxychloroquine: a meta-analysis date: - - journal: int j antimicrob agents doi: . /j.ijantimicag. . sha: doc_id: cord_uid: jg twk introduction: hydroxychloroquine (hcq) has been proposed as sars-cov- treatment but the frequency of long qt (lqt) during use is unknown. objective: conduct a meta-analysis of the frequency of lqt in patients with sars-cov- infection treated with hcq. data sources: we searched pubmed, embase, google scholar, the cochrane database of systematic reviews and preprint servers (medrxiv, research square) for studies published between december and june , . methods: effect statistics were pooled using random effects. the quality of observational studies and randomized controlled trials was appraised with strobe and the cochrane risk of bias assessment tools, respectively. outcomes: critical lqt was defined as: ( ) maximum qt corrected (qtc)≥ ms (if qrs< ms) or qtc≥ ms (if qrs≥ ms), and ( ) qtc increase of ≥ ms. results: in the studies included (n= ), the frequency of lqt during hcq treatment was . % ( % ci: . - . %). in studies (n= ), patients were also taking other qt-prolonging drugs; the frequency of lqt in the other studies (n= ) was . % ( % ci: . - . %). studies (n= ) reported hcq discontinuation due to lqt, with a frequency of . % ( % ci: . -. %). the frequency of ventricular arrhythmias during hcq treatment was . % ( / ) and that of arrhythmogenic death . % ( / ). torsades de pointes occurred in . % ( / ). the group with highest risk of hcq-associated lqt were those older than years (p< . ). conclusions: hcq-associated cardiotoxicity in sars-cov- patients is uncommon but requires ecg monitoring especially in those older than years and/or taking other qt-prolonging drugs. during the sars-cov- pandemic, hydroxychloroquine (hcq) has been used as one of the drugs in the still limited therapeutic armamentarium. hcq may block virus infection by increasing the endosomal ph required for virus/cell fusion, and by interfering with the glycosylation of cellular receptors of sars-cov [ ] . hcq is a safe drug in rheumatoid arthritis and systemic lupus erythematosus; however, adverse reactions in patients with sars-cov- are currently under scrutiny. in the midst of the pandemic, controversy has arisen regarding the cardiotoxicity of hcq, especially hcq-induced long qt (lqt) [ ] . in the electrocardiogram (ecg), the qt interval is the time between the start of the qrs complex and the end of the t wave. physiologically, it represents the sum of the action potential (ap) of ventricular myocytes, and the total duration of the depolarization phase and cardiac repolarization [ ] . normally, the clinical reading of the qt interval takes into account a correction for the heart rate (qtc). the mechanism of hcq-induced lqt is by blocking the rapidly activating delayed rectifier k + current encoded by the human ether-a-go-go-related gene (herg) and also by blocking sodium, calcium and other potassium channels. inhibition of the herg channel can cause prolongation of the action potential duration and consequently, of the qt interval [ ] . when a qt prolongation occurs, there is a potential risk of causing one-way block, recurrent extrasystoles, re-entry, and torsades de pointes ("twisting of the points", tdp). tdp is a form of ventricular tachycardia, which clinically presents as reversible syncope or may degenerate into ventricular fibrillation, cardiac arrest, and sudden death [ ] . drugs used in the treatment of sars-cov- infection that are associated with drug-induced lqt include chloroquine (cq), hcq, azithromycin (azi), and lopinavir/ritonavir. a criterion for the diagnosis of drug-induced lqt is a qt prolongation to values ≥ milliseconds (ms) [ ] . the association between hcq use and drug-induced lqt in patients with sars-cov- infection is unknown. the objective of the present meta-analysis was to evaluate the frequency of lqt in patients with sars-cov- infection treated with hcq. data were also collected on discontinuation of hcq, simultaneous use of other qt-prolonging medications, frequency of arrhythmias during hcq treatment, and arrhythmogenic death. this study was conducted following the guidelines of the preferred reporting items for systematic reviews and meta-analyses (prisma) [ ] . two independent investigators performed a systematic search in pubmed, embase, google scholar, preprint servers (medrxiv, research square) and the cochrane database of systematic reviews for studies published between december and june , . in addition, we conducted a secondary search based on the references lists of retrieved articles. the pubmed search strategy is detailed in supplementary table a. we searched for randomized controlled trials (rcts) or observational studies reporting data on lqt in patients with sars-cov- infection taking hcq. we included studies in english or other languages (all ages) meeting the following criteria: a) covid- patients were diagnosed according to the interim guidance of the world health organization [ ] ; b) studies assessing the risk of hcq-associated lqt in sars-cov- infection in which ecgs were recorded at documented timepoints before and after drug administration; c) critical qtc prolongation was defined as: ( ) maximum qtc ≥ ms (if qrs < ms) or qtc ≥ ms (if qrs ≥ ms) and ( ) qtc increase of ≥ ms [ ] ; d) sufficient data was reported to calculate frequency of hcq-induced lqt, arrhythmias during treatment, arrhythmogenic death, discontinuation of hcq, and simultaneous use of other qt-prolonging agents. the quality of observational studies (cohort, case-control, cross-sectional studies) and randomized controlled trials (rcts) were appraised according to the strengthening the reporting of observational studies in epidemiology (strobe) [ ] and the cochrane risk of bias assessment tool [ ] , respectively. two investigators evaluated the quality of the studies independently. conflicting results were resolved by discussion and involvement of a third reviewer if necessary. the following data were extracted from each study: authors, study location, year of publication, study design, number of participants, sex, age at baseline, outcome definition, exposure definition, follow-up, effect estimates and % cis. we also collected: a) anti-covid- treatment indication, participants' inclusion and exclusion criteria, and number of study participants who had ecg monitoring; b) ecg measurement methodology (e.g. centralized or study site-based, manual or automated, cardiologist or other physician reader, intermittent or continuous, any other relevant details); and c) cardiovascular adverse events: sudden cardiac death, life-threatening ventricular tachyarrhythmias (ventricular fibrillation, ventricular tachycardia, tdp), any other clinically significant arrhythmias or cardiovascular adverse events. the frequency of lqt during hcq treatment, arrhythmogenic death, discontinuation of hcq, frequency of simultaneous use of other qt-prolonging agents and frequency of arrhythmias during treatment were calculated. we used random effects with an inverse variance method to calculate the pooled risk ratios (rrs) and % confidence intervals (ci) according to the heterogeneity between studies [ ] . the overall estimates in the pooled analysis were obtained using stata software (stata corp lp, college station, tx). after screening citations, studies ( observational and rct) [ , were included (figure ) , with a total sample of participants. the characteristics of included studies are summarized in table . eleven studies were from usa, with the other being from france ( ), china ( ), india ( ), tunisia ( ) brunei ( ), italy ( ), malaysia ( ), cameroon ( ) and spain ( ); one study included patients from usa and italy. overall, mean (sd) age was . ( . ) years and . % were men. in studies, the proportion of men was more than %. in the studies ( patients) where the dose of hcq administered and the duration of treatment were reported the mean (sd) total cumulative dose of hcq was ( ) mg with a mean exposure duration of ( ) days. the three most frequent comorbidities were hypertension ( %), diabetes mellitus ( %) and chronic obstructive pulmonary disease ( %) ( table ). the mean strobe score of included studies was . (sd . ). in the studies included (n= ), the frequency of lqt during hcq treatment was . % ( % ci: . - . %) (figure ). in studies (n= ), patients were also taking other qt-prolonging drugs as well as hcq. in studies (n= ), patients were reported to be taking azithromycin (azi) and in of those studies, patients were also on other qt-prolonging drugs, with the three most frequent being lopinavir/ritonavir, propofol and amiodarone [ , , [ ] [ ] [ ] , , ] . in the subgroup analyses (table ) , those older than years had a higher risk of hcq-associated lqt (p < . ). the frequency of lqt also seemed higher in the studies that reported hcq combination with azi and/or other qt-prolonging agents (p= . ). no significant difference was evidenced when the total hcq dose was greater than mg. in / studies, a subgroup analysis was performed that suggested several risk factors related to the frequency of hcqassociated lqt. the main risk factor was simultaneously taking other qt-prolonging agents, among which were propofol and amiodarone [ , , , ] . other statistically significant risk factors were renal failure or increased creatinine [ , ] , structural heart disease [ ] and ≥ points in systemic inflammatory response syndrome score [ ] . the frequency of lqt in sars-cov- patients treated with hcq was . %. however, most patients were also taking other qt-prolonging drugs. in the minority of studies where hcq was the only qt-prolonging drug, the frequency of lqt seemed smaller ( . %). overall, the frequency of hcq discontinuation due to lqt was . %. during hcq treatment, the frequency of torsades de pointes, ventricular arrhythmias and arrhythmogenic death were very low. the risk of lqt in covid- patients treated with hcq seemed higher in patients older than years. our findings can be compared with those of four related types of studies, namely: similar systematic reviews in sars-cov- infection, studies with hcq used for short periods against malaria, studies of hcq used chronically for rheumatologic diseases, and studies with other drugs that cause lqt. currently, three meta-analytic studies on cardiotoxicity of antimalarials used in sars-cov- infection have been published [ ] [ ] [ ] and the differences with the present study (which reports lower risk frequencies) may be due to the fact that our study had the largest numbers of subjects and we only included studies with hcq. indeed, studies with cq [ , ] were purposefully excluded after borba et al.'s study [ ] was prematurely discontinued due to high mortality associated with high doses of cq. since then, clinical guidelines for treatment for sars-cov- include hcq and not cq, and there is evidence that the cardiotoxicity of cq is greater than hcq in patients with sars-cov- infection [ ] . other than drugs, risk factors that cause repolarization reserve reduction and increase the risk of tdp include drug interactions affecting drug serum levels, sex, structural heart disease, genetic polymorphisms, electrolyte disturbances, bradycardia, and hepatic disease [ ] . in the present study, the majority of patients were simultaneously taking other qt-prolonging agents including azi, lopinavir/ritonavir, propofol or amiodarone. azi is known to be associated with qt interval prolongation [ ] [ ] [ ] , tdp [ ] and polymorphic ventricular tachycardia in the absence of qt interval prolongation [ ] . the proarrhythmic mechanism of azi is that it potentiates cardiac na+ current to promote intracellular na+ loading [ ] ; however, the frequency of this is low, as azi has been calculated to cause additional cardiovascular deaths per million treatments [ ] . antimalarials are also associated with lqt, especially quinidine and halofantrine [ ] [ ] [ ] . in the pre-covid- era, sudden cardiac death with cq has been reported only by rapid intravenous administration or by self-inflicted overdose, causing hypotension due to vasodilation and negative inotropy [ ] . mortality associated with the administration of cq (not hcq) in the treatment of plasmodium falciparum and vivax malaria has been reported to be . % ( / ) and % ( / ), respectively [ ] . hcq is used in the treatment of rheumatoid arthritis and systemic lupus erythematosus. a study [ ] found a . % frequency of hcqinduced lqt in rheumatoid arthritis and systemic lupus erythematosus patients who received - mg/day of hcq for a mean of . years; it should be noted that these patients were simultaneously taking more than one medication that could prolong the qt interval. chatre et al. investigated cases published until of cq and hcq-induced cardiac adverse reactions and found cases of hcq-induced lqt ( . %) [ ] . in a study of a cohort of systemic lupus erythematosus patients on antimalarial-induced ecg abnormalities, . % frequency of hcq-induced lqt and . % of ventricular bigeminy were found [ ] . recently, hooks et al studied patients with rheumatologic diseases and found a frequency of . % of hcq-induced lqt; additionally, they found that chronic kidney disease (ckd), history of atrial fibrillation (af), and heart failure were independent risk factors for lqt; the median dosage of hcq was mg daily and duration of hcq therapy was ( - ) days [ ] . the detection of drug-induced lqt is important because it increases the risk of tdp, which in turn can degenerate into ventricular fibrillation in % of cases [ , ] . medications that increase qtc > ms are generally discontinued because they increase the risk of tdp by to -fold [ , ] . of all the drugs that are associated with lqt, antiarrhythmic drugs are those that have the highest risk of tdp with an incidence of to %, while in non-cardiovascular drugs the incidence is much less ( . %) [ ] . the present study has limitations, among which is the design of the included studies (mostly observational) and the strategies used to detect hcq-induced lqt by these studies. many studies where hcq has been used for covid- infection have not been able to measure qtc because this requires special devices (e.g. mobile cardiac outpatient telemetry) to avoid exposure to the virus, or the use of special personal protective equipment (ppe) for its measurement [ ] . on the other hand, cipriani et al. reported -h qtc dynamics in covid- patients versus controls and reported that the former had higher qtc values with no significant hourly variability, recommending that there is no need to preform multiple daily ecgs for the monitoring of possible treatment toxicity [ ] . given the very low number of studies including a majority of women, the sex differences between the risk of hcq-associated lqt warrant further investigation. in conclusion, hcq-associated cardiotoxicity in sars-cov- patients is uncommon but requires ecg monitoring especially in those older than years and/or taking other qt-prolonging drugs. ethical approval: not required a global treatments for coronaviruses including covid- urgent guidance for navigating and circumventing the qtc-prolonging and torsadogenic 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and torsade de pointes in germany predicting drug-induced qt prolongation and torsades de pointes long qt syndrome and sinus bradycardia-a mini review high prevalence of the risk factors for qt interval prolongation and associated drug-drug interactions in coronary care units table . characteristics of the studies included in the meta-analysis key: cord- -kl wh zg authors: xu, h; zhang, xy; wang, ww; wang, js title: hydroxychloroquine increased psychiatric-like behaviors and disrupted the expression of related genes in the mouse brain date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: kl wh zg hydroxychloroquine (hcq), which has been proposed as a therapeutic or prophylactic drug for sars-cov- , has been administered to thousands of individuals with varying efficacy; however, our understanding of its adverse effects is insufficient. it was reported that hcq induced psychiatric symptoms in a few patients with autoimmune diseases, but it is still uncertain whether hcq poses a risk to mental health. therefore, in this study, we treated healthy mice with two different doses of hcq that are comparable to clinically administered doses for days. psychiatric-like behaviors and the expression of related molecules in the brain were evaluated at two time points, i.e., h and days after drug administration. we found that hcq increased anxiety behavior at both h and days and enhanced depressive behavior at h. furthermore, hcq decreased the mrna expression of interleukin- beta and corticotropin-releasing hormone (crh) in the hippocampus and decreased the mrna expression of brain-derived neurotrophic factor (bdnf) in both the hippocampus and amygdala. most of these behavioral and molecular changes were sustained beyond days after drug administration, and some of them were dose-dependent. although this animal study does not prove that hcq has a similar effect in humans, it indicates that hcq poses a significant risk to mental health and suggests that further clinical investigation is essential. according to our data, we recommend that hcq be carefully used as a prophylactic drug in people who are susceptible to mental disorders. at present, tens of millions of people worldwide have been infected with coronavirus disease (sars-cov- ). among the many potential therapeutic drugs for sars-cov- , society has perhaps paid the most attention to hydroxychloroquine (hcq). although emerging evidence does not suggest that it is efficacious in treating sars-cov- ( - ), it has already been administered to thousands of individuals. furthermore, more than hundred clinical trials of hcq involving tens of thousands of potential participants have been registered. hcq is not only being used to treat patients with sars-cov- but is also being used as a pre-exposure or postexposure prophylactic drug for sars-cov- in healthy populations who are at high risk of exposure to sars-cov- ( , ) . although hcq has been used clinically for decades to treat malaria and autoimmune diseases, such as systemic lupus erythematosus (sle), sjögren's syndrome (ss) and rheumatoid arthritis (ra) ( ), our understanding of its potential adverse effects, especially in patients with sars-cov- and uninfected individuals, is insufficient. there have been reports of serious adverse effects related to hcq, including retinopathy, qt interval prolongation, hypoglycemia, cardiomyopathy, neuropsychiatric effects and so on ( , ) , and qt interval prolongation is the most closely monitored adverse effect in patients with sars-cov- . less attention has been paid to the effects of hcq on mental health, although multiple studies have reported severe psychiatric symptoms, including agitation, hallucinations, anxiety, depression, and suicidal ideation, in patients treated with hcq or chloroquine ( ) ( ) ( ) ( ) . however, whether these neuropsychiatric symptoms are caused by hcq alone or by the interaction between hcq and the disease is not well studied, possibly due to the limited number of reported cases. therefore, whether hcq poses a serious risk to mental health is still not clear. in addition to acting as an antimalaria drug, hcq is an immunomodulator that exerts broad anti-inflammatory effects, including inhibition of antigen presentation to dendritic cells, reduced signaling of both b and t cells and reduced cytokine production in macrophages ( , ) . microglia, as macrophages that reside in the brain and play critical roles in neuronal functions, are also potential targets of hcq ( ) . however, the ability of hcq to cross the blood-brain barrier (bbb) is quite limited ( , ) ; however, considering that hcq has a very long half-life in vivo ( ) , its direct effect on microglia should not be underestimated. in addition, hcq may affect the cns via its inhibitory on peripheral cytokines, such as il- β or tnf-α, which can bind to the vascular endothelium of the brain or directly entering the cns ( ) , resulting in a reduction in the production of corresponding cerebral cytokines and induction of a neuroinflammatory deficit in the brain. although hypo-neuroinflammation is not as concerning as hyperneuroinflammation, and the latter has been indicated as a potential pathology of multiple psychiatric disorders ( ) , emerging evidence suggests that maintaining the balance of the immune system is more important than preventing inflammation in the cns ( ) and that low levels of inflammation in the brain can also have negative effects on cognition and behaviors ( , ) . therefore, the immunosuppressive effect of hcq may have negative effects on the cns, especially in healthy individuals who do not suffer from hyperinflammation induced by infection. therefore, in this study, the potential effects of hcq on mental health were investigated in healthy mice. the animals were administered hcq for days, and psychiatric-like behaviors related to anxiety, depression, and cognitive impairment were evaluated. furthermore, the expression of genes, including brain-derived neurotrophic factor (bdnf), corticotropin-releasing hormone (crh) and interleukin- beta (il- β), which are closely related to mental health, were also measured in two brain areas, namely, the hippocampus and amygdala, which play critical roles in mental health and in which cytokines are produced notably ( ) . in this study, we calculated appropriate doses of hcq for mice based on clinically administered doses of hcq according to the average body surface area of humans and mice. the clinical dosages used for the calculations were and mg on the first day followed by and mg daily for the next days. we evaluated behavior and gene expression at two time points, i.e., h and days after drug administration, to evaluate the immediate and lasting effects of the drug. the male c bl/ j mice used in this study were purchased from vital river laboratory animal center (beijing, china) at the age of weeks. after a week of adaptation, the mice were randomly divided into two groups: the immediate group and the lasting group. the mice in each group were further randomly divided into three subgroups, i.e., the high-dose, low-dose and vehicle groups; there were ~ animals per subgroup. all animals were maintained under controlled environmental conditions ( h light/ h dark cycle, lights on at am; ambient temperature - °c; humidity ± %) with free access to food and water. all procedures were approved by the institutional review board of the institute of psychology, chinese academy of sciences and performed in compliance with the national institutes of health guide for the care and use of laboratory animals. hydroxychloroquine sulfate (ark pharm, chicago, usa) was dissolved in sterile phosphate-buffered saline (pbs), ph= . . the drug was intraperitoneally (i.p.) injected twice per day (every hours) for consecutive days. on the first day, the high dose of hcq was mg/kg/injection, and the low dose was mg/kg/injection; hcg was given at half of these doses for the next days. the body weight of each mouse was recorded every two days during administration and every five days after administration. to test the immediate/lasting effects of hcq on behavior and the expression of molecules in the brain, behavioral tests were performed h and days after the last injection. the interval between each test was h. for each test, the apparatus was cleaned with % ethanol to eliminate the influence of excrement or odor on subsequent animals. the tests were performed in the following order. the nor test was used to evaluate the locomotor activity and working memory of the mice. the test consisted of three phases, including the adaptation phase (phase , min), the training phase (phase , min) and the test phase (phase , min). in phase , the mice were placed in the apparatus (l*w*h: cm* cm* cm, with dim light), allowed to explore freely for min and then returned to their home cages. during the exploration period in phase , the trajectory of each mouse was captured with a camera and automatically tracked and recorded with software (anilab, suzhou, china). the distance traveled every min throughout the -min period was recorded as a measure of the locomotor activity of the mice. in phase , two identical objects (lego bricks, cm* cm* cm) were placed in opposite corners of the apparatus cm from either side of the wall. the mice were introduced from the corner not containing an object, allowed to explore for min and then returned to their home cages for h. in phase , one of the two objects were replaced with a novel object of similar size but of a different color and shape. the mice were then placed in the apparatus again and allowed to explore for an additional min. the exploration of each mice was recorded with an overhead camera and then hand-scored by a pretrained independent investigator blinded to the experimental groups. the total sniff time and the discrimination index ((sniff time for novel object-sniff time for familiar object)/(total sniff time)) ( ) were the main parameters measured in this test. discrimination index data for a few animals that showed a lack of exploratory activity (< s for one object) were excluded. the day after the locomotor and novel object recognition tests were performed, the mice were subjected to the epm test in a quiet dim room to evaluate anxiety-like behaviors. the mice were placed in the center of an elevated maze facing the open arms at the beginning of the test. during the test, the mice were allowed to freely explore the maze for min, and the movement trajectory and time spent in each area were automatically recorded by software. the duration of time spent in the open arms and in the closed arms was reported. the fst was performed h after the epm test to measure depression-like behavior. the mice were forced to swim individually for min in a glass cylinder with a diameter of cm filled cm deep with water at a temperature of ± °c. swimming behavior was recorded using a horizontal video camera, and immobility time (during which the mice exhibited no movement except for that of the whiskers) was hand-scored by two independent observers blinded to the experimental groups. the immobility time of each mouse was calculated as the average of the times measured by the two observers. the mice were sacrificed h after all the behavioral tests were completed. the hippocampus and amygdala were quickly extracted from each brain by using a mouse brain slicer. the tissues were immediately frozen on dry ice and then stored at - °c. rna was extracted from the tissues by using the rnaprep pure tissue kit (tiangen, beijing, china) according to the standard protocol. the rna concentration was quantified using the nanodrop system (thermo fisher scientific, ma, usa), and the quality of the rna was evaluated by gel electrophoresis. one microgram of total rna from each sample was reverse transcribed into first-strand cdna by using the m-mlv kit (promega corporation, madison, wi, usa) following the manufacturer's protocol. mrna expression levels were determined by real-time pcr using sybr™ green pcr master mix (life technologies, california, usa) on an abi system, and dissociation curve analysis was performed at the end of each run. each sample was individually amplified and duplicated, and then the average value was used for subsequent analyses. relative mrna expression was calculated by the standard −ΔΔct method using glyceraldehyde -phosphate dehydrogenase (gapdh) as a housekeeping gene. the following forward (f) and reverse (r) primers (bgi tech, beijing, china) were used: il- β: forward: tgtctgaagcagctatggcaac; reverse: ctgcctgaagctcttgttgatg; bdnf: forward: tggctgacacttttgagcac; reverse: aagtgtacaagtccgcgtcc; crh: forward: gatctcaccttccaccttctg; reverse: cgcaacatttcatttcccga; gapdh: forward: caagctcatttcctggtatgac; reverse: ctgggatggaaattgtgagg. the data are presented as the mean ± sem and were analyzed by graphpad . . comparisons between the three groups were performed using one-way or two-way anova, and lsd post hoc multiple comparisons were carried out. statistical significance for all analyses was set at p≤ . . as shown in figure a , two-way anova indicated that there was a significant effect of time (f ( . , . ) = . , p< . ) and a significant time × drug interaction effect (f ( , ) = . , p= . ) but no significant effect of drug treatment (f ( , ) = . , p= . ). post hoc tests indicated that the rate of increase in body weight, which was significantly lower in the low-dose group (p= . ) and high-dose group (p= . ) than in the pbs group, was significantly different between groups h after injection, but the difference between the low-dose group and high-dose group was not significant (p= . ). the rate of increase in weight over subsequent days were not significant. ± . , p= . , respectively) anxiety-like behavior seemed to become more obvious over time. for the novel object recognition test ( figure d, e) , one-way anova indicated that there was no significant effect of hcq treatment on the discrimination index at days after the final injection (f ( , ) = . , p= . ). however, there was a significant difference in total sniff time (f ( , ) = . , p= . ). post hoc analysis revealed that mice in the high-dose group spent less time sniffing ( . ± . vs. . ± . , p= . ) than mice in the pbs group. for the fst (figure f), one-way anova indicated that there was no significant effect of hcq treatment on immobility time at days after the final injection (f ( , ) = . , p= . ). as shown in figure , hcq rapidly altered the mrna expression levels of bdnf, il- β, and crh in the hippocampus and amygdala. one-way anova indicated that there was a significant effect of drug treatment on the expression of il- β in the hippocampus (f ( , ) = . , p= . ) but not in the amygdala (f ( , ) = . , p= . ). post hoc analysis revealed that the mrna expression of il -β in the high-dose group was significantly lower than that in the low-dose group (p= . ) and pbs group (p= . ). there was a significant effect of drug treatment on the expression of crh in the hippocampus (f ( , ) = . , p= . ) and amygdala (f ( , ) = . , p= . ). post hoc analysis revealed that the mrna expression of crh in the high-dose group was significantly lower than that in the low-dose group in the hippocampus and amygdala (p= . , p= . , respectively) and that the mrna expression of crh in the high-dose group was significantly lower than that in the pbs group (p= . ) in the hippocampus. there was a significant effect of drug treatment on the expression of bdnf in the hippocampus (f ( , ) = . , p= . ) and amygdala (f ( , ) = . , p= . ). post hoc analysis revealed that in the hippocampus, the mrna expression level of bdnf was significantly lower in the low-dose (p= . ) and high-dose (p= . ) groups than in the pbs group. in the amygdala, the mrna expression level of bdnf was significantly lower in the high-dose (p= . ) group than in the pbs group ( . ). as shown in figure , hcq persistently altered the mrna expression levels of bdnf, il- β, and crh in the hippocampus and amygdala. one-way anova indicated that there was a significant effect of drug treatment on the expression of il- β in the hippocampus (f ( , ) = . , p< . ) but not in the amygdala (f ( , ) = . , p= . ). post hoc analysis revealed that the mrna expression of il- β in the high-dose group and low-dose group was significantly lower than that in the pbs group (p< . for both). there was a significant effect of drug treatment on the expression of crh in the hippocampus (f ( , ) = . , p= . ) but not in the amygdala (f ( , ) = . , p= . ). post hoc analysis revealed that the mrna expression of crh in the high-dose group and low-dose group was significantly lower than that in the pbs group (p= . and p= . , respectively). there was a borderline significant effect of drug treatment on the expression of bdnf in the hippocampus (f ( , ) = . , p= . ) and a significant effect of drug treatment on bdnf expression in the amygdala (f ( , ) = . , p= . ). post hoc analysis revealed that in the hippocampus, the mrna expression level of bdnf was lower in the low-dose (p= . ) and high-dose (p= . ) groups than in the pbs group. in the amygdala, the mrna expression level of bdnf was significantly lower in the high-dose (p= . ) group than in the pbs group. the present study found that short-term repeated administration of hcq led to a significant and persistent increase in anxiety-like behaviors in healthy mice and had an effect on depression-like behavior in the short term. hcq also caused a sustained decrease in the mrna expression of il- beta, crh and bdnf in the hippocampus; however, in the amygdala, hcq temporarily affected the expression of crh and induced a lasting significant decrease in the expression of bdnf. some of these behaviors and molecular changes, including the changes in anxiety-like behavior, the expression of crh in the hippocampus and the expression of bdnf in the amygdala, were dose-dependent, as the changes in the high-dose group were greater than those in the low-dose group. to the best of our knowledge, this study is the first to test the psychiatric effect of hcq in an animal model, and our results suggest that, in healthy mice, hcq administration can induce persistent behavioral changes and disrupt gene expression in the brain. the hcq treatment regimen used in this study mimics the current short-term regimen used for the treatment of sars-cov- . according to current clinical studies, most patients take hcq for to days at a dose of mg to mg on the first day followed by half of the original dose on the remaining days; in most studies, doses from mg to mg are administered on the first day. in this study, mice were given hcq at one of two doses for days. hcq was administered at a dose of mg/kg or mg/kg on the first day and at a dose of mg/kg or mg/kg once daily for the next days; these doses corresponded to mg or mg on the first day followed by mg or mg per day on the subsequent days in humans. the drug dose for mice was calculated based on the average body surface area ratio of mice to humans (mouse/human= . ) ( ). it should be noted that for this calculation, the dose per kilogram for humans was calculated based on the global average body weight for adults, which is kg; however, the body weight of many people who take the drug may be higher. therefore, the calculated dose administered to the animals may be higher than the corresponding dose administered to humans. because the purpose of this study was to determine the potential risk of this drug, we thought it was important to choose a slightly higher dose and longer period of administration. in this study, hcq was administered by intraperitoneal (i.p.) injection because approximately %~ % of hcq can be absorbed upon oral administration ( ); thus, there would not be a large difference in drug absorption when the drug is administered orally and by i.p. injection . however, the speed of drug entry into tissues may be faster following i.p. injection than following oral administration. a previous study also indicated that the tolerated dose of i.p. injected hcq is lower than that of orally administered hcq ( ); thus, we chose to administer the drug in two injections per day (one injection every h). the body weight data showed that drug tolerance was basically good; except for a dose-dependent decrease in body weight on the first day, the rate of increase in body weight was not significantly different between the hcq groups and the vehicle group. additionally, evaluation of the locomotor activity of the animals did not show a significant difference between groups except for a hcq-induced decrease in the locomotor activity of mice in the lasting group during the first min of the test, which may have been a consequence of the anxiety-induced decrease in exploration in these animals. two time points were examined in this study, i.e., h and days after administration, and animal tissues were extracted h after the completion of all the behavioral tests; thus tissues were collected and days after administration. previous studies have shown that hcq persists for a long time in vivo, with a half-life of - days in humans ( ) ; however in rats, % of hcq is eliminated by the th day, and % is cleared after days ( ) . the metabolic rate of mice is approximately twice that of rats and times that of humans ( ) , indicating that the direct effect of hcq in the lasting group ( days after administration) was likely quite small. therefore, these two time points can effectively distinguish the immediate and lasting effects of hcq. in the current study, anxiety-like behavior was measured by using the epm test, depression-like behavior was measured by using the fst, and working memory was measured by using the nor test. the results showed that hcq had a significant and sustained effect on anxiety, as indicated by decreased exploration time in the open arms. depression-like behavior was also affected in the immediate group, as indicated by a significantly longer immobility time in the high-dose group than in the low-dose group but not in the vehicle group; these results suggest that hcq has an effect on depression-like behavior. this increase in depression-like behavior is consistent with the changes in the expression of crh in the amygdala. our results did not reveal a significant effect of hcq treatment on working memory, but total exploration time was decreased after hcq administration, which also suggests that the drugs increased anxiety. previous human studies have reported that hcq can induce anxiety-and depression symptoms ( , ) , but no reports have shown that it impairs cognitive function. however, there have been only a few reports in humans, and there is a close relationship between cognitive impairment and psychiatric disorders ( ) . furthermore, while we only measured working memory, the proteins in the brain that were affected by hcq, such as bdnf, have been indicated to be closely related to cognitive functions ( ) ; therefore, our results cannot rule out the possibilty that hcq affects cognitive functions. the mrna expression of three genes, namely, il- β, crh and bdnf, was measured in this study; these three genes are related to neuroinflammation, the hpa axis and neuroplasticity, respectively, and all of them are closely associated with the pathogenic mechanisms of mental illness ( , , ) . our results showed that hcq has a significant effect on all three molecules. at both time points, hcq significantly decreased the expression of il- β in the hippocampus but had little effect on il- β in the amygdala, possibly because the hippocampus is more susceptible to changes in peripheral cytokine levels, as it is closer to the choroid plexus than the amygdala ( ) . moreover, hcq significantly decreased the expression of crh in the hippocampus at both time points in a dose-dependent manner. furthermore, hcq had little effect on the expression of crh in the amygdala; a significant effect was only observed in the immediate group, but the difference was mainly between the high-dose group and the low-dose group. in addition, hcq significantly decreased the expression of bdnf in both brain regions at both time points. it has been reported that these genes interact with one another; for example, it was reported that il- β can regulate the expression of crh and bdnf ( , ) . however, the widespread influence of hcq on these genes is still beyond our initial expectation. considering the importance and extensive influence of these genes on the cns, the influence of hcq on the cns may be quite serious. additionally, hcq has a much longer half-life in humans than in rodents, and our results suggest that hcq has significant effects on anxiety and gene expression more than days after administration. moreover, based on the expression pattern of il- β, the effect of hcq on il- β increases after drug withdrawal. this implies that the influence of hcq on human emotions and cns functions may last for months. previous case reports have also indicated that stopping drug administration cannot resolve its psychiatric effect quickly ( ) . it should be noted that, as a preclinical study, the present study cannot prove that the clinical use of hcq induces anxiety or depression in humans. the results of this study also do not clarify the mechanisms underlying the effect of hcq on behavior or brain function. based on these preliminary results, we speculate that the effect of hcq on the cns is mainly due to its peripheral immunosuppressive effects, which induce hypo-neuroinflammation in the brain, impairing the functions of immune cells in the brain, such as microglia. if our hypothesis is true, healthy individuals and patients with mild disease who do not suffer from infection-induced hyperinflammation would be at a higher risk of psychiatric symptoms than patients with severe sars-cov- . however, much more work is needed to prove this speculation. our results prove that hcq poses a serious risk to mental health. given the current pandemic, we hope that based on the results reported in this study, scientists and clinical doctors will pay more attention to this potential adverse effect of hcq and start to monitor the mental health status of patients in clinical trials. furthermore, we suggest that individuals who are susceptible to psychiatric disorders should not take hcq, especially as a prophylactic drug for sars-cov- . figure : effects of hcq on body weight and locomotor activity. (a) the rate of increase in body weight of mice in the lasting group. (b) the locomotor activity of mice in the immediate group. 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hosseini, mohammad mahdi; rasoulinejad, ahmad; akhavan-niaki, haleh title: molecular effects and retinopathy induced by hydroxychloroquine during sars-cov- therapy: role of cyp isoforms and epigenetic modulations date: - - journal: eur j pharmacol doi: . /j.ejphar. . sha: doc_id: cord_uid: nav g k antimalaria drugs such as chloroquine (cq) and hydroxychloroquine (hcq) have been administered to several inflammatory diseases including rheumatoid arthritis and systemic lupus erythematosus, and infectious diseases such as acquired immune deficiency syndrome and influenza. recently, several patients infected with novel severe acute respiratory syndrome coronavirus (sars-cov- ) were given hcq, and showed a discrepant response. hcq inhibits sars-cov- cell entry, and inflammatory cascade by interfering with lysosomal and endosomal activities, and autophagy, impeding virus-membrane fusion, and inhibiting cytokine production resulted from inflammatory pathways activation. despite ongoing administration of hcq in a wide spectrum of disorders, there are some reports about several side effects, especially retinopathy in some patients treated with hcq. cytochrome p (cyp ) and its isoforms are the main metabolizers of hcq and cq. pharmacokinetic properties of cyp enzymes are influenced by cyp polymorphism, non-coding rnas, and epigenetic mechanisms such as dna methylation, and histone acetylation. accumulating evidence about side effects of hcq in some patients raise the possibility that different response of patients to hcq might be due to difference in their genome. therefore, cyp genotyping especially for cyp d might be helpful to refine hcq dosage. also, regular control of retina should be considered for patients under hcq treatment. the major focus of the present review is to discuss about the pharmacokinetic and pharmacodynamic properties of cq and hcq that may be influenced by epigenetic mechanisms, and consequently cause several side effects especially retinopathy during sars-cov- therapy. potential antimalarial drugs such as chloroquine (cq) and hydroxychloroquine (hcq) have been long used to prophylaxis and treatment of malaria (warhurst et al. ) . although, administration of cq and hcq have extended to several inflammatory diseases such as rheumatoid arthritis (ra), systemic lupus erythematosus (sle), and against some viruses including human immunodeficiency virus (hiv), and influenza (chiang et al., ; ooi et al., ) . the current pandemy of novel severe acute respiratory syndrome coronavirus (sars-cov- ) caused a high morbidity and mortality with , , confirmed cases and , deaths until july, , , deaths until july, , ( a . recently several studies suggested that hcq j o u r n a l p r e -p r o o f could be an effective agent to decrease the viral load, and ameliorate clinical manifestations of patients infected with sars-cov- gautret et al., ) . even though, clinical trial studies of hcq in patients with sars-cov- showed inconsistent results about the effect of hcq on decreasing patients' infection gautret et al., ) . there is some evidence that administration of hcq gives rise to retinopathy in some patients affected with autoimmune diseases such as ra and sle (kobak & deveci, ; lee et al., ) . it's noteworthy that several risk factors including duration of use, exceeding of recommended dose (> mg/kg per day ( mg in an kg person)), and pre-existing retinal disease have been identified for ocular toxicity in patients who were under hcq therapy. correspondingly, there is also evidence that prolonged use of hcq increases the risk of ocular toxicity, and high doses could lead to rapid changes in vision and consequently retinopathy development. the much higher doses of hcq used in combat against viral infections such as sars-cov- ( or mg daily dose) in comparison with its recommended doses for treatment of ra and sle ( - mg/day) have raised the possibility of more remarkable retinopathy progression in sars-cov- patients under hcq therapy (mahase, ; gautret et al., ; pereira, ) . furthermore, growing body of evidence demonstrated that several factors including cyp single nucleotide polymorphisms (snps), and epigenetic molecules such as non-coding rnas (ncrnas), dna methylation and histone acetylation influenced the expression levels of cyp , and consequently might influence hcq metabolism. (li et al., ; zeng et al., ; wang et al., ) . the major purpose of this review is to discuss the pharmacokinetic and pharmacodynamic characteristics of cq and hcq that may be influenced by epigenetic mechanisms including ncrnas and cyp d snps, and thereby cause several side effects such as cardiotoxicity, prolonged qt interval, gastrointestinal problems (like dyspepsia and abdominal cramps), central nervous system or skin disorders, and especially retinopathy. also, we review the advantage of personalized medicine to determining hcq dosage regarding the unique genotype of patients for sars-cov- treatment. properties (rainsford et al., ) . both drugs are appropriately distributed to aqueous cellular and intercellular compartments after oral administration, and have a prolonged residence time (one month in average) (augustijns et al., ; schrezenmeier & dörner, ) . table summarizes the pharmacokinetic of these drugs. hcq similar to cq is absorbed by upper intestinal tract cells, and its renal clearance is . times less than cq (hcq: % versus cq: %). the apparent volumes of distribution of hcq are , l (calculated from plasma) and , l (calculated from blood), respectively which would indicate that hcq accumulates in other compartments. cq has an apparent volume of distribution equal to , l (calculated from plasma), and , l (calculated from blood) (cutler et al, ; tett et al., ) . also, un-metabolized excretion of hcq ( %) is approximately similar to cq ( %) (collins et al., ; cutler et al., ) . accumulating evidence revealed that hcq similar to cq is excreted into breast milk, and moves across the placenta to exert its pharmacokinetic effects on fetus (law et al., ; motta et al., ) . recently, hcq was mostly applied to treat a wide spectrum of disorders including connective tissue diseases, and autoimmune disorders in comparison with cq due to lower toxicity, and side effects stokkermans & trichonas, ) . previous studies on animal models revealed that hcq may be distributed in a wide spectrum of tissues including adrenal, spleen, lung, liver, kidney, heart, retina-choroid, skin, pituitary, muscle, brain, fat, and bone (mcchesney, ; mcchesney et al., ) . several studies conducted on pharmacodynamic of hcq proposed a multi-pathway model for hcq effects (fox, ; kyburz et al., ) . obtained results showed that the major mechanism of hcq action is modulation (elevation) of cellular ph ( b; fox, ) . hcq properly passes through the lysosomal membrane, and inhibits several processes including protein degradation, antigen presentation, and toll-like receptor (tlr) signalling by increasing the ph of lysosome and endosome from to (fox, ; kyburz et al, ) . elevated levels of ph in lysosome lead to metabolites deposition, and impediment of antigen presentation mediated by major histocompatibility complex (mhc) class ii through lysosomal acidic hydrolysis blockage (fox, ; schrezenmeier & dörner, ) . inhibition of antigen presenting cells including dendritic cells , macrophages, and b cells prevents the process of t cells activation mediated by mhc ( fig a) (ireland & unanue, ) . several studies reported that lysosomal enzymes blockage prolongates the degradation of macromolecules in lysosome, and thereby progressively converts them into lipofuscin (ding j o u r n a l p r e -p r o o f sundelin & terman, ) . lipofuscins are brown-yellow accumulated metabolites such as inclusions which appear gradually with age (terman & brunk, ) . on the other hand, hcq prevents from autoantigen presentation mediated by mhc ii on antigen presenting cells due to obstruction of autoantigenic proteins degradation (yoon et al., ) . furthermore, hcq exerts its anti inflammatory effects via increasing the ph levels of endosome which is essential in tlr signaling in innate immunity (thwaites et al., ) . also, hcq could inhibit the innate immunity by binding to tlr ligands including dna and single strand rna (ssrna) which interact with tlr and tlr , respectively (schrezenmeier & dörner, ; thwaites et al., ) . strinkingly, several recent studies suggested that hcq acts as an anti inflammatory agent through disruption in cyclic gmp-amp (cgamp) synthase (cgas) binding with its ligand (cytosolic dna) an et al, ) . activated cgas by binding to cytosolic dna leads to production and binding of cgamp to stimulator of interferon genes (sting), and thereby production of several cytokines especially type i interferons zhang et al., ) . recent findings revealed that palmitoyl-protein thioesterase (ppt ) that is localized in lysosome, is a central target of hcq to modulating of ph (rebecca et al., ) . ppt enzyme cleaves and removes long-chain fatty acids called palmitate from proteins that are no longer required. ppt facilitates tumor growth via two downstream pathways including mtor and autophagy (rebecca et al., ) . increasing body of evidence illustrated that hcq potentially prevents tumor growth and inflammatory pathways (e.g ra) via inhibiting ppt ( fig b) (cook et al., ; schrezenmeier & dörner, ) . recently, organic anion transporter family member a (oatp a ) was identified as a novel target of hcq (xu et al., ) . oatp a , is a solute carrier transporter, expressed in a wide spectrum of tissues such as brain, liver, breast, testis, lung, kidney, and in retinal pigment epithelial (rpe) cells to regulate the transport and uptake of multiple substrates especially drugs and xenobiotics (lee et al., ) . collectively, hcq by influencing multiple pathways supresses inflammation in several inflammatory diseases, infectious diseases, cancers, and recently sars-cov- infection. it is becoming increasingly evident that hcq may probably be a potential agent to combat sars-cov- . previously, several studies revealed the antiviral effects of hcq against some viruses such as hiv and influenza (chiang et al., ; ooi et al., ) . recently, various studies demonstrated that hcq prevents sars-cov- from cell entry via several processes (fig ) . hcq interferes with enzymes involved in glycosylation and activation of angiotensin converting enzyme (ace) including glycosyltransferases and sugarmodifying enzymes, and consequently blocks virus-membrane fusion. also, hcq interferes with spike (s) protein glycosylation, and disrupts endosome-mediated virus cell fusion. moreover, hcq impedes s protein cleavage which is a key point for cell fusion of sars-cov- by increasing lysosomal ph levels, and inhibiting its proteases (gautret et al., ; millet & whittaker, ; vincent et al., ; yao et al., ) . also, recent in vitro study showed that hcq and cq could decrease the viral replication in a concentration-dependent manner. moreover, hcq showed a higher safety and anti-sars-cov- efficacy relative to cq (yao et al., ) . hcq may also prevent from sars-cov- spreading after cell entry through elevating `endosomal and lysosomal ph, and thereby preventing from activation of inflammation cascades (srivatsan, ; vincent et al., ) . furthermore, pseudovirion colocalization with niemann-pick c (npc )-positive endolysosomes (le/lys) disclosed that hcq could exert its antiviral effects through disturbance in sars-cov- replication (srivatsan, ). intriguingly, a study performed on hcq mechanism of action illustrated that hcq inhibits viral genome release by changing the number, size, and morphology of early endosomes or endolysosomes mingo et al., ) . furthermore, hcq could diminish inflammation responses through several pathways including inhibiting mhc ii-mediated t cell activation, inhibiting the release of cytokines such as il- , il- , tnf-α induced by t-cell activation, blockage of tlr signaling, stimulation of interferon genes (the sting pathway) by cgas and thereby inhibiting cytokines release including i interferon, il- and tnf-α induced by tlr and sting signaling ( fig a and b) (fox, ; schrezenmeier & dörner, ; ireland & unanue, ; thwaites et al., ; an et al., ) . therefore, anti-inflammatory mechanism of hcq action created this hypothesis that it might be able to halt cytokine release syndrome, and reduce consequently organ damage especially lung, induced by covid- . there are some reports about the remarkable association between cytokine storm especially il- and clinical and pathological manifestations of patients with sars-cov- , serum sars-cov- viral load (rnaaemia), and mechanical ventilation requirement gao et al., ; herold et al., ; . collectively, hcq could exert its anti-covid- effects via impeding virus' cell entry and post cell entry events such as viral replication, and inflammatory responses. j o u r n a l p r e -p r o o f hcq is structurally different from cq only by a hydroxyl group in its n-ethyl side chain (finbloom et al., ) . cyp and its isoforms play a crucial role in hcq and cq metabolism (stokkermans & trichonas, ) . both of them undergo some changes especially dealkylation by cyp isomers, and become converted into active metabolites (furst, ; projean et al., ) . several investigations suggested that multiple cyp isoforms are involved in hcq and cq metabolism including cyp c , cyp a , cyp d , and cyp a although the first three of them are more important relative to cyp a (fig ) ( li et al., ; projean et al., ) . there is some evidence that administration of hcq could interfere with other cyp-metabolized drugs such as metoprolol (somer et al., ) . metoprolol is a beta blocker drug metabolized through cyp d that is administered to prevent angina (chest pain) and manage high blood pressure. during hcq and metoprolol combination therapy, hcq interferes with metoprolol metabolism by competing for cyp d (somer et al., ) . another drug-drug interaction that could take place is simultaneous administration of hcq and methotrexate in management of ra. hcq decreases the gastrointestinal absorption and bioavailability of methotrexate by modulating ph levels (bannwarth et al., ) . concurrent administration of low dose of cq with cyclosporine was shown to cause a better treatment in patients with ra via synergic effects with cyclosporine, although significant renal dysfunction was observed (landewé et al., ) . it's noteworthy to mention that hcq could interfere with those drugs elevating gastric ph levels (e.g. h + /k + atpase pump inhibitors) including omeprazole, lansoprazole, pantoprazole, esomeprazole, and rabeprazole (namazi, ) . therefore administration of hcq in patients with covid- that are simultaneously taking other mentioned drugs could lead to unwanted side effects. ncrnas such as mirnas and lncrnas affect post-transcriptionally the expression levels of several cyp genes, and thereby influence the metabolism, and bioavailability of cyp mediated drugs (jilek et al, ; jin et al., ) . moreover, dna methylation and histone acetylation could affect the cyp-metabolized drugs through modulating cyp expression. j o u r n a l p r e -p r o o f mirnas are a novel class of non-coding transcripts with - nucleotides length, and play a crucial role in a broad spectrum of disorders (tiwar et al., ; vishnoi & rani, ) . in silico and in vitro (human embryonic kidney (hek) t cells) investigation on effect of hsa-mir- - p on expression levels of cyp b showed that hsa-mir- - p suppresses cyp b expression by binding to cyp b mrna ' utr (jin et al., ) . furthermore, a study carried out on patients with breast cancer (breast tumor and paired-normal tissue) showed a negative association between expression levels of mir- b and cyp b . also, it showed that cyp b contrary to mir- b is highly expressed in cancerous tissues. cyp b is expressed in estrogen-regulated tissues through catabolism of various procarcinogens, and -hydroxylation of β-estradiol (tsuchiya et al., ) . accumulating evidence showed that the expression levels of cyp d which have a fundamental role in hcq metabolism is influenced by several mirnas (pan et al., ; zeng et al., ) . surprisingly, a study performed on sh-sy y, and u cell lines demonstrated that testosterone could decrease cyp d activity by increasing the level of mir- and mir- - which bind to the ′ utr of cyp d mrna (li et al., ) . (li et al., ) . the high levels of cholesterol in rat induce the expression and binding of lnc-hc, a hepatocyte-specific lncrna, to hnrnpa b . lnc-hc/ hnrnpa b complex inhibits cholesterol-bile acid conversion by decreasing cyp a (fig b) (lan et al., ) . dna methylation and histone acetylation are the important epigenetic modifications that cells use to control gene expression of large number of genes (verdone et al., ; razin & kantor, ) . in this view, cyp might be influenced by other epigenetic mechanisms such as dna methylation, and histone modification . isoniazid induced promoter methylation of cyp a and cyp b resulted in their downregulation and liver injury promotion in rat. indeed, liver injury occurs probably due to upregulation of tlr , extracellular-signal-regulated kinase (erk), peroxisome proliferator-activated receptor (ppar)-γ, il- , and tnf-α, caused by decreasing levels of cyp a and cyp b (li et al., ) . human hepatoma cell line hepg were treated with -aza- '-deoxycytidine that inhibits dna methylation, and trichostatin a that inhibits histone deacetylation caused expression changes in a wide spectrum of genes such as xenobiotic metabolism (cyp a , cyp a , and cyp a ), and steroid biosynthesis (cyp a and cyp a ) genes (dannenberg & edenberg, ) . trichostatin a is an antitumor drug that was shown to induce apoptosis in hepg cells through histone h acetylation of cyp e promoter. trichostatin a-mediated cyp e upregulation promotes apoptosis and antitumor effects by producing mitochondrial reactive oxygen species (fig c) (yang et al., ) . astonishingly, the pro-inflammatory cytokine il- that plays a fundamental role in promoting colorectal cancer, was shown to increase the expression levels of cyp b in vitro and therefore mediates the metabolic activation of pro-carcinogens, which may lead to generation of reactive metabolites capable of damaging dna by genome-wide methylation of mir- b promoter (patel et al., ) . it seems that epigenetic mechanisms including light sensitivity is an essential process to intact vision (garway-heath et al., ) . permanent sensitivity to light is provided by ongoing generation and condensation of rhodopsin on the photoreceptor outer segment discs by rough endoplasmic reticulum and golgi apparatus (papermaster & schneider, ; ritter et al., ) . photoreceptor inner segment supports the outer segment by supplying necessary substances. also, the photoreceptor inner segment facilitates the elongation of photoreceptor outer segment toward rpe by continuous synthesis of discs (chen et al., ) . a study conducted on retinal photoreceptors of multiple animal models including rhesus monkey, eastern gray squirrels, and california ground squirrels showed that photoreceptor outer segments are growing and extending continuously (steinberg et al., ) . rpe cells protect photoreceptor outer segments via phagocytosis of the outer segment redundancies of photoreceptors (chen et al., ). rpe cells are playing a crucial role in maintenance of photoreceptors stability by transporting nutrients, electrolytes, ions, and water to the retina, and choroid capillaries . shown to be correlated with dystrophic retina . rpe cells prevent the accumulation of debris in photoreceptor outer segments by scavenging debris, and thereby causing rhodopsin cells renewal. rpe cells transfection with mer tyrosine kinase (mertk) sirna resulted in phagocytosis inhibition, due to downregulation of mertk which is involved in rpe phagocytosis activity (strick et al., ) . correspondingly, mertk mutated royal college of surgeons rats were diagnosed with autosomal recessive retinitis pigmentosa which emphesized on the fundamental role of phagocytosis activity of rpe cells in visual intact function . the -aminoquinoline derivatives such as hcq and cq might cause retinopathy by affecting the lysosomal function, digestion ability of phagocytes, and binding to the melanin (browning, ; lee et al., ) . hcq and cq enter the lysosome and inactivate its enzymes including n-acetyl-β-glucosaminidase, and cathepsin d through alteration of its j o u r n a l p r e -p r o o f vital acidic condition (toimela et al., ) . moreover, hcq and cq cause massive aggregation of lysosomal associated organelles, membranous cytoplasmic bodies, and lipofuscin in rpe neuron cells by reducing the lysosomal acidic potency (mahon et al., ) . ongoing accumulation of lipofuscin in the rpe cells induces a positive feedback resulting in more accumulation of lipofuscin. progressive aggregation of lipofuscin gradually damages rpe cells, and may cause several diseases such as age-related macular degeneration, and stargardt disease (dorey et al., ; wolf, ) . in addition, lipofuscins aggregation in rpe cells may reduce the normal supportive function of rpe cells by disrupting organelle's cytoplasmic trafficking (r sparrrow et al., ; terman & brunk, ) . growing evidence suggest that hcq promotes retinopathy by binding tightly to melanin in rpe. furthermore, the strong binding of hcq to melanin in other tissues including skin and ciliary bodies was shown to cause severe disorders such as skin hyperpigmentation (fig ) (ding et al., ; tracy et al., ) . visual cycle is a vital process to visual perception that is conducted by engaging transporters and binding proteins (thompson & gal, ) . visual cycle exchanges vitamin a compounds (retinol) and facilitates signal transduction via providing substrates to the photoreceptors (bok, ) . carrier organic anion transporter a (slco a ) encodes oatp a which participates in retinol circulation (kalliokoski & niemi, ) . increasing evidence suggest that oatps have a fundamental role in the endogenous and exogenous substances influx into several organs including liver (oatp b ), kidney, intestine, brain, and liver (oatp a ) (zaïr et al., ) . oatp b is the most important transporter expressed in hepatocytes and is localized in the sinusoidal membrane of hepatocytes. oatp a is located in rpe apex, and mediates the transport of all-trans-retinol (atrol) from the interphotoreceptor matrix to the rpe (chan et al., ) . interestingly, hcq and cq probably disrupt the activity of oatp a in transporting of atrol influx by competing with atrol to occupation of oatp a . consequently, atrol accumulates within the interphotoreceptor matrix, and results in deficiency of -cis-retinal ( cral) and rhodopsin, and thereby caused impaired vision (xu et al., ) . j o u r n a l p r e -p r o o f the current novel sars-cov- has been widely spread throughout the world ( countries) with high mortality and morbidity ( a). a global effort is underway to find a potential drug to counter sars-cov- ( b). recently, growing body of evidence showed that hcq might be a potential drug to counteract the novel coronavirus (gautret et al., ; yao et al., ) . hcq and cq were administrated to patients affected with infectious diseases (hiv and influenza), and immune diseases (ra and sle) (chiang et al., ; ooi et al., ; rainsford et al. ) . several investigations have reported that hcq has a higher safety and anti-viral activity to combat covid- in comparison with cq (yao et al., ; . in this contex, an in vitro study carried out on african green monkey kidney vero cells infected by sars-cov- and treated with hcq and cq showed that both of them decreased the viral replication in a concentration-dependent manner although hcq showed a larger in vitro antiviral effects relative to cq (yao et al., ). an investigation conducted in shanghai, china, on confirmed cases with covid- including cases that were given conventional treatment and cases that were given mg per day hcq along with conventional treatment for days revealed the similar frequency of negative patients for nasopharyngeal swabs test of covid- on day after treatment between two groups. also, the median time of normalization of body temperature was similar in both groups, whereas worsening computerized tomography findings of lung were seen in patients ( . %) of case group and subjects ( . %) of control group . a clinical trial study performed on french confirmed sars-cov- cases ( patients left the study) including cases that were given hcq ( mg/day) ( of them received azithromycin ( mg on day followed by mg/day, the next four days) along with hcq), and cases receiving neither hcq nor azithromycin illustrated a positive effect of combination therapy on ameliorating clinical manifestations. surprisingly, their findings demonstrated that the nasopharyngeal pcr test at days after treatment was negative for % of cases that underwent combined therapy whereas only % of patients that received hcq only, and . % of control group patients showed a negative nasopharyngeal pcr test (gautret et al., ) . the discrepancy between results of shanghai and french study may be due to small sample size in both study or combination of hcq and azithromycin in the french study in comparison with shanghai study which used conventional and hcq treatment. strikingly, the achieved results from study performed on patients affected with j o u r n a l p r e -p r o o f sars-cov- ( men and women) that had significant comorbidities associated with poor outcomes (obesity: ; solid cancer: ; hematological cancer: ; hiv-infection: ) have shown an inconsistent finding about co-administration of azithromycin and hcq (the dosage was similar to french study) in comparison with french study. it was revealed that one patient died, two were transferred to the icu, and therapy was discontinued in one patient due to prolongation of the qt interval. also, pcr test from nasopharyngeal swabs of patients who were alive showed a positive sars-cov- finding in of them on day to post treatment (molina et al., ) . different response of patients affected with covid- to hcq supports the hypothesis that different cyp snps and epigenetic mechanisms might be responsible for different hcq metabolism, and therefore different response. the cyp and its isoforms have a key role in metabolism and conversion of hcq into pharmacologically active substances (stokkermans & trichonas, (lee et al., ) . besides, several studies reported multiple drug-drug interactions for hcq and other drugs including metoprolol, methotrexate, cyclosporine, and h + /k + atpase pump inhibitors (omeprazole, lansoprazole, pantoprazole, esomeprazole, and rabeprazole) (bannwarth et al., ; landewé et al., ; namazi, ; somer et al., ) . cramps, and dysgeusia), central nervous system disorders (headaches, dizziness, anxiety, and rarely psychosis and convulsions), skin disorders (rash and discoloration), and retinopathy (ali & jones, ; joyce et al., ; pelle & callen, ) . administation of higher dosage of hcq in patients with sars-cov- raised the possiblity that hcq induced-side effects may probably take place more rapidly. on the other hand, studies showed that retinopathy induced by hcq could progress and become irreversible even after drug cessation in some patients 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cytosolic dna sensor cgas forms an oligomeric complex with dna and undergoes switch-like conformational changes in the activation loop this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. authors declare no conflict of interest. hcq: hydroxychloroquine; cq: chloroquine. j o u r n a l p r e -p r o o f key: cord- -n juf tw authors: kalligeros, markos; shehadeh, fadi; atalla, eleftheria; mylona, evangelia k.; aung, su; pandita, aakriti; larkin, jerry; sanchez, martha; touzard-romo, francine; brotherton, amy; shah, rajeev; cunha, cheston b.; mylonakis, eleftherios title: hydroxychloroquine use in hospitalized patients with covid- : an observational matched cohort study date: - - journal: j glob antimicrob resist doi: . /j.jgar. . . sha: doc_id: cord_uid: n juf tw • the efficacy of hcq in patients with covid- is currently evaluated. • in our center the use of hcq did not decrease the risk of in-hospital death. • hcq use did not decrease the time to clinical improvement and the hospitalization length. • asps can allow for the safe evaluation of agents and treatments against covid- . this is a pdf file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. this version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. hydroxychloroquine (hcq) and chloroquine (cq) were among the first drugs that emerged as potential therapeutic options against covid- . these two antimalarial drugs, also commonly used as immunomodulators in the setting of rheumatologic diseases, showed in vitro activity against sars-cov- [ , ] . our aim was to assess the efficacy and safety of hydroxychloroquine (hcq) with or without azithromycin (az) in hospitalized adult patients with covid- . we utilized data from the largest healthcare network in rhode island, usa, with the aim of exploring the efficacy and safety of hcq in hospitalized patients with covid- . the primary end point was to assess the impact of hcq with or without az, on mortality, length of hospitalization, and time to clinical improvement. we utilized treatment effects with inverseprobability-weighting and cox proportional hazards models. all analyses accounted for age, gender, race, severity on admission, days from symptoms onset and chronic comorbidities. there were no significant differences between the two groups in terms of illness severity at the time of hospital admission (table ). in our inverse probability analyses we we utilized cox proportional hazards models to estimate in-hospital risk of death due to covid- . in our unadjusted model, compared to the supportive care arm, hcq did not decrease the risk of in-hospital death (hr: . ; % ci: . - . ). moreover, hcq did not seem to decrease the risk of death even after adjusting for age, gender and race (ahr . ; % ci: . - . ) as well as days from symptoms onset, news and van walraven scores (ahr . ; % ci: . - . ). finally, using schoenfeld residuals method we tested proportionality and we didn't identify violations of the proportional hazard assumption in any of our models. among patients who received hcq, patients received hcq along with azithromycin, while patients received hcq monotherapy due to prolonged qtc on baseline. five patients did not complete their -day regimen. the reasons behind discontinuing hcq were as follows: patient developed qtc prolongation ( ms) (day ), patient died due to covid- (day ), patient developed bradycardia (day ), in patient the treating team discontinued the drug due to possible contribution to patient's altered mental status (day ), while in patient the treating team decided to discontinue the drug due to seizure activity during hospitalization (day ). in addition, although not included in our analysis, due to the receipt of < doses, one patient discontinued hcq after doses due to qt prolongation ( ms) and the development of premature ventricular contractions. after performing a paired ttest we observed a qtc prolongation from a baseline of ± ms (mean ± sd) to ± ms (mean ± sd) (p < . ). a comparison between hcq monotherapy and hcq with azithromycin was not performed due to the small number of patients receiving hcq monotherapy. finally, among patients who completed their -day regimen patients developed a qtc > ms. none of the included patients developed torsades de pointes. overall, this study did not yield definite benefits from hcq use in patients with covid- , but the results should be interpreted with caution due to the limited sample size. a recent report from the us with patients, of whom ( . %) received hcq, found no j o u r n a l p r e -p r o o f remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) observational study of hydroxychloroquine in hospitalized patients with covid- outcomes of hydroxychloroquine usage in united states veterans hospitalized with covid- no evidence of clinical efficacy of hydroxychloroquine in patients hospitalized for covid- infection with oxygen requirement: results of a study using routinely collected data to emulate a target trial key: cord- - qdjea f authors: sbidian, e.; josse, j.; lemaitre, g.; mayer, i.; bernaux, m.; gramfort, a.; lapidus, n.; paris, n.; neuraz, a.; lerner, i.; garcelon, n.; rance, b.; grisel, o.; moreau, t.; bellamine, a.; wolkenstein, p.; varoquaux, g.; caumes, e.; lavielle, m.; mekontso dessap, a.; audureau, e. title: hydroxychloroquine with or without azithromycin and in-hospital mortality or discharge in patients hospitalized for covid- infection: a cohort study of , in-patients in france date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: qdjea f objective to assess the clinical effectiveness of oral hydroxychloroquine (hcq) with or without azithromycin (azi) in preventing death or leading to hospital discharge. design retrospective cohort study. setting an analysis of data from electronic medical records and administrative claim data from the french assistance publique - hopitaux de paris (ap-hp) data warehouse, in public hospitals, ile-de-france, france. participants all adult inpatients with at least one pcr-documented sars-cov- rna from a nasopharyngeal sample between february st, and april th, were eligible for analysis. the study population was restricted to patients who did not receive covid- treatments assessed in ongoing trials, including antivirals and immunosuppressive drugs. end of follow-up was defined as the date of death, discharge home, day after admission, whichever occurred first, or administrative censoring on may , . intervention patients were further classified into groups: (i) receiving hcq alone, (ii) receiving hcq together with azi, and (iii) receiving neither hcq nor azi. exposure to a hcq/azi combination was defined as a simultaneous prescription of the treatments (more or less one day). main outcome measures the primary outcome was all-cause -day mortality as a time-to-event endpoint under a competing risks survival analysis framework. the secondary outcome was -day discharge home. augmented inverse probability of treatment weighted (aiptw) estimates of the average treatment effect (ate) were computed to account for confounding. results a total of , patients (mean age: . +/- ; males: , ( %)) were included, of whom ( . %) received hcq alone, ( . %) received hcq plus azi, and , ( . %) neither drug. patients receiving "hcq alone" or "hcq plus azi" were more likely younger, males, current smokers and overall presented with slightly more co-morbidities (obesity, diabetes, any chronic pulmonary diseases, liver diseases), while no major difference was apparent in biological parameters. after accounting for confounding, no statistically significant difference was observed between the "hcq" and "neither drug" groups for -day mortality: aiptw absolute difference in ate was + . % (- . to . ), ratio in ate . ( . to . ). -day discharge rates were statistically significantly higher in the "hcq" group: aiptw absolute difference in ate (+ . % [ . to . ]), ratio in ate ( . [ . to . ]). as for the "hcq+azi" vs neither drug, trends for significant differences and ratios in aiptw ate were found suggesting higher mortality rates in the former group (difference in ate + . % [- . to . ], ratio in ate . [ . to . ];p= . ). conclusions using a large non-selected population of inpatients hospitalized for covid- infection in hospitals in france and robust methodological approaches, we found no evidence for efficacy of hcq or hcq combined with azi on -day mortality. our results suggested a possible excess risk of mortality associated with hcq combined with azi, but not with hcq alone. significantly higher rates of discharge home were observed in patients treated by hcq, a novel finding warranting further confirmation in replicative studies. altogether, our findings further support the need to complete currently undergoing randomized clinical trials. what is already known on this subject -the use of hydroxychloroquine (hcq) or hcq with azithromycin (azi) has been associated with viral load reduction at days in covid- infected patients -no difference between hcq and no-hcq groups in terms of risk of death or need for mechanical ventilation was found in two large cohorts of hospitalized covid- infected patients -using a large non-selected population of inpatients hospitalized for covid- infection in hospitals in france and robust methodological approaches, we found no evidence for efficacy of hcq on -day mortality -our results suggest an excess risk of mortality in patients treated by a combination of hcq and azi, but not with hcq alone -significantly higher rates of discharge home were observed in patients treated by hcq, a novel finding warranting further confirmation in replicative studies . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint the covid- pandemic due to the sars-cov- coronavirus started in wuhan, china last december, . the covid- epidemic is a worldwide pandemic with more than . million cases reported up to may , , of whom nearly , have died. because effective treatments are urgently needed, more than clinical trials are currently ongoing in a worldwide effort to fight the coronavirus. the -aminoquinolines chloroquine (cq) and hydroxychloroquine (hcq) are synthetic antimalarials drugs (amd). due to their anti-inflammatory and immunomodulatory properties, synthetic amds are the standard treatment in autoimmune diseases such as lupus. in vitro data schowed a non-specific antiviral action of synthetic amds against emerging viruses such as hiv, dengue, hepatitis c, chikungunya, influenza, ebola, severe acute respiratory syndrome, and middle east respiratory syndrome and human immunodeficiency viruses. the use of hcq or hcq with azithromycin (azi) has arisen as a promising treatment or combination of treatment for covid- infection. first, in vitro data have shown the effectiveness of hcq (and to a lesser extent cq) in reducing the viral load of cells infected with sars-cov- . then, a chinese clinical trial showed that cq had a significant effect, including a better clinical outcome, when compared to control groups. a french research team suggested that hcq, at a dose of mg/day, was associated with viral load reduction in twenty covid- patients and its effect was strengthened by azi. these preliminary results were further backed up by two prospective cohorts from the same team of and , participants, suggesting good clinical outcomes in ( %) and ( %) patients, respectively, with lower frequency of aggressive clinical course requiring oxygen therapy, fewer transfers to the intensive care unit (icu), or death after at least days of treatment and a viral load reduction at day . , more recently, several published or preprint publications have raised the question of hcq efficacy for covid- infection. four observational studies with diverse cohort sizes ( , , , , and , participants) failed to find a difference between hcq and no-hcq groups in terms of risk of death or need for mechanical ventilation. [ ] [ ] [ ] [ ] preliminary results from the uk recovery randomized trial have been communicated, but not yet published, concluding that there was no beneficial effect of hcq on -day mortality in hospitalized patients with covid- . data from large health care databases provide a unique opportunity to assess the potential effectiveness and harm of hcq in a real-world setting, including unselected population. because some variation has been reported between studies, replicated analyses minimizing selection and confounding biases are crucially needed to disentangle current evidence on the actual risks and benefits of hcq-based treatments. we consequently aimed to assess the clinical effectiveness of oral hcq in preventing death or allowing to hospital discharge using a large, exhaustive, nonselected population of in-patients hospitalized for covid- infection in hospitals in france, accounting in detail for patients characteristics. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint we performed a retrospective cohort study using the corona omop database, which combines electronic medical records and administrative claim data from the greater paris public hospitals (assistance publique -hôpitaux de paris (ap-hp) data warehouse, called 'entrepôt de données de santé' (eds). this study was approved by the french data protection agency commission nationale de l'informatique et des libertés (regulatory decision de- - ), irb . patients and the public were not involved in the present study. the eds currently collects data on more than million patients treated in the hospitals of the ap-hp, ile-de-france, france. the warehouse contains medico-administrative data from the medical information system program (pmsi), the french national hospital database which gathers information from standardized discharge reports on diagnoses and procedures performed in all medical units involved in patient management during his/her hospital stay. primary and associated diagnoses are recorded using the international classification of diseases, th edition (icd- ) and therapeutic procedures using a national standardized classification system (classification commune des actes médicaux, ccam, th edition). since discharge reports are mandatory and used for hospital fund allocation, the pmsi database contains exhaustive information on all admissions to hospitals in france, including proprietary and public hospitals. in addition to pmsi data, the eds gathers information from multiple electronic health record databases, including biology and imaging results, drug prescriptions (stored within the orbis medication database and coded according to the international anatomical therapeutic chemical (atc) classification system) and medical text reports associated with hospital visits, including emergency department data and outpatient visits. because pmsi coding requires human-based and time-consuming processing, complete pmsi information was not available for a number of patients being still hospitalized or for whom discharge reports were not yet processed at the time of analysis. likewise, information on drugs prescriptions were not directly available in some icu not using the orbis medication system. consequently, data acquisition for the present study relied on both structured data (i.e. pmsi pertaining to past hospitalizations, if any, biological results, orbis medication system) and unstructured data (i.e. medical text records). for the latter, we used artificial intelligence algorithms based on natural language processing (nlp), to extract information on patients diagnoses (including comorbidities, see below) and drugs prescriptions (including hcq+/-azi), considering contexts where mentions of drugs by name do not correspond to actual prescriptions (i.e. when the drug is mentioned in a negative context), and considering both international non-proprietary name (inn) and trade-marks. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint all adult (≥ years of age) inpatients with at least one polymerase chain reaction-documented sars-cov- rna from a nasopharyngeal sample between february st , and april th , were eligible for the present analysis. the date of inclusion in the study cohort (index date) was defined as the date of admission. we restricted the study population to previously hydroxychloroquine-and azithromycin-naive inpatients, defined as those who had not received a prescription before the index date, and excluded those having received specific covid- treatments, i.e. treatments assessed in ongoing trials: remdesivir, lopinavir-ritonavir (atc j ar ), favipiravir (j ax ), anti-interleukin -i.e., anakinra (atc l ac ), canakinumab (atc l ac ) -anti-interleukin -i.e., tocilizumab (atc l ac ), sarilumab (atc l ac ). when patients were transferred between hospitals for the same stay, several discharge reports were available which were analyzed as a single hospital stay until first discharge home. patients who died or were discharged within hours following their admission were excluded. the end of follow-up was defined by the time of death, discharge home, day (d ) after admission, whichever occurred first, or administrative censoring on may , . patients transferred to hospitals outside ap-hp or to follow-up care and rehabilitation services before day were considered as censored. the study's primary outcome was all-cause -day mortality, assessed as a time-to-event endpoint under a competing risks survival analysis framework. for patients discharged home before day , we looked at subsequent re-admissions to determine vital status on day . the secondary outcome was -day discharge home, also assessed as a time-to-event endpoint. while there was no overarching recommendation regarding hcq+/-azi prescription at the ap-hp level, guidelines were nonetheless proposed at local level in several individual hospitals, suggesting hydroxychloroquine to physicians as a therapeutic option for patients with moderateto-severe covid- infection, i.e. requiring oxygen. the suggested hcq regimen was a loading dose of mg on day , followed by mg daily for additional days. azi at a dose of mg on day and then mg daily for more days in combination with hcq was an additional suggested therapeutic option. prescription of hcq or hcq together with azi was at the discretion of the physicians. using data acquisition procedures previously detailed, we identified patients with a prescription of hcq (atc p ba ), azi (atc j fa ), steroids (atc h ab), and antithrombotic agents (heparin group, atc b ab) usually used for acute respiratory distress syndrome. , exposure to a hcq/azi combination was defined as a simultaneous prescription of the two treatments (within one day). based on previous possible combinations, patients were further classified into three groups: (i) receiving hcq alone, (ii) receiving hcq together with azi, and (iii) receiving neither hcq nor azi. patients receiving azi alone were excluded, in accordance with our objective to assess clinical effectiveness of hcq with or without azi vs. neither specific treatment. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint for each patient, age, sex, hospital-admission location, icu admission, icu stay length and hospital stay length were recorded. co-morbidities and risk factors (smoker status, obesity, hypertension, diabetes, dyslipidemia, ischaemic or rhythmic heart diseases, heart failure, renal disease, presence of chronic respiratory insufficiency or asthma or cystic fibrosis, and cancer) were recorded for the two-year period before the index date. clinical severity features within h after admission were also recorded: icu transfer within the first h, oxygen saturation, partial pressure of oxygen (pao mmhg), and carbon dioxyde (pco ). lastly, biologic values were also recorded at the index date using their loinc (logical observation identifiers names and codes). biological values were recorded for neutrophils, lymphocytes, c reactive protein, d-dimer, prothrombin time, creatine, and lacticodesydrogenase. detailed definitions of the covariates are available in supplemental table . all analyses were performed considering the three main treatment modalities of interest, as previously described (hcq/hcq+azi/neither drug), in the entire population or after stratifying by the level of severity of covid- at admission, considering (i) early icu admission as defined as occurring within the first hours of admission or (ii) not. descriptive results are given as medians [interquartile range (iqr)] for continuous variables, and counts (%) for categorical variables. unadjusted between-groups comparisons were performed using the chi-square test for comparing categorical variables, the kruskal-wallis (three-groups comparisons) and mann-whitney rank sum tests (pairwise comparisons, correcting for test multiplicity with the benjamini-hochberg procedure) for continuous variables, as appropriate. for time-to-event analyses, non-parametric nelson-aalen estimates of the cumulative cause-specific hazards were plotted for the occurrence of death or hospital discharge. due to the influence on treatment assignment of baseline characteristics of patients included in non-randomized observational studies, it is essential to account for such differences when estimating treatment effects to address bias arising from confounding. under assumptions of unconfoundedness (i.e., potential outcomes are independent of the treatment assignment conditionally on a vector of covariates) and its extension in the presence of missing values, the average treatment effect (ate) can be identified. for the present analysis, causal inference modeling relied on doubly robust estimators, combining an outcome regression with a model for the treatment (i.e., propensity score) to derive augmented inverse probability of treatment weighting (aiptw) estimators, a more effective approach in minimizing bias due to model misspecification than only iptw. the selection of the relevant covariates to be used in causal inference modeling was based on available published data at the time of analysis and expert a priori knowledge on key prognostic factors and determinants of treatment assignment, including patients' demographics, comorbidities, hospital and time period of admission. supplemental figure generated using . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint dagitty, shows the causal inference model we applied, differentiating between variables assessed as predictors of the treatment assignment, unrelated to the outcome (brown), predictors of the outcome, unrelated to the treatment assignment (blue), predictors of both treatment and outcome (violet). as the primary analysis, we constructed cause-specific cox proportional hazards regression models to account for the competing risk between all-cause death and hospital discharge. doubly-robust estimation equations were derived based on regression models for the outcome and the censoring (using cox modeling), and the treatment distribution (using a generalized linear model with a logit link function), conditional on baseline covariates. ates were calculated as the absolute difference and ratios in the standardized absolute risks, along with their % confidence intervals. we conducted several sensitivity analyses to check the stability of our results under varying approaches. first, we assessed -day mortality as a binary endpoint, considering patients discharged home before day as alive at that date and excluding patients transferred to hospitals outside ap-hp or to follow-up care and rehabilitation services before day . to do so, we used the causal forest implementation based on the generalized random forests (grf) method to compute forest-based weighting functions and derive aiptw estimates for doubly robust inference of the average treatment effect (ate). second, conventional multivariable and ipt-weighted analyses ('singly robust') using cause specific cox models and fine-gray competing risks analyses were also performed, computing ipt-weighted and adjusted hazard ratios (hr) and subhazard ratios (shr), respectively, and plotting raw and adjusted cumulative incidence curves to illustrate the associations. for all ipt-weighting based analyses, standardized differences of the means were computed before and after iptw to assess imbalance of the covariates between treatment groups. standardized differences less than % were considered negligible following common practice when using iptw to estimate causal treatment effects in observational studies. to account for the potential influence of missing data on causal inference procedures, we used single imputation with a (regularized) iterative factorial analysis for mixed data model (famd), accounting for similarities between both individuals and relationships between covariates, treatment assignment and the outcome. variables showing departure from normality using graphical methods and kurtosis/skewness statistics were log-transformed prior to imputation. for the grf method, the grf-mia approach which enables the computation of ate without any imputation of the data was used for missing data handling. a two-tailed p-value of less than . was considered significant. statistical analyses were performed using r v . . (r foundation for statistical computing, vienna, austria; packages grf, riskregression). , . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint from february st to april , , , adult patients were hospitalized at ap-hp for a covid- infection and did not receive specific covid- treatments other than hcq or azi. patients who died (n= ) or who were discharged (n= ) within hours after their admission were excluded, as well as patients receiving azi alone (n= ) or patients who did not initiate hcq and azi the same day (more or less hours, n= ). thus, a total of , patients (mean age: . ± ; males: , ( %)) were included in the study population, of whom ( . %) received hcq alone, ( . %) received hcq plus azi, and , ( . %) neither hcq nor hcs plus azi (figure ) . in the 'hcq alone' and 'hcq plus azi' groups, median timing of the first dose of hcq after the admission was . days, iqr ( to . ). the main characteristics of the study population are summarized in table . patients receiving 'hcq alone' or 'hcq plus azi' were more likely younger, males, current smokers and overall presented with slightly more co-morbidities (obesity, diabetes, any chronic pulmonary diseases, liver diseases) than the 'neither drug' group, while no major difference was apparent in biological parameters. table shows unadjusted clinical outcomes by treatment group. raw -day mortality rates statistically differed between the three groups (number of deaths: ( . %), ( . %) and ( . %) for 'hcq alone', 'hcq plus azi' and 'neither drug' groups, respectively; p< . ). of the , patients, ( . %) were transferred to icu within h after the admission, more markedly so in the 'hcq plus azi' group ( . %; p< . ). groups main characteristics stratified by early icu transfer are summarized in supplemental tables s and s . discharge rates at day significantly varied across groups, ranging from . % (neither drug) to . % (hcq alone; p< . ), with corresponding lengths of stay among patients who lived ranging in median (iqr) from . [ . - . ](hcq+azi) to . [ . - . ](neither drug; p= . ) and in mean±sd from . ± (hcq+azi) to . ± . (neither drug, p< . ). results from competing risks multivariable analyses for -day mortality and hospital discharge are displayed in table , showing both raw unadjusted estimates for the average treatment effect of 'hcq alone 'or 'hcq plus azi', and aiptw results from double robust estimation accounting for confounders for the outcome and the treatment allocation. in the whole population, the raw difference in average treatment effect for 'hcq' versus 'neither drug' comparison was - . % (- . to - . ); in other words, there was a . % absolute reduction in the -day mortality rate in the 'hcq alone' group when compared to patients in the 'neither drug' group, with a corresponding ratio in ate of . ( . to . ). after accounting for confounding, no significant difference was observed between the 'hcq alone' and 'neither drug' groups: aiptw difference in ate of + . % (- . to . ), ratio in ate of . ( . to . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . in subgroup analyses stratified by early icu tranfer or not, results were close to those described above, with no statistically significant difference observed in aiptw ate between 'hcq alone' and 'neither drug' groups for the -day mortality, a trend towards higher discharge rates for the 'hcq alone' group in the not-early icu transfer subpopulation (ratio in aiptw ate . [ . to . ]; p= . ), and apparent worse mortality rates in the 'hcq plus azi' group but without reaching statistical significance. results from double robust analyses considering -day mortality as a binary endpoint analyzed at a fixed timepoint are shown in supplemental table s , with corresponding balance plots of covariates before and after ipt-weighting depicted in supplemental figure s . these analyses confirmed those obtained for 'hcq alone' group from the main analysis with no statistically significant difference in mortality, but found no excess of mortality for the 'hcq plus azi' group compared with 'neither drug' group. results from multivariable analyses using conventional adjustment and/or ipt weighting are shown in figure for the cause specific cox proportional hazards models. using these approaches, results essentially confirmed those obtained from double robust estimates, illustrating for the 'hcq alone' vs. 'neither drug' comparison ( figure a ) the influence of confounders on estimations as indicated by the progressive alignment of death incidence curves according to treatment after adjustement and/or ipt weighting, and the persistence of statistically significant differences in discharge rates after adjustment, but not when using ipt weighting (p= . ) or combining both approaches (p= . ). regarding the 'hcq plus azi' vs. 'neither drug' comparison ( figure b) , a trend for a statistically significant difference was found for mortality after multiple adjustment and ipt weighting (hr= . ; p= . ), but not for discharge (hr= . ; p= . ). results from fine-gray models identified similar but not significant trends for the excess risk of mortality in the 'hcq plus azi' group. (supplemental figures s and s ) . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint using a large non-selected population of , hospitalized for covid- infection in hospitals in france, we found no evidence for efficacy of hcq or hcq combined with azi on -day mortality. our findings suggest that patients treated by association of hcq and azi are at greater risk of mortality compared with the 'neither drug' group. interestingly, significantly higher rates of discharge home were observed in patients treated by hcq when using competing risks survival analyses, a finding whose statistical significance persisted after multivariable adjustment and propensity-score weighting. these results were found to be robust to a variety of methodological approaches conducted regarding missing data handling and causal inference modeling to properly account for potential confounders. the absence of difference on mortality between hospitalized patients receiving hcq and those receiving neither drug is consistent with previous observational studies led in hospitalized patients. [ ] [ ] [ ] [ ] similarly to two recent studies with a > sample size, patients from the present report had a median age around years, with substantial differences at baseline between patients being prescribed hcq and those being not, including higher prevalence in the former group of comorbidities and risk factors such as obesity, diabetes, hypertension, chronic lung and cardiovascular diseases. however, patients with either drug in our study were remarkably younger ( [hcq], [hcq+azi], [neither drug]) and fared significantly better in raw analyses, in contrast with findings from the two previous studies where worse raw outcomes were observed in the groups receiving hcq. yet in all studies, including ours, no statistically significant difference was eventually found after accounting for confounding by multivariable analyses potentially combined with propensity score weighting, matching or adjustment methods. finally, it should be stressed out that findings from these reports pertain to hospitalized patients and do not provide information on the efficacy of either drug when administered in earlier forms of the disease. regarding the association of hcq and azi, our findings indicate a trend towards higher risk of death for the 'hcq plus azi' group compared with the 'neither drug' group (ratio in ate at . [ . to . ]). this finding is consistent with the results from rosenberg et al study (adjusted hr for mortality . [ . to . ]). because of a limited sample size for this subgroup in our study, our results should be taken with caution. however, among possible hypotheses that could be discussed, an increased risk of serious adverse events such as arrhythmia has been advocated in several studies. , among consecutively included patients receiving hcq for covid- infection in israel, mercuro et al detected change in qtc in patients ( %), and more evidently so in the 'hcq plus azi' subgroup. hcq is already known to inhibit voltage-gated sodium and potassium channels, prolonging the qt interval and increasing the risk of torsades de pointes, syncope and sudden cardiac death. azithromycin has also been implicated in qtc prolongation and proarrhythmic events. in addition to hcq and azi interaction, patients hospitalized with severe covid- pneumonia are also at risk to present clinical characteristics leading to qt prolongation such as hypokalemia or congestive heart failure. one original finding of the present paper lays in the assessment of discharge and in-hospital death in a competing events survival analysis framework. this analysis allowed us to investigate both events separately, to provide insights beyond that provided by mortality rates alone. in our study, . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint we identified increased discharge rates at day- in the hcq group, corresponding to a ratio in average treatment effect of . in favor of hcq, with corresponding predicted day- discharge rates of % [hcq] vs. % [neither drug] in the whole study population. to our knowledge, this is the first report from a large observational study specifically addressing this issue in addition to mortality and icu-related outcomes, [ ] [ ] [ ] [ ] whereas a similar endpoint was used for remdesivir randomized controlled trials for covid infection as a primary outcome. while mortality remains the ultimate clinical endpoint, assessing discharge rates and determinants may inform decision making for optimizing organization of inpatient services in the context of a pandemic. our results could be supported by in vitro and in vivo studies which established a decrease of the viral load in patients receiving hcq and hcq plus azi. , causal interpretation should here again remain cautious, but should such results be replicated and validated in randomized studies, it would provide useful information to draw a balanced assessment of the risks and benefits associated with hcq-based regimens. from a methodological standpoint, the concomitant examination of inhospital mortality and discharge and length of stay through survival analyses require to account for the effect of competing risks to avoid overestimation of the probability of the events of interest. with that regard, we used two approaches to competing risks, primarily using cause specific cox models for causal inference, and further confirming our results by using the fine-gray approach with ipt weighting and multivariable adjustment. among the strengths of our study is the use of advanced causal inference approaches both considering time-to-event survival analyses and binary endpoints at a single timepoint. because inappropriately accounting for confounders can drastically modify results, we performed several sensitivity analyses to check the stability of our results under varying approaches, including so called double robust estimations relying on both multivariable modeling of the outcome and of the treatment (including propensity score-based approaches), which offer better robustness to model misspecification, and use of varying missing data imputation techniques confirming the stability of our findings. other strengths of this work include the assessment of a large, representative sample study population in the greater paris area from hospitals. study's limitations include the absence of direct, clinical information on regimen duration and dosages, and respiratory parameters of covid- infection, including oxygen requirement, non-invasive or mechanical ventilation, which are potential confounders. however, we used biological parameters proxy to assess the severity of the covid- infection including creatine, lymphocyte count and inflammatory markers (d-dimer and c-reactive protein) well known to be associated with severity of covid- . yet, causal interpretation of our findings relying on retrospective evaluation of medical records should remain cautious considering the observational nature of the study design. using a large non-selected population of inpatients hospitalized for covid- infection in hospitals in france and robust methodological approaches, we found neither evidence for reduced or excess risk of -day mortality with the use of hcq alone. our findings suggest a possible higher risk of death for patients receiving hcq combined with azi. significantly higher rates of discharge home were observed in patients treated by hcq, a novel finding warranting further confirmation in replicative studies. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint we thank susan wilkinson for editorial assistance. data used in preparation of this article were obtained from the ap-hp covid cdr initiative database. a complete listing of ecai members can be found at (https://eds.aphp.fr/covid- ). . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint figure . death and discharge cumulative incidence curves: results from cause specific cox competing risks analysis. adjusted results are based on the adjustment strategy for the outcome detailed in figure s . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint clinical characteristics of coronavirus disease in china an endocytosis blocking agent, inhibits zika virus infection in different cell models in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) efficacy of hydroxychloroquine in patients with covid- : results of a randomized clinical trial. medrxiv hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in covid- patients with at least a six-day follow up: a pilot observational study full-length title: early treatment of covid- patients with hydroxychloroquine and azithromycin: a retrospective analysis of cases in marseille, france clinical efficacy of hydroxychloroquine in patients with covid- pneumonia who require oxygen: observational comparative study using routine care data outcomes of hydroxychloroquine usage in united states veterans hospitalized with covid- . medrxiv observational study of hydroxychloroquine in hospitalized patients with covid- association of treatment with hydroxychloroquine or azithromycin with in-hospital mortality in patients with covid- in new york state linguistic approach for identification of medication names and related information in clinical narratives dexamethasone treatment for the acute respiratory distress syndrome: a multicentre, randomised controlled trial anticoagulant therapy in acute respiratory distress syndrome an introduction to propensity score methods for reducing the effects of confounding in observational studies reducing bias in observational studies using subclassification on the propensity score comparing methods for estimation of heterogeneous treatment effects using observational data from health care databases doubly robust estimation of causal effects clinical characteristics of covid- in new york city dagitty: a graphical tool for analyzing causal diagrams generalized random forests on the estimation of average treatment effects with right-censored time to event outcome and competing risks estimates of absolute cause-specific risk in cohort studies moving towards best practice when using inverse probability of treatment weighting (iptw) using the propensity score to estimate causal treatment effects in observational studies risk of qt interval prolongation associated with use of hydroxychloroquine with or without concomitant azithromycin among hospitalized patients testing positive for coronavirus disease (covid- ) drug-induced prolongation of the qt interval remdesivir for the treatment of covid- -preliminary report competing risk survival analysis of time to inhospital death or discharge in a large urban neonatal unit in kenya evaluating mortality in intensive care units: contribution of competing risks analyses creatine (mg/dl), median key: cord- -eqxn auk authors: garcia‐cremades, maria; solans, belen p.; hughes, emma; ernest, jacqueline p.; wallender, erika; aweeka, francesca; luetkemeyer, anne f.; savic, radojka m. title: optimizing hydroxychloroquine dosing for patients with covid‐ : an integrative modeling approach for effective drug repurposing date: - - journal: clin pharmacol ther doi: . /cpt. sha: doc_id: cord_uid: eqxn auk hydroxychloroquine (hcq) is a promising candidate for coronavirus disease of (covid‐ ) treatment. the optimal dosing of hcq is unknown. our goal was to integrate historic and emerging pharmacological and toxicity data to understand safe and efficacious hcq dosing strategies for covid‐ treatment. the data sources included were (i) longitudinal clinical, pharmacokinetic (pk), and virologic data from patients with severe acute respiratory syndrome‐ (sars‐cov‐ ) infection who received hcq with or without azithromycin (n = ), (ii) in vitro viral replication data and sars‐cov‐ viral load inhibition by hcq, (iii) a population pk model of hcq, and (iv) a model relating chloroquine pks to corrected qt (qtc) prolongation. a mechanistic pk/virologic/qtc model for hcq was developed and externally validated to predict sars‐cov‐ rate of viral decline and qtc prolongation. sars‐cov‐ viral decline was associated with hcq pks (p < . ). the extrapolated patient half‐maximal effective concentration (ec( )) was . µm, comparable to the reported in vitro ec( s). hcq doses > mg b.i.d. for ≥ days were predicted to rapidly decrease viral loads, reduce the proportion of patients with detectable sars‐cov‐ infection, and shorten treatment courses, compared with lower dose (≤ mg daily) regimens. however, hcq doses > mg b.i.d. were also predicted to prolong qtc intervals. this prolongation may have clinical implications warranting further safety assessment. due to covid‐ 's variable natural history, lower dose hcq regimens may be indistinguishable from controls. evaluation of higher hcq doses is needed to ensure adequate safety and efficacy. membrane that reduces viral entry and inhibition of later stages of replication. hcq also causes immunomodulation, and, thus, may suppress the cytokine storm associated with advanced stages of covid- illness. in a small, nonrandomized, open-label clinical trial in france, patients with covid- , who received hcq dosed as mg t.i.d. were compared with a convenience sample of patients who only received supportive care. in this study, hcq was well-tolerated, however, approximately one third of patients remained viremic by nasopharyngeal swabs after days. a small study in china reported no apparent clinical benefit of mg daily hcq administered for days compared with placebo in patients with covid- , the majority of whom had mild disease. based on in vitro data, several hcq regimens have been proposed, including mg q.i.d. and mg b.i.d. followed by mg daily for days, but none of these regimens have been evaluated clinically. the centers for disease control (cdc) also provides anecdotal dosing suggestions, but explicitly states optimal dosing and duration of hcq for covid- are unknown. with promising initial clinical and in vitro results for hcq, our goal was to integrate all available pharmacological data and mechanistic knowledge related to covid- , to date, which include novel in vitro pharmacokinetic/pharmacodynamic (pk/pd) data for sars-cov- , historical data on viral replication of similar coronaviruses (e.g., sars-cov- , first observed in in china), historical data on population pks and safety of hcq from large patient cohorts, and newly emerging clinical pk/pd data from patients with covid- . we use translational pk/pd modeling to propose optimized hcq dosing regimens, which ensure the highest likelihood of success as a covid- treatment. in vitro hcq and cq drug sensitivity data for sars-cov- , reported as percent inhibition, were obtained from -hour and -hour experiments in vero or veroe cells derived from african green monkey kidney epithelium. , , the experiments are described in detail in the original publications. estimated, apparent half-maximal effective concentration (ec ) values were reported, whereas the % effective concentration values were obtained by digitizing the graph of antiviral activity for hcq using the software webplotdigitizer version . . in vitro viral replication data were obtained from longitudinal data profiling the growth of sars-cov- in vero cells over days. rna extraction and quantitative real-time polymerase chain reaction (pcr) were performed, and viral load was reported as cycle threshold (ct). viral load was calculated from the ct reported in the original publication as / log (ct). in vivo data was obtained from a published nonrandomized single arm open label study of hcq mg t.i.d., with or without azithromycin, for treatment of sars-cov- infection in france. participants had pcr confirmed sars-cov- infection by nasopharyngeal swab and swab samples were obtained daily. controls were selected by convenience at multiple hospitals in france. viral load was calculated from ct. one sparse serum hcq concentration was reported for each patient on days , , or of treatment. sixteen patients in the control arm (samples = ), patients in the hcq arm (samples = ), and patients in the hcq with azithromycin arm (samples = ) contributed viral load samples over days. additional patient characteristics and results are reported in the original publication. an external cohort of patients receiving mg t.i.d. for days was used for external model validation. translational and clinical pk/pd-viral kinetics models a published two-compartment population pk model for hcq was used to predict plasma drug concentrations for different dosing regimens of hcq. published scaling parameters were used to simulate lung concentrations and the fraction unbound was assumed to be the same in the lung as in the plasma ( . ). in vitro viral growth, death, and saturable growth parameters were estimated using data from sars-cov- in the software nonlinear mixed effects modeling version . (nonmen icon development solutions, ellicott city, md). the drug effect over time on viral replication rate was established by simulating unbound plasma concentrations or unbound lung tissue concentrations using a previously defined partition coefficient ( . ; hcq unbound fraction assumed to be ~ %) and using the established in vitro sigmoidal efficacy parameters. , , regimens of hcq included - mg b.i.d. for days. the drug effect on the viral replication rate was simulated by fixing ec to one of the reported in vitro values, ranging from . µm to . µm. , , each regimen was simulated with each ec values times. the pk/pd analysis of the in vivo viral load data was performed sequentially. the control patients' longitudinal viral load was analyzed to inform the unperturbed viral growth. a previously published population pk model was used to define the individual pk profile for each patient treated with hcq (with and without azithromycin). plasma and serum concentrations were assumed to be comparable. subsequently, longitudinal plasma pk and viral load were linked to predict the impact of time varying hcq concentrations on viral replication. viral dynamics (vl) were described using the viral kinetic model (eq. ), where k g and k d represent first-order growth and death rates, respectively. max virus stands for the saturated maximal viral load. drug (pk/pd) effect (edrug) was included as a linear function increasing k d (eq. ), where the slope (sl) of the function is estimated, and c p represents the drug concentration predicted with the pk model. a function (eq. ) independent of the drug effect was added during the model validation stage to improve predictions at later time points (beyond day ). this function might mimic the immune response (ir), which comes on board once the viral load is reduced to a certain level, described as a function of the immune effect (ie) and time. finally, participants' sex, age, baseline viral load, clinical status time between onset of symptoms and inclusion, and the use of concomitant azithromycin were tested as potential covariates. pk/pd parameters were estimated using the laplace estimation method with interaction, and the m method was applied to handle data below the limit of quantification by estimating the likelihood of the predicted viral load being less than the lower limit of assay quantification. , interindividual variability parameters were modeled exponentially and the residual variability was described using proportional error. model selection and evaluation were done by the likelihood ratio test, goodness of fit plots, and visual predictive checks. the final model was used to externally predict %pcr-negative patients from the new patient cohort who received mg t.i.d. with azithromycin for days. simulated regimens of hcq included , , , and mg b.i.d. for , , and days with and without loading dose. all simulations included virtual patients, simulated , times. large variation in the basal viral load range was assumed. the pk model was used to generate the virtual individual pk profiles. the percentage of patients with positive pcr results was computed longitudinally throughout and at the end of the treatment. regimens were evaluated based on the proportion of patients with viral loads above the lower limit of assay quantification. all model simulations were performed using nonmem and the pkpdsim package in r software version . . . to predict the risk of corrected qt (qtc) prolongation associated with hcq, we used a published pk-qtc model established for high-dose cq in children, describing a linear pk-qtc relationship where for every nm increase in cq concentration there was a . ms ( % confidence interval . - . ) increase in qt interval. a conservative upper boundary estimate ( . ms/nm) was used assuming that hcq and cq have a similar pk-qtc relationship. this relationship was validated as it successfully predicted qtc prolongation in adults, after a single dose of mg cq reported by mzayek et al. a conservative assumption was made that the pk-qtc relationship remains linear and it does not reach a maximal effect. we assumed a trial population of , patients with a baseline qtc of ± sd. we conducted simulations of each hcq dosing regimen in this population using the established population pk model (including intersubject variability) and predicted the peak hcq concentrations during treatment. we assumed all changes in qtc were attributed to pk variability. the maximum delta qtc during treatment was predicted using the linear pk-qtc relationship for cq ( . ms/nm); the maximum predicted qtc was the sum of the baseline and the delta qtc. although the baseline qtc distribution contained variability, the slope used ( . ms/nm) did not. the proportion of patients with a delta qtc > ms or predicted peak qtc on treatment > ms were reported. the natural course of disease progression is variable and might impact the benefit of hcq therapy. we explored various models of natural history for covid- based on the viral load distribution from the control arms in two recently reported studies. , three natural history scenarios were explored for several plausible control arms with low, high, and mixed baseline viral load. viral load decline of various hcq doses was compared with the control arm under different assumptions. translational pk/pd model the pk/pd viral kinetic model structure is shown in figure . the translational pk/pd model included the unperturbed growth rate of sars-cov- in vitro, the clinical population pk model, and the hcq in vitro efficacy parameters in sars-cov- . , , , , the in vitro viral kinetic model included a first-order growth ( . day - ) and death rate ( . day - ) and saturated maximal viral load ( table s ). the simulated unbound lung concentrations were above the ec for all evaluated dosing regimens, including a subtherapeutic mg dose. for unbound plasma concentration, the impact of the drug effect on viral replication increased with the dose but was highly sensitive to the in vitro ec values ( figure ). for all reported ec s , higher doses increased the rate of viral decline. clinical pk/pd model hcq concentrations from the clinical cohort fell within the range expected from historical population profiles (figure a left). , figure b shows the reconstructed individual pk profiles in the treatment group. the patients' viral load is displayed in figure a (right). pharmacokinetic/pharmacodynamic (pk/pd)-viral kinetics model diagram. a previously published two-compartment plasma pk model was used to simulate plasma concentration. pd compartments included a one-compartment model describing viral growth, death, and drug effect, and a model describing drug effect on qtc prolongation. e drug represents the pd relationship for hydroxychloroquine (hcq) plasma concentration (c p ). in the in vitro model, e drug is characterized by a maximum effect (e max ) function e drug =e max × cp cp +ec , whereas for the clinical model it is described using a linear function (e drug = sl × c p ). cl, clearance; ec , half-maximal effective concentration; qtc, corrected qt; v, volume of distribution. article viral response to treatment was significantly associated (p < . ) to hcq pk concentrations using a linear effect model ( figure and table s ). each μm increase in plasma hcq is associated with a % decrease in viral load per day. the pk/pd model was significantly better compared with a drug effect model (p < . ), confirming a significant concentration-effect relationship. in addition, a maximum effect (e max ) model to link drug concentrations to effect was also tested, however, it was not identifiable. this is most likely due to the limited clinical pk concentration range. the effect of azithromycin on viral load decline was not significant. among the covariates explored, two asymptomatic patients in the treatment group seemed to have a lower baseline viral load and cleared the virus by the second day ( figure s ). the visual predictive check (figure c) shows good agreement between the raw data and model simulations, both for the longitudinal viral load dynamics and for the proportion of censored pcr samples, associated with undetectable viral load. the estimated plasma concentrations for % viral inhibition in patients was . . this value is in closer agreement with the in vitro values reported after a -hour incubation (ec = . µm) compared with -hour incubation (ec figure ). , , pharmacokinetic simulations hcq pks, relative to in vitro and extrapolated in vivo ec , are summarized in figure and figure s (with population variability). simulated unbound lung concentration are shown in figure s . optimal dosing regimens are those that, to the extent possible, are close or above the clinical ec values and below . µm, identified as the mean concentration associated with > % of patients having an increase in > ms qtc while on treatment. regimens that give ~ mg/day either loaded upfront, or as mg b.i.d., figure translational pharmacokinetic/pharmacodynamic (pk/pd) simulation. simulated scenarios of drug effect on in vitro replication rate based on reported hydroxychloroquine (hcq) efficacy. , , viral kinetics were estimated from in vitro replication rate of severe acute respiratory syndrome-coronavirus (sars-cov)- and unbound drug concentration in plasma and lungs were simulated with hcq pk model. , solid continuous line represents the th percentile of the simulated data and shaded areas represent the % prediction intervals for median, . th, and . th percentiles obtained from simulated datasets. dotted horizontal lines represent the baseline level, whereas dashed vertical lines indicate the start of treatment. seem to have good efficacy and safety, and hcq is detectable in plasma for up to days (figure c ). for pk/pd-viral kinetics simulations, a median baseline ct of . ( . - . %; . - . ) was assumed (figure a) . first, we predicted the longitudinal viral loads of an external study of patients receiving mg t.i.d. of hcq with azithromycin ( figure a) . the model predicted the viral decline in the first week, however, it overpredicted later time points. after incorporating a time varying function to mimic a delayed immune effect, predictions aligned well with the data throughout treatment (figure a) . the simulations performed in order to obtain the predicted percentage of patients with positive pcr accounted for interindividual variability in the pk and growth rate kinetics. the data were not sufficient to identify variability in the drug effect and it was not included in our simulations. hcq mg b.i.d. for days was predicted to produce the lowest percentage of patients with detectable viral loads ( %), however, it was predicted to result in a significant probability of qtc prolongation (data not shown). both mg b.i.d. for or days, and mg b.i.d. for , , or days were predicted to have lower detectable viral loads than those previously studied (figure b,c) . dosing regimens that included loading doses mg b.i.d. and mg b.i.d. for or days followed by a maintenance dose of mg b.i.d. or mg t.i.d. are shown in figure s . by comparison, mg b.i.d. or t.i.d. regimens showed modest efficacy. reaching ec levels and viral load data. in the pk graph, raw data is shown in red, whereas black and grey lines represent the typical and population plasma pk simulation (n = ) using the pk model. in vitro half-maximal effective concentration (ec s ) indicated in the graph were calculated considering total drug using the values reported in yao et al. in the viral load graph (left), thick lines represent the mean profiles of each group, whereas the thin ones represent the individual profiles. (b) individual pk plasma profile predicted with the pk model for each patient treated with hydroxychloroquine (hcq). (c) visual predictive check of population pk/pharmacodynamic model. the solid continuous line represents the th percentile of the observations, dashed lines represent . th and . th percentiles of observations, and shaded areas represent the % prediction intervals for median, . th, and . th percentiles obtained from , simulated datasets. the lower panel shows the proportions of below the limit of quantification values observed (solid line), with % prediction variability shown by shaded area. ct, cycle threshold; lloq, lower limit of quantification. faster by using higher doses seems to offer more benefit compared with extending treatment duration. both longer durations and higher doses of hcq resulted in greater qtc prolongation (figure d ; table s ). an average patient with baseline qtc value of ms or less could receive higher doses of mg b.i.d. over or days with minimal risk ( . % and . %) of qtc prolongation (table s ) . given the number of assumptions, reported numbers should be interpreted more as an indication of risk, which remain to be determined in clinical trials. the viral load decline in the control arms was substantially different in the two reported clinical studies (figure a) . , we explored hcq efficacy under different control arm scenarios. different viral growth and death values were obtained when the model was fit to each study's control arm. this represents the intrinsic variability of disease progression, and, accordingly, resulted in different viral kinetics over time, suggesting large uncertainty and variability in the natural history of disease. two dosing regimens ( mg hcq daily and mg hcq b.i.d.) were simulated and overlaid for comparison with the different natural history scenarios (figure b) . this sensitivity analysis revealed that the low-dose regimen might be indistinguishable from placebo under different control group scenarios. however, higher hcq doses (≥ mg b.i.d.) are likely to show efficacy in viral clearance regardless of the control arm. for hcq to maximally suppress sars-cov- replication in vivo, the hcq dose may need to be optimized. to best define the effective hcq concentrations for treatment of covid- , all available data from in vitro and clinical studies using hcq for sars-cov- were pooled to quantify the relationship between hcq pk and sars-cov- viral decline in patients with covid- . we predicted that higher hcq daily doses (e.g., as high as mg b.i.d.), were associated with rapid rates of viral decline and increased the percentage of pcr-negative patients but could result in increased risk of qtc prolongation. regimens that give ~ mg/day either loaded upfront or as mg b.i.d., could be safely tolerated and would reduce the time with a detectable sars-cov- viral load, and, thus, improve treatment outcomes. higher hcq doses of up to mg b.i.d. could result in even faster rates of viral decline but there is limited safety information for these high doses. hcq pharmacology is complex; hcq distributes extensively into erythrocytes (whole blood to plasma ratio ~ . , exhibits a long half-life ( hours) and a large volume of distribution, all attributed to extensive tissue uptake, clearly important for treatment of covid- systemic illness. , hcq and cq are diprotic weak bases (with pka of . and . vs. . and . for hcq and cq, respectively). interestingly, both drugs experience ion-trapping in which the drug becomes ionized in acidic environments like the lysosome (ph ~ . ). this causes an irreversible accumulation, explains the large volume of distribution, and potentially impacts the amount of free drug available in tissues. , hcq is converted into at least three metabolites (desethylhydroxychloroquine, desethylchloroquine, and bidesethylhdroxychloroquine). desethylhydroxychloroquine hcq, the primary metabolite, is pharmacologically active for some nonviral illnesses, and formed by various cytochrome p isozymes. for our analysis, we focused on the parent hcq, as potent in vitro activity against sars-cov- has only been described for the parent compound. for derivation of our dosing rationale, we have utilized hcq levels in plasma, instead of the lungs. lung accumulation has been observed for hcq and cq in animal pk studies and reported to be substantial (a partition coefficient of ( . )). the partition coefficient ratio enables quantification of the total drug concentration in the tissue, and by assuming the same fraction of unbound drug in plasma and tissue, one can further estimate unbound concentrations in the tissue. by using this approach, a wide range of doses, including doses as low as mg, seem to be potentially therapeutic. the drug efficacy at the site of action is determined by the fraction of drug unbound in the tissue, which has not been studied for hcq, and, thus, the amount of free drug in tissue remains unknown. highly lipophilic drugs for other infectious diseases, like bedaquiline and clofazimine, accumulate in lungs as well, however, the accumulation correlates with binding to macromolecules in tissue, not necessarily to the free fraction. , based on the physicochemical parameters of hcq (log p of . and pka of . , . ), the fraction unbound in tissue is likely low. therefore, in our study, we conservatively assume that the free fraction in plasma equilibrates between plasma and tissue and consider that to be the fraction of drug that can contribute to drug effect. tissue binding studies using a rapid equilibrium dialysis assay with lung homogenate should be performed to define an accurate fraction unbound in the tissue. using a mechanistic pk/pd modeling approach, we were able to quantify a relationship between hcq concentration and sars-cov- viral decline. however, we were not able to differentiate if azithromycin offered any additional benefit. the group receiving hcq and azithromycin had the lowest baseline viral load and showed a similar rate of viral decline compared with the hcq group. therefore, it remains unclear if azithromycin offers any additional benefit. clinically significant qtc prolongation associated with hcq have been reported. [ ] [ ] [ ] only two small observational studies have reported associations between hcq doses of - mg daily and qtc prolongation , and a concentration-dependent qtc relationship is not available. as a result, we used cq as a model to predict qtc prolongation risk. hcq and cq have an identical structure with the substitution of a hydroxyl group for hcq, and both have been found in vitro to inhibit the inward rectifier k+ channels. , this has been associated with qtc prolongation, and docking studies suggest nitrogen in the alkylamine and quinoline ring found in both compounds are responsible for binding with potassium channels. although a dedicated study is needed, the hydroxyl group in hcq is unlikely to affect rectifier k+ channels binding as the pka for the alkylamine nitrogen is similar to that of chloroquine's. in vitro data from cq identified an herg ic of , nm. we leveraged a recent study of high-dose cq for malaria treatment to predict potential risk of qtc prolongation with hcq. in support of our findings, a maximum dose of , mg daily for - weeks has been well-tolerated without reported cardiac toxicity. , based on this evidence, and the pk-qtc relationship for cq presented here, we expect a hcq course of - mg b.i.d. for days or less is unlikely to be associated with clinically significant cardiac toxicity in patients without a known risk factor for qtc prolongation. as data for hcq and qtc prolongation are limited, we recommend the highest doses of hcq be reserved for study in dose escalation studies. additional toxicities associated with hcq include retinopathy and gastrointestinal adverse events. , the mechanism of irreversible retinal damage associated with hcq is unknown, but it has been associated with hcq doses > mg/kg and in patients who receive hcq for > years. retinopathy associated with use < month of hcq has not been reported, and this side effect is less likely in the acute setting. , gastrointestinal toxicity with hcq is concentration-related and could be a limiting factor to dosage of hcq but doses up to , mg have been reported to be well-tolerated without adverse events in patients with cancer and rheumatologic disease in other studies. there were a few limitations to this study. first, clinical hcq data are limited to nonrandomized studies, and a clear model for the natural rate of viral decline is not well defined. to explore this effect, we compared viral kinetic trends on treatment to the extracted baseline data from cao et al. (n = hospitalized patients who received supportive care). second, the translational viral replication was obtained from sars-cov- data. sars-cov- and sars-cov- share an estimated . % sequence homology. third, we imputed the pk profiles for hcq using population pk parameters derived from a pool of both healthy and malaria-infected patients. fourth, we were not able to predict how concomitant hcq and azithromycin may impact the risk of qtc prolongation or anticipate how underlying risk factors for qtc prolongation could impact the pk-qtc relationship due to the lack of available data. closely monitored clinical trials will be needed to confirm that high-dose hcq is safe with or without azithromycin. finally, our model used plasma hcq concentrations to predict nasopharyngeal viral loads, which may not fully correlate with clinical improvement or viral load measured at different sites, however, it has generally been accepted that viral decline is a desirable marker leading to clinical improvement. [ ] [ ] [ ] [ ] [ ] [ ] in addition, all relevant assumptions made during the analysis are summarized in supplementary table s . treatment options for covid- can most effectively be advanced by utilizing all available data and pharmacologically driven drug repurposing. suboptimal dosing can result in wasted time and resources. even more problematic is the potential to declare a drug ineffective because of misdosing. using pk-exposure modeling, we predict that higher doses of hcq will be needed to achieve cure within days for all patients. given the observed prolonged viral shedding in patients with covid- , these data support the possibility that early treatment with high-dose hcq could reduce transmissibility and potentially reduce the risk of late clinical decompensation. however, given the possibility of qtc prolongation with high-dose regimens, rigorous trials must precede widespread clinical usage. we predict that higher hcq doses, (> mg b.i.d.) are most efficacious for viral suppression and should be further examined in clinical trials to evaluate safety and efficacy. supplementary information accompanies this paper on the clinical pharmacology & therapeutics website (www.cpt-journal.com). coronavirus disease (covid- ) outbreak situation <https design and synthesis of hydroxyferroquine derivatives with antimalarial and antiviral activities in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial a pilot study of hydroxychloroquine in treatment of patients with common coronavirus disease- (covid- ) information for clinicians on therapeutic options for covid- patients <https hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro in vitro screening of a fda approved chemical library reveals potential inhibitors of sars-cov- replication sars-associated coronavirus replication in cell lines a randomized, controlled trial of ebola virus disease therapeutics clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in covid- patients with at least a six-day follow up: an observational study pharmacokinetics of hydroxychloroquine and its clinical implications in chemoprophylaxis against malaria caused by plasmodium vivax plasma or serum in therapeutic drug monitoring and clinical toxicology likelihood based approaches to handling data below the quantification limit using nonmem vi ways to fit a pk model with some data below the quantification limit high-dose chloroquine for uncomplicated plasmodium falciparum malaria is well tolerated and causes similar qt interval prolongation as standard-dose chloroquine in children randomized dose-ranging controlled trial of aq- , a candidate antimalarial, and chloroquine in healthy volunteers a trial of lopinavir-ritonavir in adults hospitalized with severe covid- pharmacokinetics of hydroxychloroquine in pregnancies with rheumatic diseases on the mechanism of chloroquine resistance in plasmodium falciparum hydroxychloroquine: a physiologically-based pharmacokinetic model in the context of cancer-related autophagy modulation the contribution of lysosomal trapping in the uptake of desipramine and chloroquine by different tissues prediction of drug penetration in tuberculosis lesions tuberculosis drugs' distribution and emergence of resistance in patient's lung lesions: a mechanistic model and tool for regimen and dose optimization hydroxychloroquine is much less active than chloroquine against chloroquine-resistant plasmodium falciparum, in agreement with its physicochemical properties toxicokinetics of hydroxychloroquine following a massive overdose life threatening severe qtc prolongation in patient with systemic lupus erythematosus due to hydroxychloroquine chronic hydroxychloroquine use associated with qt prolongation and refractory ventricular arrhythmia long qt and hydroxychloroquine; a poorly recognised problem in rheumatology patients voltagedependent biphasic effects of chloroquine on delayed rectifier k(+)-channel currents in murine thymocytes urgent guidance for navigating and circumventing the qtc-prolonging and torsadogenic potential of possible pharmacotherapies for coronavirus disease (covid- ) the molecular basis of chloroquine block of the inward rectifier kir . channel pka values of medicinal compounds in pharmacy practice cardiotoxicity of antimalarial drugs hydroxychloroquine concentration-response relationships in patients with rheumatoid arthritis phase i trial of hydroxychloroquine with doseintense temozolomide in patients with advanced solid tumors and melanoma development and validation of a risk score to predict qt interval prolongation in hospitalized patients the risk of toxic retinopathy in patients on long-term hydroxychloroquine therapy accidental hydroxychloroquine overdose resulting in neurotoxic vestibulopathy a pneumonia outbreak associated with a new coronavirus of probable bat origin sars-cov- viral load in upper respiratory specimens of infected patients viral load of sars-cov- in clinical samples virological assessment of hospitalized patients with covid- detection of sars-cov- in different types of clinical specimens single-cell rna-seq data analysis on the receptor ace expression reveals the potential risk of different human organs vulnerable to -ncov infection high expression of ace receptor of -ncov on the epithelial cells of oral mucosa the investigators thank the savic laboratory for all their help and support during this project. we would also like to thank tia tummino for her help addressing reviewer comments. no funding was received for this work. the authors declared no conflict of interest. this is an open access article under the terms of the creative commons attribution-noncommercial license, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. key: cord- - hbcbvt authors: lane, j. c. e.; weaver, j.; kostka, k.; duarte-salles, t.; abrahao, m. t. f.; alghoul, h.; alser, o.; alshammari, t. m.; biedermann, p.; burn, e.; casajust, p.; conover, m.; culhane, a. c.; davydov, a.; duvall, s. l.; dymshyts, d.; fernandez bertolin, s.; fister, k.; hardin, j.; hester, l.; hripcsak, g.; kent, s.; khosla, s.; kolovos, s.; lambert, c. g.; ver der lei, j.; londhe, a. a.; lynch, k. e.; makadia, r.; margulis, a. v.; matheny, m. e.; mehta, p.; morales, d. r.; morgan-stewart, h.; mosseveld, m.; newby, d.; nyberg, f.; ostropolets, a.; park, r. w.; prats-uribe, a.; rao, g. a.; reich, title: safety of hydroxychloroquine, alone and in combination with azithromycin, in light of rapid wide-spread use for covid- : a multinational, network cohort and self-controlled case series study date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: hbcbvt background: hydroxychloroquine has recently received emergency use authorization by the fda and is currently prescribed in combination with azithromycin for covid- pneumonia. we studied the safety of hydroxychloroquine, alone and in combination with azithromycin. methods: new user cohort studies were conducted including severe adverse events (saes). rheumatoid arthritis patients aged + and initiating hydroxychloroquine were compared to those initiating sulfasalazine and followed up over days. self-controlled case series (sccs) were conducted to further establish safety in wider populations. separately, saes associated with hydroxychloroquine-azithromycin (compared to hydroxychloroquine-amoxicillin) were studied. data comprised sources of claims data or electronic medical records from germany, japan, netherlands, spain, uk, and usa. propensity score stratification and calibration using negative control outcomes were used to address confounding. cox models were fitted to estimate calibrated hazard ratios (calhrs) according to drug use. estimates were pooled where i < %. results: overall, , and , users of hydroxychloroquine and sulfasalazine, and , and , users of hydroxychloroquine-azithromycin and hydroxychloroquine-amoxicillin were included. no excess risk of saes was identified when -day hydroxychloroquine and sulfasalazine use were compared. sccs confirmed these findings. however, when azithromycin was added to hydroxychloroquine, we observed an increased risk of -day cardiovascular mortality (calhr . [ . - . ]), chest pain/angina (calhr . [ % ci . - . ]), and heart failure (calhr . [ % ci . - . ]) conclusions: short-term hydroxychloroquine treatment is safe, but addition of azithromycin may induce heart failure and cardiovascular mortality, potentially due to synergistic effects on qt length. we call for caution if such combination is to be used in the management of covid- . as the severe acute respiratory syndrome coronavirus (sars-cov- ) pandemic exerts an unprecedented pressure on health care systems worldwide, there remains a paucity of evidence surrounding the safety and effectiveness of potential treatments. several existing drugs have been postulated to be effective against sars-cov- . these include conventional synthetic disease modifying anti-rheumatic drugs (csdmards), which are most commonly used as the first line treatment of autoimmune diseases such as rheumatoid arthritis (ra) and systematic lupus erythematosus (sle). , hydroxychloroquine (hcq) has been proposed as potential treatment options for covid- based on its mechanism of action. accumulating in the acid vesicles (endosome, golgi vesicles, lysosomes), hcq causes alkalinisation, leading to enzyme dysfunction and preventing endosome mediated viral entry to the cell. [ ] [ ] [ ] [ ] it is also suggested in vitro that hcq can prevent glycosylation of virus cell proteins including the ace receptor, inhibiting virus entry and replication, and that similar compounds like chloroquine can specifically inhibit sars-cov- . , [ ] [ ] [ ] in clinical studies, the addition of hcq has shown increased early virological response to treatment for chronic hepatitis c, and reduced viral load in patients with hiv infection, compared to placebo. , treatment with hcq also lowered il- level in hiv patients, suggesting the agent may have immunosuppressive properties helpful in the prevention or treatment of cytokine storm associated with severe covid- disease. , as of th march , there are over registered ongoing clinical trials and prophylactic studies assessing the efficacy of hydroxychloroquine hcq for the treatment of sars-cov- . - early results from randomised controlled trials conducted in china have shown reduced severity and course of the disease with hydroxychloroquine hcq, compared with placebo, without detecting serious adverse effects, although others have suggested no difference in outcome from conventional treatment. , of those studies that have reported more detailed results and received significant media attention, hcq . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint has been proposed at higher doses than used in the treatment of auto-immune disorders and alongside azithromycin (azm), a macrolide antibiotic. results from this open label observational study suggest that the combination of hcq and azithromycin azm might lead to a faster recovery and reductions in viral load in the treatment of covid- . however, many authors have criticised the study due to lack of low power, limited follow-up, confounding by indication, and lack of adherence to the allocated treatment arm. the efficacy of hcq in combination with azm is therefore yet to be established, but approval for compassionate use by regulators and media attention will likely lead to an increase in use of this combined therapy for the management of covid- worldwide. in preparation for our study, we systematically searched the literature (pubmed, embase), clinical trial registries (clinicaltrials.gov, ictrp and chinese clinical trial registry) and preprint servers (biorxiv and medrxiv) from inception until / / (supplementary appendix section ) . no contemporary large-scale evidence was found to identify the real-world comparative safety of hcq compared to other first line dmards, especially in combination with macrolide antibiotics such as azm that are being considered for use in treating covid- . sepriano et al. led a systematic review to inform eular recommendations for the safety of ra medications, but little high-level evidence focussed on hcq. another recent review of the comparative risks of non-serious and serious adverse events (saes) associated with dmards predominantly focussed upon biologic therapies. there is little good high quality evidence quantifying saes risk in the literature with several studies suggesting no increased infection risk with any nonbiologic dmards, including hcq. , the safety profile of hcq is described in its summary of products characteristics, with adverse drug reactions including severe cardiac disorders as qt segment prolongation that could lead to arrhythmia, myocardial arrest or cardiovascular death. azithromycin (azm, and macrolides in general) . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint are known to induce cardiotoxicity when used alone, and to also increase the risk of other drugs that prolong qtc interval. [ ] [ ] [ ] [ ] it is therefore of utmost importance that we understand the safety implications of the proposed combination of hcq and azithromycin azm before this becomes standard practice in the management of covid- globally. in light of the current global pandemic, information regarding the safety of hcq in worldwide real-world practice is vital to inform policy. , we aimed to assess the safety of hydroxychloroquine (hcq) alone and in combination with azm to help guide decisions in the face of the growing covid- pandemic. two study designs were developed and executed across a multinational, distributed database network. first, new user cohort studies were used to estimate the safety of hcq compared to sulfasalazine (ssz), and to assess the risks associated with the addition of azm compared to amoxicillin (amx) amongst users of hcq in patients with rheumatoid arthritis (ra). ssz and amx were chosen as active comparators as they have similar indications as the target treatments (hcq and azm respectively). as a secondary analysis, self-controlled case series (sccs) was used to estimate the safety of hcq in the wider population, including uses for non-ra indications. electronic health records and administrative claims databases from primary care and secondary care containing participants from germany, japan, netherlands, spain, the uk, and the usa were analysed in a distributed network, and are detailed in the supplementary appendix, table s . . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint observational healthcare databases mapped to the observational medical outcomes partnership (omop) common data model collaborated in an international effort with the observational health data science and informatics (ohdsi) community. , de-identified or pseudonymised data were obtained from routinely collected records from clinical practice in germany, spain, the uk, japan, and the usa. studies were performed locally and no patient level data shared using the following databases: iqvia disease analyser germany emr (ambulatory emr from germany); jmdc (japanese claims); ipci (primary care emr from netherlands); sidiap (primary care emr from spain); cprd and imdr (primary care emrs from uk); and ccae, optum, mdcr, mdcd, panther, iqvia openclaims, veteran affairs (va), and iqvia us ambulatory emr (usa). sccs were conducted on a subset of these as a secondary analysis: ccae, cprd, optum, mdcd, and mdcr. rather than pooling these data assets, all analyses were conducted in a distributed network, where analysis code was sent to participating sites and only aggregate summary statistics were returned, with no sharing of patient-level data between organizations. the study period started from / / and ended at the latest available date for all data sources in . follow-up for each of the cohorts started at an index date defined by the first dispensing or prescription of the target/comparator drug as described in the cohort definitions (supplementary table . ). two periods were considered to define time-at-risk. first, for an intention-to-treat analysis, followup started one day after the index date and continued up until the first of: outcome of interest, loss to follow-up, or days after the index date to resemble the likely duration of covid- treatment regimens. secondly, for an on-treatment analysis, follow-up started one day after the index date and continued until the earliest of: outcome of interest, loss to follow-up, or discontinuation, with an added . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint washout time of days. continued use of a same treatment was inferred by allowing up to -day gaps between dispensing or prescription records. in the hcq versus ssz study, the index event was defined as the first recorded dispensing or prescription of the drug in a patient's history. for the study of hcq combined with azm, follow up started when the second of the two co-administered treatments was initiated while still exposed to the first treatment (e.g. when azm started during a period of hcq use, or when hcq started during a period of azm use). hcq use was assumed to be chronic in the management of ra, and azm was assumed an acute prescription for infection treatment, and therefore inferred persistent exposure to azm was assessed by allowing up to days between dispensing or prescription records. cohorts of combined hcq and amoxicillin were generated using these same rules as an active comparator. for sscs, periods of inferred persistent exposure to hcq were generated by allowing up to -day gaps between dispensing or prescription records. individual sccs analyses were executed separately for each of the proposed study outcomes, including both safety events and negative control outcomes. patients were followed for their entire observation time (e.g. from enrolment to disenrollment in each database), and incidence rates of each of the study outcomes calculated in periods of inferred persistent exposure to hcq and non-exposure periods. for the new user cohorts, participants included those with a history of ra (a condition occurrence or observation indicating ra any time before or on the same day as therapy initiation), aged years or over at the index event, with at least days of continuous observation time prior to index event. inclusion and start of follow-up started at the time one of the drugs of interest (hcq, ssz, or addition of . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint azm or amx amongst users of hcq) was initiated after a diagnosis of ra. for the sccs study, all prevalent users of hcq were included, regardless of ra history or indication for hcq therapy. participants were identified using pre-specified code lists reviewed by a core team of clinicians, epidemiologists, vocabulary experts, and health data scientists with extensive expertise in the use of the omop cdm and the ohdsi tools. the code lists in the omop cdm used to identify participants are listed in supplementary table . . the proposed code lists for the identification of the study population and for the study exposures were created by clinicians with experience in the management of ra using atlas , and reviewed by clinicians and epidemiologist (supplementary table . ). a total of severe adverse events (saes) were analysed. hospital-based events, not available in primary care records (cprd, imrd and sidiap), included gastrointestinal bleeding, acute renal failure, acute pancreatitis, myocardial infarction, stroke, transient ischaemic attack, and cardiovascular events (composite). additionally, angina/chest pain, heart failure, cardiac arrhythmia, bradycardia, venous thromboembolism, end stage renal disease, and hepatic failure were analysed from both primary and secondary care data. mortality outcomes were obtained only from data sources with reliable information on death date (cprd, imrd, ipci, optum, sidiap, va) and cardiovascular events preceding death records (cprd, imrd, optum, va), with the former contributing to informing all-cause mortality, and the latter also used to assess to cardiovascular death. all codes for the identification of the proposed study outcomes were based on a previously published paper, and are detailed in supplementary table . . face validity for each of the outcome cohorts was further reviewed by . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint exploring age-and sex-specific incidence rates compared to previous clinical knowledge and/or existing literature. two active comparator analyses were conducted in the cohort studies: first, incident users of hcq were compared to new users of ssz; second, new use of azm amongst prevalent users of hcq was compared to incident use of amx during ongoing hcq use. exposure commenced on the first day of dispensing or prescription recorded with at least days of prior observation period to increase confidence that the exposure was incident. exposure interval gaps of ≤ days (hcq and ssz) and of ≤ days (azm and amx) between drug dispensing or prescription records were allowed and inferred as persistent exposure. drug discontinuation was considered in the hcq study if a patient switched from one study drug to another. patients who switched from target exposure to comparator exposure, or vice versa, contributed follow-up time to the exposure cohort that they entered first, and were censored at the time of switching in the 'on treatment' analysis. a list of negative control outcomes was also assessed for which there is no known causal relationship with any of the drugs of interest. these outcomes were identified using a semi-automatic process based on data extracted from literature, product labels, and spontaneous reports, and confirmed by manual review by clinicians. a full list of codes used to identify negative control outcomes can be found in supplementary table , and details on covariate/confounder identification are provided in supplementary table . this study was undertaken using routinely collected data and all patients meeting the eligibility criteria above during the study observation period were included. no a priori sample calculation was performed; instead, a minimum detectable rate ratio (mdrr) was estimated for each drug-outcome pair . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint in each of the available databases. the mdrrs for each of the databases for each drug pair-outcome analysis, as well as sample size for each of the comparisons are reported in full in an interactive web app (https://data.ohdsi.org/covid estimationhydroxychloroquine/. only analyses with counts in either treatment group were excluded based on power, with all others contributing to meta-analytic estimates where applicable. ps stratification was used as the analytical strategy to adjust for imbalance between exposure cohorts in a comparison, using a large-scale regularized logistic regression fitted with a lasso penalty and with the optimal hyperparameter determined through -fold cross validation. baseline patient characteristics were constructed for inclusion as potentially confounding covariates. from this large set of tens of thousands of covariates, key predictors of exposure classification were selected for the propensity score. the predictor variables included were based on all observed patient characteristics and covariates available at each data source, including conditions, procedures, visits, observations and measurements. all covariates that occur in fewer than . % of patients within the target and comparator cohorts were excluded prior to propensity score model fitting for computational efficiency. patients in the target and comparator cohorts were stratified into propensity score quintiles. plotting the propensity score distribution and assessment of covariate balance expressed as the standardized difference of the mean was undertaken for every covariate before and after propensity score adjustment. a standardized difference > . indicated a non-negligible imbalance between exposure cohorts. the target and comparator cohort were compared using a univariate cox proportional hazards model conditioned on the propensity score strata with treatment allocation as the sole explanatory variable. negative control outcomes analyses and empirical calibration were used to . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint further minimise potential unresolved confounding with calibrated hrs (calhrs) and % confidence intervals estimated. , for sccs, safety of hcq therapy was assessed separately as a secondary analysis, regardless of indication, comparing exposed and unexposed time periods within the same individuals. the method is self-controlled in that it makes within-person comparisons of event rates during periods of hypothesized increase risk with other periods of baseline risk, with eliminates all time-invariant confounding. because we do not compare between persons, the sccs is robust to between-person differences, even including unmeasured differences (like genetics). however, the method is vulnerable to time-varying confounders: the time of exposure may be incomparable to the time when not exposed. to adjust for this, we included many time-varying co-variates in the models, including age, season, and other drug exposures. the effects of age and season were assumed constant within each calendar month and were modelled using bicubic splines with knots. a conditional poisson regression was used to fit the outcome model using the cyclops package, with a hyperparameter selected through -fold crossvalidation. study diagnostics (power, propensity score distribution, covariate balance, empirical null distribution) were evaluated by clinicians and epidemiologists to determine which database-target-comparatoroutcome-analysis variants could produce unbiased estimates. database-target-comparator-analysis variants with zero event outcomes in the time-at-risk window or contained analyses with baseline covariate with standardized mean difference> . after stratification were excluded from analysis. study diagnostics for all database-target-comparator-outcome-analysis will be provided as part of study, regardless of which effect estimation results are unblinded. all the proposed analyses were conducted for each database separately, with estimates combined in fixed effects meta-analysis methods where i . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint is <= %. no meta-analysis was conducted where i for a given drug-outcome pair is > %. of note, when running analysis in a distributed network, it was not possible to link across datasets, and to know the extent of overlap between data. all analytical code is available at https://github.com/ohdsistudies/covid estimationhydroxychloroquine, with study diagnostics considered prior to the unblinding of estimation results. all study diagnostics are available for exploration at https://data.ohdsi.org/covid estimationhydroxychloroquine/. all statistical analyses were conducted using tools previously validated by the ohdsi community. for the cohort analysis, the cohortmethod package was used (https://ohdsi.github.io/cohortmethod/) using a large-scale propensity score (ps) constructed through the cyclops package (https://ohdsi.github.io/cyclops). all sccs were run using the freely available package (https://ohdsi.github.io/selfcontrolledcaseseries/). a total of , hcq and , ssz users were identified, with , and , contributing to the analyses of combination therapy of hcq with azm compared to hcq with amx respectively. participant counts in each data source are provided in appendix s . users of hcq were more likely female (e.g. . % vs . % in ccae) and less likely to have certain comorbidities like inflammatory bowel disease (e.g. prevalence of crohn's disease . % vs . % in ccae) or psoriasis (e.g. . % vs . % in ccae). all these differences were however minimised after propensity score stratification, with all reported analyses balanced on all identified confounders including sociodemographics, comorbidities and concomitant drug/s use. similarly, users of combination hcq+azm differed from those of hcq+amx, with a prevalence of acute respiratory disease appearing higher . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint amongst azithromycin users ( . % vs . % in ccae). again, propensity score methods resolved these differences, and comparison groups became balanced for all observed confounders after stratification. detailed baseline characteristics for hcq vs ssz and for hcq+azm vs hcq+amx after propensity score stratification in ccae are detailed in table we report here (table ) on database-specific counts and rates of key outcomes (cardiovascular mortality, chest pain/angina and heart failure) observed in the proposed -day intention-to-treat analysis. . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint database-specific counts, incidence rates (ir) of all study outcomes stratified by drug use are detailed in full in supplementary table s . least common outcomes included bradycardia (e.g. ir . / , . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint person-years (py) amongst hcq users in ccae) and end-stage renal disease (e.g. ir < . / , py amongst hcq users in ccae), whilst most common ones were chest pain/angina (e.g. ir . / , py amongst hcq users in ccae) and composite cardiovascular events (e.g. ir . / , py amongst hcq users in ccae). as expected, most irs appeared higher in data sources which included older populations (e.g. ir of composite cardiovascular events in hcq users in mdcr of . / , py). mortality rates ranged from . / , person-years in hcq users in optum to . / , py amongst hcq users in va, with cardiovascular-specific mortality ranging from ir . / , py in hcq users in va to < . / , person-years in ssz users in the same data source. . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint figure . meta-analytic risk estimates for hydroxychloroquine vs sulfasalazine and azithromycin vs amoxicillin new users during on-treatment during -day and on-treatment follow-up hcq=hydroxychloroquine; ssz=sulfasalazine; azm=azithromycin (plus concurrent hydroxychloroquine exposure); amx=amoxicillin (plus concurrent hydroxychloroquine exposure); calhr=calibrated hazard ratio; ci=confidence interval; i =estimate heterogeneity statistic. . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint consistent findings were seen with the long-term (on treatment) use of hcq vs ssz (figure ) , with the exception of cardiovascular mortality, which appeared inconsistent in the available databases, but overall increased in the hcq group when meta-analysed: pooled calhr . ( . - . ). similar results were obtained in sccs analyses, which looked at the effect of hcq use (on-vs offtreatment) on all outcomes except mortality regardless of indication, and therefore included non-ra patients (tables s . to . for database-specific results). all the obtained database-and outcome-specific calhrs for the association between short-term ( month) use hcq+azm vs hcq+amx are depicted in the form of forest plots in supplementary figure . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint (figure ). despite a lack of evidence on efficacy, hcq and hcq+azm have become the most popular treatment/s for covid- . this is the largest ever analysis of the safety of such treatments worldwide, examining over , hcq and more than , hcq+azm users respectively. the results on the risk of saes associated with short-term ( month) hcq treatment as proposed for covid- therapy are reassuring, with no excess risk of any of the considered safety outcomes compared to an equivalent therapy (ssz). however, long-term treatment with hcq as used for ra is associated with a % increase in cardiovascular mortality. worryingly, significant risks are identified for combination users of hcq+azm even in the short-term as proposed for covid management, with a - % increased risk of angina/chest pain and heart failure, and a two-fold risk of cardiovascular mortality in the first month of treatment. a systematic review of the cardiac side effects of chloroquine and hcq identified articles reporting short series or individual cases. in the included patients, cardiac side effects occurred in mainly women ( . %) who had a median age of years. conduction disorders were the main side effect reported ( %), with heart failure ( . %), ventricular hypertrophy ( %), hypokinesia ( . %), valvular dysfunction ( . %) and pulmonary arterial hypertension ( . %) being the other reported side effects. when drugs were withdrawn, . % of patients recovered normal cardiac function; . % sustained irreversible damage, and % died. it should be noted that cardiac toxicity was induced by a high cumulative dose of chloroquine or hcq in most patients, although some studies identified by this . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint our results suggest that long-term use of hcq leads to an increased risk of cardiovascular mortality, with no observable excess risk of major cardiovascular events or diagnosed bradycardia. considering the current evidence, this may relate to cumulative effects of hcq leading to an increased risk of qt lengthening or relate to the moderately increased risk of angina and heart failure seen. however, as the strong association observed with cardiovascular death is not observed with diagnosed arrhythmia or bradycardia in this study, sudden cardiovascular death here is more likely due to qt lengthening and undetected and/or sudden torsade-de-pointes. although long-term treatment with hcq is not expected for the management of covid- , some research suggests that higher doses as prescribed for covid- can, even in the short-term, lead to equivalent side effects given the long half-life of hcq. qt lengthening is a known effect of all macrolides including azm and physicians already use caution when prescribing macrolides concurrently with other medications that can also increase the qt interval. [ ] [ ] [ ] in this study, the elevated risk of cardiovascular death with combined hcq +azm therapy may arise through their synergistic effects of inducing lethal arrhythmia. . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint as with all observational data, this study is limited by its ability to appropriately identify exposure and outcome. due to the nature of sudden cardiac death, capturing the true cause of cardiovascular related mortality is difficult. we therefore have explored cardiovascular related outcomes other than mortality to determine if deterioration in these pathophysiological processes led to increased mortality. since this is not seen, and sudden cardiac death in association with prolonged qt interval is described in the literature, our conclusions are drawn from these assumptions. it should be acknowledged that misclassification can occur due to non-adherence or non-compliance with exposure medication, and incomplete lack of recording of saes may lead to underestimation of these outcomes. another potential limitation in this study is the potential for patients to be included in more than one dataset in the us. whilst we ran meta-analysis, which assume populations are independent, we wish to highlight we are likely to under-estimate variance in our meta-analytic estimates. the comparative new user cohort studies are anchored in patients using hcq for ra, who therefore are likely to be using hcq at a lower dose than is currently being proposed for use in the treatment of covid- . we have taken into consideration that patients with ra taking hcq may also have further auto-immune conditions such as systemic lupus erythematosus (sle) and therefore generate the potential for confounding by indication. we therefore ensured that when investigating covariate balance after propensity score stratification and matching and before unblinding study results, that we did not see unbalanced proportions of patients with a diagnosis of sle between the groups. negative control outcome analyses also did not identify any residual unobserved confounding in the ps analysis. whilst patients with ra may have greater levels of comorbidities than the general population, the age and demographic profile of patients developing cardiovascular complications described in both the systematic review and faers database suggests that complications are not only restricted to those with . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint multimorbidity. however, absolute risk in our study should be interpreted cautiously since patients with ra are likely different from those with covid- . as the world awaits the results of clinical trials for the anti-viral efficacy of hcq in the treatment of sars-cov infection, this large scale, international real-world data network study enables us to consider the safety of the most popular drugs under consideration. hcq appears to be largely safe in both direct and comparative analysis for short term use, but when used in combination with azm this therapy carries double the risk of cardiovascular death in patients with ra. whereas we used the collective experience of a million patients to build our confidence in the evidence around the safety profile, the current evidence around efficacy of hcq+azi in the treatment of covid- is quite limited and controversial. all data partners received irb approval or waiver in accordance to their institutional governance guidelines. this is a retrospective database study on de-identified data and is deemed not human subject research. approval is provided for ohdsi community studies. ccae new england institutional review board (irb) and was determined to be exempt from broad irb approval, as this research project did not involve human subject research. cprd approval for cprd was provided by the independent scientific advisory committee (isac). this study is based in part on data from the full feature general practice research database obtained under license from the uk medicines and healthcare products regulatory agency. however the interpretation and conclusions contained in this report are those of the author/s alone. the protocol for this study ( _ r) was approved by the independent scientific advisory committee (isac). . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint da germany this is a retrospective database study on de-identified data and is deemed not human subject research. approval is provided for ohdsi community studies. the present study is filed and under review for scientific review committee for institutional adjudication. due to the public health imperative of information related to these data, approval is provided for this publication. ipci the present study was approved by the scientific and ethical advisory board of the ipci project (project number: / ). jmdc new england institutional review board (irb) and was determined to be exempt from broad irb approval, as this research project did not involve human subject research. mdcd new england institutional review board (irb) and was determined to be exempt from broad irb approval, as this research project did not involve human subject research. mdcd new england institutional review board (irb) and was determined to be exempt from broad irb approval, as this research project did not involve human subject research. open claims this is a retrospective database study on de-identified data and is deemed not human subject research. approval is provided for ohdsi community studies. optum new england institutional review board (irb) and was determined to be exempt from broad irb approval, as this research project did not involve human subject research. panther new england institutional review board (irb) and was determined to be exempt from broad irb approval, as this research project did not involve human subject research. the use of sidiap data base was approved by the sidiap scientific committee and the idiapjgol clinical research ethics committee. va the use of va data was reviewed by the department of veterans affairs central institutional review board (irb) and was determined to meet the criteria for exemption under exemption category ( ) and approved the request for waiver of hipaa authorization. the va privacy office certified the release of aggregate analysis results for the meta-analysis. all authors have completed the icmje uniform disclosure form at www.icmje.org/coi_disclosure_.pdf author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint tremendous work and dedication of the participants from nations in the march ohdsi covid- virtual study-a-thon (https://www.ohdsi.org/covid- -updates/), without whom this study could not have been realized. who. report of the who-china joint mission on coronavirus disease (covid- ) eular recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: update chloroquine and hydroxychloroquine as available weapons to fight covid- effects of chloroquine on viral infections: an old drug against today's diseases? confronting an influenza pandemic with inexpensive generic agents: can it be done? virus glycosylation: role in virulence and immune interactions new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid- ? new insights into the antiviral effects of chloroquine remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro hydroxychloroquine augments early virological response to pegylated interferon plus ribavirin in genotype- chronic hepatitis c patients hydroxychloroquine treatment of patients with human immunodeficiency virus type comparison of hydroxychloroquine with zidovudine in asymptomatic patients infected with human immunodeficiency virus type study for the efficacy of chloroquine in patients with novel coronavirus pneumonia (covid- ). . . chictr . efficacy of therapeutic effects of hydroxycholoroquine in novel coronavirus pneumonia (covid- ) patients(randomized open-label control clinical trial). . . chictr , the first hospital of peking university y. efficacy of therapeutic effects of hydroxycholoroquine in novel coronavirus pneumonia (covid- ) patients(randomized open-label control clinical trial). . . chictr , renmin hospital of wuhan university n. therapeutic effect of hydroxychloroquine on novel coronavirus pneumonia (covid- ). . . chictr . therapeutic effect of hydroxychloroquine on novel coronavirus pneumonia (covid- ). . . chictr , the fifth affiliated hospital sun yat-sen university y. efficacy of chloroquine and lopinavir/ ritonavir in mild/general novel coronavirus (covid- ) infections: a prospective, open-label, multicenter randomized controlled clinical study. . . chictr , the fifth affiliated hospital of sun yat-sen university y. a prospective, open-label breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies hydroxychloroquine and azithromycin as a treatment of covid- : preliminary results of an open-label non-randomized clinical trial chloroquine for influenza prevention: a randomised, double-blind, placebo controlled trial no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe covid- infection safety of synthetic and biological dmards: a systematic literature review informing the update of the eular recommendations for the management of rheumatoid arthritis impact of adverse events associated with medications in the treatment and prevention of rheumatoid arthritis use of nonbiologic disease-modifying antirheumatic drugs and risk of infection in patients with rheumatoid arthritis long-term safety of methotrexate monotherapy in patients with rheumatoid arthritis: a systematic literature research the cardiotoxicity of macrolides: a systematic review azithromycin and the risk of cardiovascular death azithromycin/chloroquine combination does not increase cardiac instability despite an increase in monophasic action potential duration in the anesthetized guinea pig cardiac risks associated with antibiotics: azithromycin and levofloxacin covid- : chloroquine and hydroxychloroquine only to be used in clinical trials or emergency use programmes request for emergency use authorization for use of chloroquine phosphate or hydroxychloroquine sulfate supplied from the strategic national stockpile for treatment of coronavirus disease comprehensive comparative effectiveness and safety of first-line antihypertensive drug classes: a systematic, multinational, large-scale analysis accuracy of an automated knowledge base for identifying drug adverse reactions evaluating large-scale propensity score performance through realworld and synthetic data experiments balance diagnostics for comparing the distribution of baseline covariates between treatment groups in propensity-score matched samples robust empirical calibration of p-values using observational data interpreting observational studies: why empirical calibration is needed to correct p-values massive parallelization of serial inference algorithms for a complex generalized linear model multiple self-controlled case series for large-scale longitudinal observational databases data mining and safety analysis of drugs for novel coronavirus pneumonia treatment based on faers: chloroquine phosphate herald of medicine cardiac complications attributed to chloroquine and hydroxychloroquine: a systematic review of the literature electrocardiogram abnormalities related to anti-malarials in systemic lupus erythematosus key: cord- - a zv xl authors: o’connell, thomas f.; bradley, christopher j.; abbas, amr e.; williamson, brian d.; rusia, akash; tawney, adam m.; gaines, rick; schott, jason; dmitrienko, alex; haines, david e. title: hydroxychloroquine/azithromycin therapy and qt prolongation in hospitalized patients with covid- date: - - journal: jacc clin electrophysiol doi: . /j.jacep. . . sha: doc_id: cord_uid: a zv xl abstract background hydroxychloroquine and azithromycin (hcq/azm) are being widely used to treat covid- despite the known risk of qt interval prolongation and unknown risk of arrhythmogenesis in this population. objective the study aimed to characterize corrected qt (qtc) prolongation in a cohort of hospitalized covid- patients treated with combination hcq/azm. methods a retrospective cohort of covid- hospitalized patients treated with hcq/azm was reviewed. the qtc interval was calculated prior to drug administration and for the first days following initiation. the primary end point was the magnitude of qtc prolongation, and factors associated with qtc prolongation. secondary endpoints were incidences of sustained ventricular tachycardia or ventricular fibrillation and all-cause mortality. results among patients receiving concomitant hcq/azm, the mean qtc increased from ± msec to a maximum of ± msec ( ( %) had a qtc ≥ msec). factors associated with qtc prolongation ≥ msec were age (p < . ), body mass index < kg/m (p = . ), heart failure (p < . ), elevated creatinine (p = . ), and peak troponin (p < . ). the change in qtc was not associated with death over the short period of the study in a population where mortality was already high (hazard ratio, . , p = . ). no primary high-grade ventricular arrhythmias were observed. conclusions an increase in qtc was seen in hospitalized covid- patients treated with hcq/azm. several clinical factors were associated with greater qtc prolongation. changes in qtc were not associated with increased risk of death. in december , severe acute respiratory syndrome coronavirus- , a novel coronavirus which causes coronavirus disease (covid- ) , was first detected in wuhan, china. since then, a global pandemic of covid- has emerged with high rates of hospital admission and respiratory failure. while large multi-center clinical trials have begun evaluating potential therapies, several small studies have proposed that repurposed drugs, such as azithromycin and hydroxychloroquine, may offer benefit in the treatment of this disease. , the united states food and drug administration has provided emergency use authorization for health care providers to prescribe hydroxychloroquine to hospitalized patients with covid- , and it has been used in conjunction with azithromycin. however, both hydroxychloroquine and azithromycin (hcq/azm) are known to prolong the electrocardiographic qt interval and could increase risk of drug-induced torsade de pointes and sudden cardiac death in these patients. [ ] [ ] [ ] [ ] because risks associated with qt prolongation in this patient population are largely unknown, the present study aimed to determine the magnitude of qt prolongation and prevalence of serious arrhythmias among patients hospitalized with covid- who were treated with the combination of hcq/azm, and determine if qt prolongation was associated with adverse outcomes. this was a retrospective cohort study of consecutive patients enrolled in the beaumont health covid- database which was granted a waiver of consent by the institutional review board. all patients years of age and older with proven covid- admitted to beaumont hospital -royal oak and beaumont hospital -troy between march and april , were screened for study entry. of this population, those patients who were administered concomitant hcq/azm, had at least one day of qtc assessment following drug initiation, and an interpretable baseline ecg were included in the cohort. demographics, past medical history, relevant laboratory values, and clinical outcomes were recorded (see appendix ) . administration of drugs with known risk or probable risk of inducing torsade de pointes were documented. the pharmacodynamics of qtc prolongation after initiation of hcq/azm was assessed over the days of drug administration in a subgroup of patients. patients with qtc measurements at baseline, day , days and/or , and days and/or were included in this analysis. those patients with incomplete data due to missing or technically inadequate tracings were excluded from this analysis. patients with proven or suspected covid- infection were prescribed hcq/azm at or soon after hospital admission at the discretion of the admitting physician. the standard dose regimens prescribed were hydroxychloroquine mg twice daily for two doses then mg twice daily for days and azithromycin mg once followed by mg daily for days. patient care decisions were made by the hospital clinicians, and no attempt was made by study personnel to direct their care. decisions regarding early termination of hydroxychloroquine and/or azithromycin (in some cases due to excessive prolongation of the qtc interval) were at the sole discretion of the physicians supervising care. an institutional policy was created during the covid- pandemic to balance safe monitoring for qt interval prolongation in patients prescribed hcq/azm while also limiting ecg technician exposure to infected patients and preserving personal protective equipment ( figure ). per institutional policy, a baseline corrected qt interval (qtc) was measured from a -lead ecg prior to initiation of hcq/azm. if the patient had an ecg on file within days of drug initiation, that qtc interval value could be used as the baseline measure. due to limits on telemetry availability during the covid- pandemic, institutional guidelines recommended no further qtc monitoring for patients with baseline qtc intervals less than milliseconds (msec) unless there was another indication for telemetry monitoring present. the institutional policy recommended that patients with qtc intervals msec or greater were to be placed on telemetry for daily qtc and arrhythmia monitoring during drug initiation. multiple repeat lead ecgs to monitor the qtc interval were discouraged. it was at the discretion of the attending physician whether to initiate or discontinue hydroxychloroquine, azithromycin, and/or telemetry. available lead ii telemetry monitor strips and ecgs were reviewed for consecutive days while the patient was on combination drug therapy. the day qtc measured on telemetry was defined as the first day after initiation of both hcq/azm. the qtc data were only recorded when patients were on combination therapy. those without an interpretable baseline ecg or available telemetry/ecg monitoring for at least day after drug administration were excluded from analysis ( figure ). the ecgs and telemetry recordings were analyzed off line. the qt intervals were measured manually using the tangent method and dividing by the square root of the average rr interval (bazett's formula) to derive the qtc. for patients with intraventricular conduction delays (paced rhythms or bundle branch block), a modified qtc was calculated using the formula: modified qtc = (qt -(qrs - msec)) /√rr. the primary study endpoint was to quantitate the magnitude of qtc prolongation observed in a hospitalized cohort of patients with covid- treated with both hcq/azm, and to identify risk factors in this patient population for significant qtc prolongation. secondary endpoints were the incidences of sustained ventricular tachycardia or ventricular fibrillation, and the relationship of qtc prolongation to in-hospital sustained ventricular tachyarrhythmias and mortality during the study period. cause of death was adjudicated by review of the resuscitation records from all patients with attempted resuscitation, and the reviewers of these data were blinded to the qtc data. patients who died without monitoring because of transfer to "comfort care only" status were defined as non-arrhythmic deaths. high-grade ventricular arrhythmias were defined as ventricular tachycardia or ventricular fibrillation greater than seconds in duration or requiring urgent medical intervention. secondary ventricular tachyarrhythmias observed during the course of cardiac resuscitation subsequent to a bradycardic or pulseless electrical activity arrest were not designated as high-grade ventricular arrhythmic events. all authors participated in multiple aspects of the trial including data collection (tfo, cjb, ar, amt, rg, js), data compilation and analysis (all), statistical analysis (ad), and manuscript preparation (tfo, cjb, aea, bdw, ar, amt, ad, deh). repeated measures of % of the qtc data were performed, and the average intraclass correlation coefficient was . . descriptive analyses were performed to assess differences between groups. continuous variables were expressed as means and standard deviations. categorical variables were expressed as numbers and percentages. the differences between the groups were evaluated using the twosample t-test for continuous variables and fisher's exact test for categorical variables. a multivariate logistic regression analysis was conducted evaluating the impact of key demographic and clinical factors associated with qtc ≥ msec. a second multivariate analysis was conducted to evaluate the impact of key demographic and clinical factors associated with qtc prolongation as well as changes in the qtc interval on mortality. these analyses were performed using the cox proportional hazards model. qtc changes from baseline to days through were included as a time-varying covariate in the second analysis. the hazard ratio, % confidence interval and p-value were computed from this model to characterize the influence of each factor on mortality. a two-sided p-value less than . was considered statistically significant. no formal adjustments for multiplicity were applied. the analyses were performed using r software (version . . ) (r foundation for statistical computing, vienna, austria). a total of patients were admitted during the study period with a proven diagnosis of covid- and treated with combination therapy of hcq/azm. of those patients, met the inclusion criteria, and were excluded because of incomplete or technically limiting ecg or clinical data ( figure ). the demographics of the included and excluded patients are listed in table . the excluded group was younger, had shorter baseline qrs duration, and had shorter qtc intervals (attributable to the institutional policy that ecg telemetry monitoring was not required in patients with baseline qtc intervals < msec). in the included study population, the baseline qtc interval was ± msec (range - msec). the qtc progressively increased coincident with administration of hcq/azm ( figure a ). the maximum qtc observed over the course of monitoring for all patients receiving the drugs was ± msec (range - msec), the magnitude of qtc prolongation compared to the baseline tracing was ± msec (range - msec). the qtc was ≥ msec after drug administration in patients ( %). notably, during the hospitalization, patients ( %) received additional medications with known risk of torsade de pointes (table s in the supplementary appendix). factors associated with qtc prolongation ≥ msec are presented in table . changes in the qtc over the -day prescription duration were assessed in a subset of patients with qtc measurements at baseline prior to medication administration and on days through . in this subgroup, the average time to maximum qtc was . ± . days. the change in the qtc from baseline increased progressively with time ( figure b ). throughout the study period, no primary high-grade ventricular arrhythmias were observed in any patient in the included or excluded groups. of the entire treatment population, there were a total of deaths in hospital. twenty-six of ( %) excluded patients died, and of ( %) in the included population died. of those patients, patients had pulseless electrical activity or bradycardia at the initiation of the resuscitation and patients died off monitor because "do not resuscitate" orders were placed. the change in qtc interval from baseline was greater in patients who died during hospitalization compared to those who survived to hospital discharge or study termination ( figure b ). data from the proportional hazards model for time-varying qtc changes with concurrent mortality for the subset of patients with complete qtc data and the overall population of patients are presented in table . despite the association of qtc prolongation with mortality, the only independent predictor of mortality was age. in response to the poor outcomes observed in patients with covid- infections and lack of proven curative therapies, clinicians have turned to unproven remedies like hcq/azm, even though this drug combination is known to prolong the qt interval and has an unknown risk of proarrhythmia in this disease. the present study was performed to quantitate the magnitude of qt prolongation in a cohort of hospitalized covid- patients treated with hcq/azm and to identify qt-prolonging risk factors in this population. the qtc was found to increase over the day hcq/azm course with the average change in the baseline to maximum qtc measuring msec. this is the first study in a covid population to identify that older age, lower bmi, higher admission creatinine, higher peak troponin, and a history of congestive heart failure independently predicted potentially hazardous qtc prolongation ≥ msec. however, despite the significant increases in qtc intervals in these patients, no high-grade ventricular arrhythmias were observed and changes in qtc were not associated with increased likelihood of dying. this suggests that the actual risk of torsade de pointes in this setting is very low, or that the prescribing physicians were appropriately adjusting therapies in patients in whom excess qt prolongation was observed. hydroxychloroquine is a widely prescribed anti-malarial and anti-rheumatic medication , and azithromycin is a commonly prescribed antibiotic. they are both currently being prescribed, often in combination, as an off-label treatment for covid- . both medications increase the qtc interval by blocking the kcnh -encoded herg potassium channel. [ ] [ ] [ ] prior to the covid- pandemic, most of the clinical evidence of hydroxychloroquine's qt prolonging properties was limited to case reports. , azithromycin's qt-prolonging properties had been better studied with a large case-control study demonstrating that hospitalized patients treated with azithromycin were more likely to have severe qt prolongation. the present study confirms evidence of the qt-prolonging properties of hcq/azm, and extends these results to a covid- population. the qtc progressively and significantly increased during hcq/azm administration similar to other studies in covid- populations. [ ] [ ] [ ] [ ] confirmation of qtc prolongation in this population better informs physicians of this important side effect of hcq/azm that will necessitate qtc assessment prior to drug initiation, and qtc monitoring during treatment for many patients. identifying qtc prolonging risk factors will be important before prescribing hcq/azm and when deciding on a monitoring strategy. genetics, medications, underlying medical conditions, and other metabolic factors predispose patients to qt prolongation which can lead to torsade de pointes. one out of people are genetically predisposed to have a long qt interval due to congenital long qt syndrome. hospitalized patients are particularly susceptible to drug-induced qt prolongation due to concomitant proarrhythmic risk factors, and those on non-antiarrhythmic qt prolonging medications have twice the risk of in-hospital cardiac arrest. this risk further increases with prescription of multiple qtc prolonging medications. , while no qtc value can predict the onset of torsade de pointes with certainty, data from the congenital long qt syndrome and druginduced torsade de pointes populations have shown patients with qtc > msec are at increased risk. the united states food and drug administration has discouraged use of hcq/azm outside of clinical trials or hospitalized settings with monitoring , and cardiovascular leaders have issued guidance to clinicians to consider withholding and withdrawing hcq/azm for a qtc> msec. over % of the patients in our cohort had a qtc ≥ msec after hcq/azm administration. cohort characteristics which predicted qtc prolongation were similar to those predicting significant qt interval prolongation in a validated risk score model in hospitalized patients. elevated troponin was independently associated with qtc prolongation ≥ msec which does not rule out ischemia as a mechanism for qtc prolongation in this cohort. while other known precipitants of qtc prolongation such as hypokalemia and hypomagnesemia were not significant in our cohort, this is likely attributable to an institutional policy that recommended careful electrolyte monitoring in these patients. despite careful monitoring of patients on hcq/azm, over % of the cohort was administered at least one additional medication during hospitalization that increased the risk of torsade de pointes. this speaks to the medical complexity of this patient population, but also suggests inadequate awareness of the qt prolonging effects of many common medications. throughout the study period, no primary high-grade ventricular arrhythmias were observed. over % of the study population died which highlights the high mortality of hospitalized covid- patients who suffer significantly from severe respiratory illness. while positive changes in qtc from baseline were associated with increased morality in univariate analysis, this was not observed in multivariate analysis. qtc prolongation may be a marker of sicker patients as evidenced by the comorbid conditions and laboratory abnormalities predicting qtc prolongation ≥ msec. given the magnitude of qtc prolongation on hcq/azm, larger studies are warranted to investigate the prevalence of arrhythmias and mortality if this remains a therapy for covid- . while the world waits for the results of larger trials, proposed systems to safely monitor the qtc will remain necessary and cannot be ruled out as an explanation for the results of this study. there were several limitations to the study. although a specific dosing schedule was recommended for all patients, the decisions when and how to prescribe hcq/azm were deferred to the prescribing physicians. due to the retrospective nature of the study, it was not possible to definitively determine whether hcq/azm was discontinued by clinicians in response to qtc assessments. retrospective collection of some data and technical limitations prevented acquisition of qtc intervals from all patients on all days, so the associations between magnitude of qt prolongation and clinical and outcome data are based upon incomplete information. the initial qtc interval was obtained from a -lead ecg but subsequent qtc measurements were almost exclusively obtained from a telemetry monitoring system. this policy was instituted by the health care system in an attempt to balance monitoring for qtc interval prolongation while also protecting staff and limiting use of personal protective equipment. although the death rate was high, the rate of high-grade ventricular arrhythmias was very low. the rate of appropriate icd therapy or death in patients with congenital long qt syndrome and qtc intervals > ms was reported as % over five years, but the duration of follow up in the present study was brief and it was not adequately powered to assess the association of druginduced qt prolongation and mortality. in addition, the results of qt monitoring certainly affected clinical decisions thus confounding outcome data. also, it is possible that the covid- infection could directly prolong the qtc independent of drug effects. unfortunately, the high utilization of hcq/azm in these hospitals precluded analysis of a drug-free control arm. finally, the cohort studied was comprised of hospitalized patients, and the results may not apply to non-hospitalized patients or prophylactic therapy regimens. because decisions when and how to prescribe hcq/azm were deferred to the prescribing physicians. it is possible that the / patients prescribed hcq/azm were the sickest patients. hospitalized patients with covid- treated with hcq/azm had a significant and progressive increase in qtc during combination drug therapy. several risk factors identified patients at risk of severe qtc prolongation. despite this finding, the risk of serious arrhythmias during this brief observation period was low. competency in medical knowledge -hcq/azm are both known to prolong the qt interval and are associated with a risk of torsade de pointes. addition of these drugs to other medications with known qt prolonging effects can further increase patient risk. competency in patient care and procedural skills -patients with covid- infection are susceptible to cardiac injury and significant mortality. the value of hcq/azn in treating this infection is unknown, but their effects on qt interval prolongation are significant. even though risk of serious arrhythmia in these patients is low, it may be increased with the use of these drugs. prospective randomized trials of hcq/azm treatment in patients with covid- infections are needed to assess possible benefit and compare those benefits to the small but important risk of excess qt interval prolongation and drug-induced torsade de pointes. tacrolimus ondansetron tamoxifen propofol tizanidine sotalol tolterodine tramadol venlafaxine early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia covid- in critically ill patients in the seattle region -case series race to find covid- treatments accelerates remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro evaluation of ebola virus inhibitors for drug repurposing association of treatment with hydroxychloroquine or azithromycin with in-hospital mortality in patients with covid- in new york state guidance for cardiac electrophysiology during the coronavirus (covid- ) pandemic from the heart rhythm society covid- task force; electrophysiology section of the american college of cardiology; and the electrocardiography and arrhythmias committee of the council on clinical cardiology azithromycin and the risk of cardiovascular death urgent guidance for navigating and circumventing the qtc prolonging and torsadogenic potential of possible pharmacotherapies for coronavirus disease (covid- ) intra-and interreader variability in qt interval measurement by tangent and threshold methods in a central electrocardiogram laboratory a novel method for correcting qt interval for qrs duration, predicts all-cause mortality update of the eular recommendations for the management of systemic lupus erythematous hydroxychloroquine: an old drug with new relevance diagnosis and treatment of adults with communityacquired pneumonia: an official clinical practice guideline of the drug-induced inhibition and trafficking disruption of ion channels: pathogenesis of qt abnormalities and drug-induced fatal arrhythmias hydroxychloroquine reduces heart rate by modulating the hyperpolarization-activated current if: novel electrophysiologic insights and therapeutic potential electrophysiologic studies on the risks and potential mechanism underlying the proarrhythmic nature of azithromycin chronic hydroxychloroquine use associated with qt prolongation and refractory ventricular arrythmia suspected hydroxychloroquine-associated qt-interval prolongation in a patient with systemic lupus erythematous risk evaluation of azithromycin-induced qt prolongation in real-world practice the qt interval in patients with covid- treated with hydroxychloroquine and azithromycin the effect of chloroquine, hydroxychloroquine and azithromycin on the corrected qt interval in patients with sars-cov- infection assessment of qt intervals in a case series of patients with coronavirus disease (covid- ) infection treated with hydroxychloroquine alone or in combination with azithromycin in an intensive care unit risk of qt interval prolongation associated with use of hydroxychloroquine with or without concomitant azithromycin among hospitalized patients testing positive for coronavirus disease (covid- ) prevention of torsade de pointes in hospital settings: a scientific statement from the american heart association and the american college of cardiology foundation inhospital cardiac arrest is associated with use of non-antiarrhythmic qtc prolonging drugs fda cautions against use of hydroxychloroquine or chloroquine for covid- outside of the hospital setting or a clinical trial due to risk of heart rhythm problems. u.s. food and drug administration consideration for drug interactions on qtc in exploratory covid- (coronavirus disease ) treatment. circulation development and validation of a risk score to predict qt interval prolongation in hospitalized patients prolongation of the qtc interval is seen uniformly during early transmural ischemia who are the long-qt syndrome patients who receive an implantable cardioverter-defibrillator and what happens to them?: data from the european long-qt syndrome implantable cardioverter-defibrillator (lqts icd) registry the beaumont health covid- database was established to calculate all demographics, clinical characteristics, comorbid conditions, medications, and outcomes of patients tested for covid- . the data from the registry was abstracted through automated reports generated through a multi-platform database query tool from beaumont health's electronic medical record. demographics abstracted from the registry for the purpose of this study were age, sex, race, and body mass index. comorbid conditions abstracted were hypertension, diabetes mellitus, coronary artery disease, chronic obstructive pulmonary disease, chronic kidney disease, tobacco use, cancer, history of atrial fibrillation prior to hospitalization, and congestive heart failure. congestive heart failure was defined as a previous admission for congestive heart failure or a diagnosis of congestive heart failure in the electronic medical record. laboratory values abstracted were the first creatinine, aspartate aminotransferase, alanine transaminase, and lactic acid. the inpatient medication administration record for each patient was abstracted from the registry and compared with a list of medications available in the united states which are known or suspected to increase the risk of torsade de pointes. all ecg intervals, telemetry strip intervals, death dates, discharge dates, maximum troponin laboratory values, potassium and magnesium laboratory values immediately prior to azithromycin and hydroxychloroquine initiation, azithromycin and hydroxychloroquine initiation and completion dates, and occurrences of sustained ventricular tachycardia, non-sustained ventricular tachycardia, and ventricular fibrillation were abstracted from the electronic medical record by clinicians trained to abstract the needed data into sharepoint. all occurrences of ventricular tachycardia and ventricular fibrillation were reviewed by a board certified cardiac electrophysiologist. a random % sample of qtc intervals were re-abstracted to ascertain agreement and monitor calibration. key: cord- -gyuh fvl authors: siddiqui, arif jamal; jahan, sadaf; ashraf, syed amir; alreshidi, mousa; ashraf, mohammad saquib; patel, mitesh; snoussi, mejdi; singh, ritu; adnan, mohd title: current status and strategic possibilities on potential use of combinational drug therapy against covid- caused by sars-cov- date: - - journal: journal of biomolecular structure & dynamics doi: . / . . sha: doc_id: cord_uid: gyuh fvl the spread of new coronavirus infection starting december as novel sars-cov- , identified as the causing agent of covid- , has affected all over the world and been declared as pandemic. approximately, more than , , confirmed cases of covid- infection and , deaths have been reported globally till the end of june . until now, there is no specific drug therapy or vaccine available for the treatment of covid- . however, some potential antimalarial drugs like hydroxychloroquine and azithromycin, antifilarial drug ivermectin and antiviral drugs have been tested by many research groups worldwide for their possible effect against the covid- . hydroxychloroquine and ivermectin have been identified to act by creating the acidic condition in cells and inhibiting the importin (impα/β ) mediated viral import. there is a possibility that some other antimalarial drugs/antibiotics in combination with immunomodulators may help in combatting this pandemic disease. therefore, this review focuses on the current use of various drugs as single agents (hydroxychloroquine, ivermectin, azithromycin, favipiravir, remdesivir, umifenovir, teicoplanin, nitazoxanide, doxycycline, and dexamethasone) or in combinations with immunomodulators additionally. furthermore, possible mode of action, efficacy and current stage of clinical trials of various drug combinations against covid- disease has also been discussed in detail. communicated by ramaswamy h. sarma human coronaviruses are a group of spherical or pleomorphics medium size ( - nm), enveloped rna viruses containing petal or club shaped peplomers on the surface. they belong to the family coronaviridae. these viruses infect mammals and birds causing diseases of the respiratory tract, gastrointestinal tract, liver, kidney and nervous system. human coronavirus is responsible for human respiratory disease and a causative agent of common cold. the us national institute of allergy and infectious diseases (niaid) research efforts build on earlier research on severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers), which also are caused by coronaviruses (acter et al., ) . mers was firstly erupted in saudi arabia in september . according to who, this disease has since spread to countries (chafekar & fielding ) . patients infected with mers coronavirus (mers-cov) develop severe acute respiratory illness, consisted with shortness of breath, cough and fever. infection with sars coronavirus (sars-cov) can also cause a severe viral respiratory illness. it was first reported in china in february though cases subsequently were noticed from november (rabaan et al., ) . by the time it was being contained, sars spreaded to countries in the span of four months. the evidence of research suggested that mers-cov and sars-cov are originated from bats and in the same manner as covid- was spread as well (rabaan et al., ) . furthermore, sars-cov spreads from infected civets to the human, while mers-cov spreads from infected dromedary camels to human. scientists are still trying to find out how sars-cov- spread from an animal reservoir to human (mahanta et al., ; rabaan et al., ) . currently, the world is suffering from a pandemic disease covid- caused by a novel strain of coronavirus, called as sars-cov- (aanouz et al., ) . availability of number of covid- diagnostic test/kits or poor health services and sectors in lower income countries (mahanta et al., ) . currently till date ( june ), countries with the most number of recorded infected cases are united states ( , , cases, , deaths and , recovered), followed by brazil ( , , cases, , deaths and , recovered) , russia ( , cases, deaths and , recovered) , india ( , cases, , deaths and , recovered), united kingdom ( , cases, , deaths) , spain ( , cases, , deaths) , peru ( , cases, deaths and , recovered), italy ( , cases, , deaths and , recovered) , chile ( , cases, deaths and , recovered) , iran ( , cases, deaths and , recovered) and germany ( , cases, deaths and , recovered) (ecdc ; who ; worldometer ) (figure ). according to this data, it can be concluded that infection rate due to covid- is spreading exponentially in new hotspots like united states and europe, when compared to first epicenter china, where the number of new cases is rapidly declining. therefore, at this stage, the urge and requirement of effective drug or vaccine to control the covid- disease is at its peak (islam et al., ; k et al., ) . discovery of new drug or vaccine development after all trials for human use will take approximately further time of - years or more. however, some countries have already started efforts in making vaccine or chemoprophylaxis against covid- disease, even initiation of human trials are on track (choudhary & sharma ; khan et al., ; muralidharan et al., ) . while some drugs have shown therapeutic effect against covid- infection such as hydroxychloroquine (al-kofahi et al., ; choudhary & sharma ; liu et al., ; sinha & balayla ) , azithromycin, (andreani et al., a; choudhary & sharma ) ivermectin (caly et al., ; chaccour et al., ; choudhary & sharma ) and some other antivirals (asai et al., ; boopathi et al., ; lian et al., ) . however, it is still not clear that whether these drugs have a better therapeutic effect, when compared to other drugs or combination therapy of multiple drugs with immunomodulators will prove to provide us with better results. consequently, this review will provide an insight and comprehensive view on different therapeutic approaches including combining of different known anti-parasitic drugs, as well as proposing novel suggestions of chemoprophylaxis drug therapy, which can be used in the current treatment and vaccine development strategies against covid- disease. all peer reviewed scientific papers were used for this review article. extensive literature searches have been performed using various literature search engines, including science direct, pubmed, web of science, google scholar, scopus and researchgate with several terms: ( ) mers-cov, sars-cov- and sars-cov- related articles; ( ) antimalarial drugs usages in covid- disease; ( ) antiviral drugs; and ( ) immunomodulators and vaccine related compounds. all relevant studies meeting search criteria were included in this review. lacking specific antiviral treatment, both sars-cov and mers-cov pose major clinical management challenges (lu et al., ) . many drugs and therapies are still under clinical trials and substantial efforts are underway to discover new therapeutic agents for coronavirus infections (lin et al., ; zumla et al., ) . the isaric (international severe acute respiratory & emerging infection consortium), collected drugs list in july which are easily available for the treatment of pandemic influenza, mers-cov, sars-cov- . sars-cov- came into picture first time in southern china and quickly spreaded around the globe in - (rabaan et al., . with high rate of nosocomial transmission having symptoms of high fever and unusual epidemic of a typical pneumonia to health care workers was happened in foshan, guangdong, china on november (sims et al., ) . the most promising and clinically available drugs were ribavirin and interferon (ifn), or a combination of the two. the combination of drugs have shown the efficacy in an in vivo model for mers-cov infection ( figure ) (falzarano et al., ) . although, the combination could not fulfill the recovery criteria in the small number of severely ill mers-cov patients (dyall et al., ) . in an in vitro study, mycophenolic acid (mpa) and ifn-b were also found to be highly effective against mers-cov infection . mpa was found to be effective and specific to mers-cov, and also with some activity were detected against sars-cov infection (chan et al., ; hart et al., ) . clinical trials have demonstrated the expression of ip- , ifn-c, il- and il- and these cytokines are denoted the severity of the disease (russell et al., ) . three fda-approved broad-spectrum inhibitors (chlorpromazine, chloroquine, toremifene) that were shown to be effective against mers-cov infection in immortalized cell lines and evaluated their antiviral activities (cong et al., ) . among all three drugs, toremifene is known as an estrogen receptor modulator which has been found to restrict filoviruses and thus inhibit both mers-cov and sars-cov (cong et al., ) . previously, chloroquine (cq), a well-established anti-parasitic agent, showed strong inhibition on mers-cov and sars-cov with low toxicity (cong et al., ) . cq likely accumulates in lysosomes, where it sequesters protons and increases the ph. the drug interacts with a variety of host proteins and cellular processes, resulting in modulation of immune response (cong et al., ) . cq has also been reported to inhibit replication of multiple viruses such as flaviviruses, influenza viruses, human immunodeficiency virus (hiv), ebola and nipah viruses in vitro . however, in other study cq had showed the least anti-mers activity with very low toxicity during the treatment of coronavirus (cong et al., ) . although, another drug chlorpromazine, belongs to neurotransmitter inhibitor, considered as first established antipsychotic drug and was used for schizophrenia treatment (de wilde et al., ). in addition, the mode of action of chlorpromazine is by inhibiting the clathrin-mediated endocytosis via blocking the formulation of clathrin-coated pits at the plasma membrane (de wilde et al., ). according to earlier work, which has stated that chlorpromazine have potential to inhibit mers-cov infection of huh cells with an ec of . , vs cc of . lm (cong et al., ) . however, all these studies determined the tested compounds, each of which were shown to be efficacious in continuous cell lines, and could be a promising therapeutic drugs to treat the coronaviruses related diseases. current status of various drug therapy in use for the treatment of covid- with their possible antiviral effects and mechanism of action there are many drugs which are currently in use for the treatment of covid- disease and most of the drugs are under clinical trials (table ) . the hcq is the derivative of cq, and is the first category drug which is working as a therapeutic agent against covid- infection (beura & prabhakar ; choudhary & sharma ; sinha & balayla ) . this drug has also been previously used for the treatment of rheumatoid arthritis and systemic lupus erythematosus (adeoye et al., ; sinha & balayla ) and is a first line drug for malaria treatment (azad et al., ; bhardwaj et al., ; roesch et al., ; siddiqui et al., ) . effect of hcq on viral replication goes beyond cytokines inhibition (asai et al., ; bhardwaj et al., ; siddiqui, bhardwaj, et al., ; sinha & balayla ) . it acts as weak base and tend to increase the ph within the intracellular vacuole (choudhary & sharma ; hasan et al., ; sinha & balayla ) . due to the weak base of this medication, it may affect acid balance and can inhibit many enzymes. this specific feature of hcq possibly inhibits the viral entry to the cell (asai et al., ; sinha & balayla ) . it is also known to obstruct the viral post-translational modifications and glycosyl-transferases (choudhary & sharma ) . furthermore, it has been known to modify the processes such as degradation of protein through acidic hydrolases in the lysosome ( figure ) (choudhary & sharma ) . the impact of antiretroviral has been considered due to inhibition of viral glycosylation; a significant antiviral mechanism of hcq (asai et al., ) . additionally, it also inhibits the protein replication of viruses by blocking/distracting the pathway of endosome/lysosome or viral protein maturation. some studies have revealed the possible mechanism of hcq and its activity against covid- sinha & balayla ) . most of the countries are currently using hcq for the treatment and management of covid- . however, many recent studies showed that the use of hcq in hospitalized covid- patients had no impact on the risk of the most severe outcomes from the disease (ferner & aronson a; geleris et al., ) . furthermore, latest published data showed that, treatment with hcq in hospitalized patients of covid- , reduced the risk of mechanical ventilation. although, mortality rate was higher in patients who got the treatment with hcq alone (magagnoli et al., ) . another study also showed similar data that high dose of hcq drug is not of any apparent benefit and also the mortality rate is still high (borba et al., ) . likewise, joshua geleris et al., also treated covid- patients with hcq ( mg twice on day , then mg daily for a median of days), and the authors said the results should not be rule out either benefit or harm from hcq use, however, the finding of this study do not support continued use of hcq drug in covid- patients (geleris et al., ) . furthermore, gautret et al., study showed that hcq treatment is significantly associated with viral load reduction/disappearance in covid- patients (gautret et al., a) . similarly, zhaowei chen et. al, study showed that the use of hcq drug in patients with covid- was just providing a significant recovery in the short period . data collected by who, regarding safety and efficacy of hcq as the treatment of covid- , the team of executive group of the solidarity trial decided to implement a temporary pause on hcq trials as a precautionary measure. however, hcq has been registered for clinical trial in more than different countries and approximately randomized clinical trials started giving answer about hcq's efficacy against covid- (bienvenu et al., ) . we need to wait to shed more light, based on clinical evidences for using hcq against covid- patients. azithromycin is the second choice of antimalarial drug to use against covid- and it belongs to the class of antibiotics (andreani et al., a; bakheit et al., ; soni et al., ) . many studies have recently revealed the therapeutic effect of azithromycin against the covid- infection (andreani et al., a; asai et al., ; choudhary & sharma ) . however, the exact mechanism is still unknown, but multiple mechanisms have been proposed for the putative antiviral properties determined with azithromycin drug. some researches states that azithromycin acts as acidotropic lipophilic weak base, which changes (increase) the ph level of endosome maturation and trans-golgi network (asai et al., ; choudhary & sharma ) . moreover, it can potentially inhibit endocytosis and viral genetic shedding from lysosomes, ultimately restricting viral replication (gravesen & judy ) . similarly, influenza and hiv also needs an acidic environment for the uncoating of the envelop. however, coronavirus also belongs to enveloped virus and it might have similar mechanism (choudhary & sharma ; thanh le et al., ) . likewise, these kinds of mechanisms have also been found inhcq. furthermore, azithromycin directly acts on bronchial epithelial cells by decreasing mucus secretion to enable and enhances the lung function (choudhary & sharma ) . recently, quantum mechanical modeling proposed a possible role of azithromycin drug in interfering with viral entry through binding collaboration between the covid- spike protein and host receptor ace protein (angiotensin converting enzyme ) ( figure ) (basit et al., ; choudhary & sharma ; lobo-galo et al., ) . however, clinical trials are still necessary to confirm the role of the drug and its work as prophylaxis in reducing the infection rate. azithromycin is currently table . list of drugs used in the treatment of mers-cov, sars-cov- and sars-cov- with their chemical structure, biological activity and possible mechanism. chemical structure biological activity/therapeutic effect mode of action references hydroxychloroquine used in the treatment of malarial and inflammatory activities. it is also now often used as an antirheumatologic agent in systemic lupus erythematosis and rheumatoid arthritis. currently, this drug is used against sars-cov- . inhibits terminal glycosylation of ace , the receptor that sars-cov- target for cell entry. ace that is not in the glycosylated state may less efficiently interact with the sars-cov- spike protein, further inhibiting viral entry. (gautret et al. a; liu et al. ) azithromycin it is used orally in children for the treatment of acute otitis media, malaria and also against sars-cov- interferes with viral entry through binding collaboration between the covid- spike protein and host receptor ace and block the viral entry. (andreani et al. b; asai et al. ) ivermectin it is a macrocyclic lactone derived from streptomyces avermitilis with antiparasitic activity such as nematodes, scabies and onchocerciasis (river blindness). now this drug is also used against sars-cov- . acts and prevents the import (integrase protein and importin (imp) a/b heterodimer) through the rise in antiviral response, inhibits the nuclear import of viral and host protein under clinical trial and has reached phase iv with promising results (nct ). ivermectin is generally used for the treatment and control of filarial diseases (murthy ). recently, its usage against covid- infection showed some positive results and patients response. in vitro study on ivermectin have also been found very effective against positive-sense single-stranded rna viruses, such as dengue, zika and yellow fever, via inhibiting the viral replication (azeem et al., ; choudhary & sharma ; gotz et al., ; mastrangelo et al., ) . similarly, recent report suggested that ivermectin have a potential to inhibit covid- replication in vitro (caly et al., ) . moreover, the treatment of it is a synthetic, broadspectrum tetracycline antibiotic exhibiting antimicrobial activity. currently, it is also in use for treatment against covid- disease. chelate zinc from mmps and on the basis of chelating activity of this antibiotic, it might help in inhibiting sars-cov- . (conforti et al. ; malek et al. ; szolnoky ) ribavirin it is a nucleoside analogue and antiviral agent used in therapy of chronic hepatitis c, other flavivirus and coronavirus infections. ribavirin is incorporated into viral rna, thereby inhibiting viral rna synthesis, and inhibiting normal viral replication. (dyall et al. ; falzarano et al. ) it is an antineoplastic antibiotic derived from various penicillium fungal species. it is an active metabolite of the prodrug mycophenolate mofetil and has antibacterial, antifungal, and antiviral activities. the mechanism of action of this drug is still unknown for sars-cov- . however, in case of mers-cov, they synergistically inhibit the papain-like protease (plpr). (chan et al. ; hart et al. ) it is a phenothiazine that was once the most commonly prescribed antipsychotic agent, but it is now rarely used. coronaviruses has been using clathrin-mediated endocytosis pathway to enter human cells. this drug has potential to inhibit clathrin-mediated endocytosis pathway for entry of viruses. (cong et al. ; de wilde et al. ) it is an aminoquinoline used for the prevention and therapy of malaria. it is also effective in extraintestinal amebiasis and act as an antiinflammatory agent for therapy of rheumatoid arthritis and lupus erythematosus does not affect the level of ace expression on cell surfaces, but inhibits terminal glycosylation of ace , the receptor that sars-cov and sars-cov- target for cell entry (cong et al. ; dyall et al. ) it is a non-steroidal antiestrogen that is used in the treatment of estrogen receptor positive breast cancer. long term toremifene therapy has been associated with development of fatty liver, steatohepatitis, cirrhosis, and rare instances of clinically apparent acute liver injury inhibits the spike protein and nsp (methyltransferase non-structural protein) of sars-cov- . zhou et al. ) it is a synthetic corticosteroid with anti-inflammatory and immunomodulating properties. methylprednisolone binds to and activates specific nuclear receptors, resulting in altered gene expression and inhibition of proinflammatory cytokine production. zhu et al. ) covid- by the single dose of ivermectin was found to reduce the viral load up to fold in vitro culture within h (caly et al., ) . however, the best part of this drug is that no toxicity was observed during in vitro culture. mechanism of action of this drug against covid- is still unknown, though researchers believed that this drug is working in a similar way it acts on other viruses. single stranded rna viruses are mostly dependent on integrase protein and importin (imp) a/b heterodimer during infection, and this drug acts and prevents the import through the rise in antiviral response that ivermectin inhibits the nuclear import of viral and host protein (caly et al., ; choudhary & sharma ) (figure ). ivermectin has passed the phase i of clinical trial and now mcg/kg dose of ivermectin is under phase ii clinical trial (nct ). favipiravir is generally used for the treatment and control of influenza virus (delang et al., ; elfiky ; furuta et al., ) . this medicine was first licensed by japan and largely used as an anti-influenza drug. it's efficacy in inhibiting other viruses such as ebola, lassa and rabies have been seen effectively (asai et al., ) . this drug is a derivative of pyrazinecarboxamide and it is being developed and manufactured by fujifilm group. it targets rdrp enzymes, that is important for the transcription and replication of viral genomes (coomes & haghbayan ) . furthermore, favipiravir also has a potential to use for the treatment of avian influenza and other influenza strains that are resistant to neuramidase inhibitors (coomes & haghbayan ) . the mechanism of action of favipiravir is described previously; it acts on viral rna synthesis as a chain terminator at the specific site, where the rna is incorporated into the host cell ( figure ). furthermore, this drug is not effective against dna based viruses. conversely, this specific property of favipiravir proves to have a positive outcome in the treatment of covid- disease (asai et al., ) . currently, favipiravir is under clinical trial and it has passed phase i and ii with promising results (nct ). significantly, some study showed that the treatment of covid- patients with favipiravir showed reduction in the load of sars-cov- when compared with control groups (cai et al., ; coomes & haghbayan ; lu et al., ) . the pharmaceutical company glenmark, india launched antiviral drug favipiravir under the brand name fabiflu on th june for the treatment of mild to moderate covid- patients. however, fabiflu is the first oral favipiravir drug approved medication in india for the treatment of covid- infected patients. dose of favipiravir is administrated orally, mg twice daily on day than mg twice daily up to days. Ã sars-cov- enters the human cell and initiate its replication cycle. first stage starts with the binding of sars-cov- virus with ace receptor, followed by transfer of viral rna in to the human cell. rna-dependent rna polymerase (rdrp) enzyme initiates the production of viral rnas. during rna methylation, rna cap is formed, which protects against the innate immune responses. furthermore, during the process of viral rna synthesis, translation of proteins is associated with ph-dependent membrane stress, which possibly elicits adverse effects against immune cells and cytokines. during this stage, if the viral replication cycle is not inhibited or infected cells are not eradicated; packed/assembled viruses will get disseminated and transfect other healthy host cells. triphosphate (atp) analog. furthermore, after administration it gets metabolized into its active form gs- , . its activity against many other viruses such as lassa fever, nipah, hendra, respiratory syncytial, junin, mers and sars coronaviruses has also be seen (asai et al., ; hendaus ) . however, the mechanism of action of this drug is shown by the inhibition of rdrp enzymes, conceivably through the interruption of rna chain termination in the host cells (figure ) (augustin et al., ) . consequently, it has been suggested that, remdesivir may be one of most useful and effective drug for the treatment of covid- disease (asai et al., ; ferner & aronson b) . in vitro testing with remdesivir has showed good results with significant reduction in sars-cov- load (grein et al., ) . moreover, remdesivir has already reached phase iii clinical trials against sars-cov- (nct ) after encouraging pre-clinical results against sars-cov (agostini et al., ; sheahan et al., ) and mers-cov (de wit et al., ) . umifenovir is commonly used for the treatment of influenza a and b viruses and hepatitis c virus (pshenichnaya et al., ) . umifenovir is a derivative of indole carboxylic acid. this drug was firstly developed by russia in (pshenichnaya et al., ) . however, after in vitro demonstration, this drug showed some potential to treat ebola virus, human herpes virus, tacaribe arenavirus (pshenichnaya et al., ) . the mechanism of action of umifenovir is known, it obstructs viral cell membrane fusion as well as virus endosome fusion with the host cell membrane. it also interferes with hydrogen bonding network of phospholipid (costanzo et al., ) . however, in other diseases caused by influenza virus, umifenovir directly interacts with virus particles to stabilize hemagglutinin. blaising et.al first showed the in vitro activity of umifenovir with good results against sars-cov- and sars-cov- . currently, umifenovir is undergoing clinical trials as a single agent (nct , nct ) and also in randomized human clinical trial for comparison with favipiravir (chictr ). teicoplanin is a glycopeptide antibiotic and basically used in the prophylaxis and treatment of bacterial infections caused by gram positive bacteria such as staphylococcus aureus and enterococcus faecalis (shea & cunha ) . however, teicoplanin have also showed good efficacy against many viruses such as hiv, ebola virus, hepatitis c virus (hcv), flavivirus, influenza virus, as well as mers-cov and sars-cov infection (baron et al., ) . recently, in vitro study suggests that, teicoplanin has potential efficacy to inhibit the virus activity and reduces the load of sars-cov- (pandey et al., ; zhang, ma, et al., ) . furthermore, suggested that the concentration of teicoplanin antibiotic requires to inhibit the viral activity that is ic value of . um (zhang, ma, et al., ) . it acts on early stage of the viral life cycle by blocking or inhibiting the low-ph cleavage of the viral spike protein through cathepsin l in the late endosome. thus, this way it stops the releasing of genomic viral rna and preventing the viral replication cycle inside the host cell (pandey et al., ) . therefore, teicoplanin needs to be further investigated for its potential effect against sars-cov- by randomized clinical trials, and hopefully this antibiotic will come out with some promising results. nitazoxanide is used for the treatment of parasitic diseases (cryptosporidiosis and giardiasis) that cause diarrhea, and viral diseases (hiv, hcv, hepatitis b virus (hbv), rotavirus, influenza virus, mers-cov) (pepperrell et al., ; simsek yavuz & unal ) . previous in vitro study suggested that, tizoxanide; the active circulating metabolite of nitazoxanide, inhibits the replication of the viruses (calderon et al., ) . the amount of drug required to inhibit viral replication by % (ic s) are between . and . mg/ml in human and canine cell lines (pepperrell et al., ) . furthermore, nitazoxanide has a potential capacity to enhance the production of interferon-a and interferon-b, which has been previously shown in vitro to exhibit activity against mers-cov, rotavirus, hbv, hcv, influenza virus and sars-cov- (calderon et al., ) . this drug has been shown to selectively inhibit the maturation of the hemagglutinin glycoprotein at the posttranslation stage (calderon et al., ) . nitazoxanide is currently under clinical trial to confirm its effectiveness with a dose of mg alone or in combination with other drugs against covid- (nct ). nitazoxanide is known for its safety in humans. research showed the tolerability of single doses up to g with minimal gastrointestinal side effects (rajoli et al., ) . doxycycline belongs to tetracycline antibiotic, that work against and inhibit various bacterial infections such as urinary tract infection, intestinal infection, gonorrhea, chlamydia and others (ali et al., ) . currently, doxycycline is also under use for the treatment against covid- , because coronavirus is well-known to bind with metalloproteases (mmps) of the host cells, in particular to ensure viral survival (conforti et al., ) . furthermore, doxycycline is known to chelate zinc from mmps, and on the basis of chelating activity of this antibiotic might help in inhibiting sars-cov- (conforti et al., ; szolnoky ) . on the other hand, it is also known that these class of antibiotics have the ability to inhibit the replication of positive polarity single stranded rna viruses (szolnoky ) . it is also used for the treatment of inflammatory skin diseases for long time, due to modulatory activity of innate immune response generation by this antibiotic (malek et al., ) . due to modulating effect, it can decrease the expression of nf-jb and releases the inflammatory cytokines such as tumor necrosis factor-a, interleukin- b and interleukin- , which can inhibit granulomas inflammatory response and release free radicals (malek et al., ) . therefore, due to these properties, possibility to inhibit the viral replication of sars-cov- inside the human cells is high (malek et al., ) . currently, doxycycline has already reached phase iii clinical trials with mg of daily dose given to patients that are infected with sars-cov- (nct ). the drug dexamethasone is used since long time for the treatment of arthritis and asthma. currently, it is used in treatment of covid- infected patients in united kingdom and under clinical trials. according to one research team, they got effectual and promising results (al saleh et al., ) . however, it is known that patients with advance stage of covid- infection have severe lung inflammation, but clinicians are checking for the dexamethasone effectiveness results in last stage of infection (theoharides & conti ) . covid- infected patients were randomly selected for clinical trial and all patients were administrated with mg dexamethasone once a day for days (theoharides & conti ) . in addition, it has reduced the death rate by one third in ventilated patients, according to press release from the recovery trial organizers (nct ). more than countries have started the development strategies to make a vaccine against covid- . currently, over different vaccines are being developed by these countries using various approaches and novel technologies (mandal ; thanh le et al., ) . however, some groups are already in a stage of human trials, while others are still testing on animals (thanh le et al., ) . presently, formulation of vaccines against covid- are divided into eight categories i.e. virus-weakened form, virus-inactivated form, replicating viral vector vaccine, non-replicating viral vector vaccine, nucleic acid based vaccine (dna and rna), protein based vaccine (protein sub-unit vaccine and virus like particles vaccine) (thanh le et al., ) . currently, there are many drugs which are in use and trials for curing this pandemic disease covid- . many studies have showed and determined that covid- infection suppresses the immune system yaqinuddin & kashir ) . therefore, we need to ponder over other options which might have therapeutic potential: ( ) use of combinational therapy with effective immunomodulators with known mechanism of action, which can enhance the functioning and response of immune system. some important immunomodulators with their pharmacological effects have been listed in table ; ( ) use of some other anti-malarial drugs such as artemisinin derivative and amodiaquine, because artemisinin has also shown antiviral activity. mode of action of artemisinin is still not clear, but there is a possibility that it interferes with the viral replication, because of its usage for the treatment of severe malaria, helminths parasites and cancer. however, amodiaquine's mode of action is similar to hcq, ( ) similarly, we must think of new drug approaches which can act on ace receptor, because covid- virus uses its surface of spike protein to block onto ace receptor on the surface of host receptor, ( ) another possible suggestion is to use two or three different combination of drugs i.e. ivermectin, hcq and azithromycin antimalarial drug in combination with favipiravir or remdesivir antiviral drug with immunomodulators or bcg vaccine for the treatment of covid- infection. however, researchers have already started using combination therapy for treating the covid- patients, but new approaches and combination of drugs are still needed, which can work in multiple way to cure the patients infected with sars-cov- . clinicians all over the world using combination drug therapy for the treatment of covid- , which includes varieties of drugs in combination with different drugs and immunomodulators or natural remedies. some drugs work well, while others not. below are some drug combinations which are currently in use to treat covid- infected patients. hcq þ az based treatment showed an apparent accelerated virus load clearance in the patients. this combination is already in use with phase iii clinical trials underway (andreani et al., b; gautret et al., b) . administration of hcq þ az in the covid- patients are given for days with loading dose of mg (hcq) þ mg (az) for first day and mg (hcq) þ mg (az) for the next four days (nct ). fruitful results have been observed. another combination for the treatment of covid- patients has reached phase ii clinical trials. in this combination (hcq þ nz), both drugs have different mode of action and both works in two different sites of virus. however, the administration of hcq þ nz drugs for covid- patients are given for days, three times daily with loading dose of mg (hcq) and mg (nz) given orally twice daily for days (nct ). still, we need to wait further for the evaluation and efficacy of clinical results of this combination. this combination of drugs (nz þ iv) have also been in use for the treatment of covid- infected patients (pepperrell et al., ; simsek yavuz & unal ) , and is in phase ii of clinical trial with participants. both drugs have the potential to inhibit sars-cov- infection. in addition, mode of action of both drugs is different, they inhibit viral protein synthesis and viral replication respectively. however, the administration of these two drugs are orally with mcg/kg ivermectin on empty stomach plus mg nitazoxanide twice daily with food for days (nct ). another important combination will soon be going for clinical trials. these two drugs (az and nz) have shown good efficacy against sars-cov- ( kelleni ) , while acting on different sites of virus life cycle as described in table . administered dose is mg of nz drug alone is currently in use for the treatment of covid- (kelleni ) . this combination needs to be considered and tested for positive and effective results. iv and dx showed good results in using alone administered treatment in covid- patients. due to the effective response, this combination is under phase ii clinical trial. here, the mode of action of these two drugs is different and doxycycline also increases the pro-inflammatory response after inhibiting nf-jb pathway (malek et al., ) . administration dose of these two drugs is mcg/kg ivermectin as single dose with mg doxycycline on day , followed by mg doxycycline at every hour for days (nct ). however, this is still in very initial phase of clinical trials, thus we need to wait more for the outcome of this combination clinically. the new way of combination drug therapy in use for the treatment of covid- such as antiviral and antimalarial drugs fv þ hcq and fv þ az combinations. the mode of action of fv is different when compared with hcq and az, which is a good sign where combination will prove to provide with good results (jean et al., ) . furthermore, these combinations has already reached the phase iii clinical trials. administration of these two drugs are mg hcq on day , followed by mg doses for - days with mg fv on day , followed by mg on - days. on the other hand, the current drug combination of fv and az dose used against covid- infected patients are mg az on day , followed by mg doses for - days with mg fv on day , followed by mg on - days (nct ). another approach to create new drug combination for the treatment of covid- infected patients is by using antiviral drug plus cytokine (il- ) blocking agent such as tz. tz is currently used to treat cytokines release syndrome (crs) and arthritis . during sars-cov- infection, the researcher observed that many cytokines increases and one of them is interleukin- . the mechanism of action of tz is still not clear. however, this drug has been started using in clinical trials with combination of fv and rd and have reached phase iii of clinical trials. administration of these two drugs are mg of fv twice on day , followed by mg doses twice for - days with mg tz on day (nct ). moreover, drug combination with tz þ rd has also reached the clinical trial level (nct ). recently, antiviral drugs such as uf, lp/rt, hcq (anti-malarial) and immunomodulator interferon-b a have been used as potential effective agents against covid- . furthermore, these combinations have already reached phase iv clinical trials for the treatment of covid- patients (nct ). however, there is another drug combination ia þ lp/rt þ ribavirin, which was also used to treat covid- patients and shown good results of viral load elimination from nasopharyngeal swabs in phase ii clinical trials (jalkanen et al., ) . till date, there is no specific drug or vaccine has been developed against covid- disease. it is important to develop a specific and novel inhibitor for blocking the viral entry and echinacea species extract uses in respiratory tract infections and inflammatory conditions, including common cold, coughs, bronchitis, and inflammation of mouth and pharynx its replication on the host cells, which will essentially control this pandemic disease. as we know that, there are many clinical trials related to new drugs, drug repositioning and vaccine development studies against novel coronavirus are on track. however, novel strategies like combinational therapeutic approaches with different drugs and immunomodulators will possibly show a better path in controlling covid- infection. furthermore, at this stage, computational approaches could help and lead us in developing or designing new therapeutic drugs at rapid level. on the other hand, successful vaccine development can only be achieved by using variety of therapeutic approaches and correctly shared information. this is significant and a must requirement to develop an important vaccine against covid- and eradicate this pandemic from the world. the authors have declared no conflict of interest. arif jamal siddiqui http://orcid.org/ - - - mitesh patel http://orcid.org/ - - - mohd adnan http://orcid.org/ - - - moroccan medicinal plants as inhibitors against sars-cov- main protease: computational investigations evolution of severe acute respiratory syndrome coronavirus (sars-cov- ) as coronavirus disease (covid- ) pandemic: a global health emergency. the science of the total environment repurposing of chloroquine and some clinically approved antiviral drugs as effective therapeutics to prevent cellular entry and replication of coronavirus 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pentapati, s. s. k.; batmanabane, g.; padhy, b. m.; bal, s.; singh, s. r.; mohanty, r. r. title: role of ivermectin in the prevention of covid- infection among healthcare workers in india: a matched case-control study date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: dqhdws k background: ivermectin is one among several potential drugs explored for its therapeutic and preventive role in covid- infection. the study was aimed to explore the association between ivermectin prophylaxis and development of covid- infection among healthcare workers. methods: a hospital-based matched case-control study was conducted among healthcare workers of aiims bhubaneswar, india, from september to october . profession, gender, age and date of diagnosis were matched for case-control pairs. cases and controls were healthcare workers who tested positive and negative, respectively, for covid- by rt-pcr. exposure was defined as the intake of ivermectin and/or hydroxychloroquine and/or vitamin-c and/or other prophylaxis for covid- . data collection and entry was done in epicollect , and analysis was performed using stata version . conditional logistic regression models were used to describe the associated factors for covid- infection. results: ivermectin prophylaxis was taken by controls and cases. two-dose ivermectin prophylaxis ( . , % ci, . - . ) was associated with % reduction of covid- infection among healthcare workers for the following one month, those who were involved in physical activity ( . % ci, . - . ) for more than an hour/day were more likely to contract covid- infection. type of household, covid duty, single-dose ivermectin prophylaxis, vitamin-c prophylaxis and hydroxychloroquine prophylaxis were not associated with covid- infection. conclusion: two-dose ivermectin prophylaxis at a dose of g/kg with a gap of hours was associated % reduction of covid- infection among healthcare workers for the following one-month. further research is required before its large scale use. keywords: covid- , ivermectin, chemoprophylaxis, healthcare workers, india the sars-cov- pandemic has claimed over , , lives and affected over , , persons worldwide. meanwhile, the subcontinent of india has reported , , active covid- confirmed cases and , , deaths related to the same virus by th october . healthcare workers (hcws) worldwide, have been exposed to the infection as frontline workers in a battle to save patients affected by covid- infection, which has led to an increasing number of cases and deaths in this group. a systematic review on infection and deaths in hcws due to covid- found that the number of infected hcws workers ranged from , to , according to each country's data. another report in september stated that covid- had infected nearly , hcws while as many as , had succumbed to the disease in the region. organisations at international and national levels have shared advisories and guidelines with measures to ensure the safety of hcws while they serve amidst the covid- pandemic. provision of ppes while on duty, free-of-cost covid- testing, timely payments, support helplines, online discussions, training, and capacity building for infection prevention and control are some of the measures taken to prevent the infection and spread of covid- among hcws. [ ] [ ] [ ] for high-risk persons such as hcws, additional safety measures are necessary to prevent them from getting infected. the use of hydroxychloroquine (hcq) as chemoprophylaxis was used in india under the recommendation of a few experts with little evidence and lack of scientific data. , the who solidarity trial's executive group and principal investigators decided to stop the hydroxychloroquine arm based on evidence from the solidarity trial and uk's recovery trial which showed that hcq did not result in the reduction of mortality of hospitalised covid- patients compared with standard of care. . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint ivermectin has been used as a therapeutic treatment of mild to moderate covid- cases. it has also been found to prevent symptoms of covid- in post-exposure prophylaxis among hcws. when given to high-risk healthcare workers in contact with covid- patients in a study from egypt, it was found that compared to . % of the intervention arm, . % of participants from the control arm had symptoms suggesting of covid- infection. few other studies have also shown favourable results with the use of ivermectin as prophylaxis and treatment. , all india institute of medical sciences, bhubaneswar, is a tertiary care, government-funded teaching hospital situated in odisha which is in the eastern part of india. from august onwards, large numbers of hcws who were employees of the hospital were getting infected, which was affecting healthcare at the hospital. considering the fact that ivermectin had been shown to have diverse mechanisms by which it successfully attacks the sars-cov- and the fact that ivermectin has a proven safety profile as a safe drug which has been used for many decades and the encouraging results of the study from egypt prompted us to explore the role of ivermectin as prophylaxis for hcws for the prevention of covid- . the present study is a hospital-based matched case-control study, conducted among hcws of the all india institute of medical sciences (aiims) in bhubaneswar, odisha, india, during september-october . aiims, bhubaneswar is a tertiary care hospital located in eastern india. to calculate sample size, we assumed ivermectin in the control group to be % as there were no data available from prior studies. considering, % power, % alpha, : matching of cases to controls, minimum discordant pairs to be detected was set to , with an expected odds . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint ratio of . , the sample size was estimated to be pairs, i.e. individuals. cases and controls were identified from the existing line list, which was prepared by the contact tracing team at aiims bhubaneswar. this line list contains the list of the aiims bhubaneswar hcws' risk of exposure to covid- assessment based on world health organization(who) risk assessment guidelines. this risk assessment helped in identifying similar risk population of cases and controls. cases were hcws who were diagnosed as positive for covid- by reverse transcription polymerase chain reaction (rt-pcr). controls were defined as hcws who were diagnosed as negative for covid- by rt-pcr with a similar risk of exposure to covid- . for every enrolled case, a control was selected from the existing line list. individual matching was done for their profession, gender and age, and also an attempt was made to match for the date of diagnosis. however, when the match was not possible for the same date, we selected the control from the nearest possible date of diagnosis. in the majority of cases, it was within a week. the average number of days for a difference in date of diagnosis was . days between cases and controls. exposure was defined as the prophylaxis viz., ivermectin and or/ (hcq) and or/ vitamin c and or/ other interventions taken for the prevention of covid- . hcws of aiims bhubaneswar were advised for hcq prophylaxis as per icmr guidelines from th april in addition to the appropriate personal protective equipment (ppe) depending on the place they were posted. however, the uptake was not encouraging on account of known side-effect. further, on th september , a decision to provide all hcws with ivermectin for prophylactic use was announced, based on a consensus statement that was released. (panel ) after the selection of cases and controls, a phone call was made to each participant. the data related to covid duty, family type, history of prophylaxis intake, history of hospital admission . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint and physical activity were collected. data was entered in epicollect . data cleaning and analysis was done using stata version . the difference in characteristics of cases and controls were assessed using the chi-square test for categorical variables and t-test for continuous variables. mean and standard deviation was used for continuous variables and proportion was reported for categorical variables, proportions were reported. matched pair analysis was done using the mcnemar chi-square test. matched pair odds ratio was estimated for ivermectin, vitamin-c and hcq prophylaxis. the potential confounders which could not be matched were adjusted during analysis with conditional logistic regression models. in model , we included the variables -covid duty, family type and physical activity (a proxy for social contacts), which may be the risk factors for covid- . in model , we included the variablesivermectin, vitamin-c and hcq, which were practised as prophylaxis by hcws for prevention of covid- . the study protocol was approved by the institutional ethics committee of aiims, bhubaneswar via ref number: t/im-nf/cm&fm/ / . verbal informed consent was obtained telephonically before participation in the study. this consent procedure was approved by the ethics committee. there were a total of staffs of aiims, bhubaneswar who got tested for covid- during last one month ( th september - th october ). out of persons who tested, persons were tested positive, and persons were tested negative for covid- . after matching with the profession, gender, age and date of diagnosis, we have cases for which controls were available. out of cases, cases did not give consent for the participation. therefore, we finally included matched pair or participants in our study. participants is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint had a mean (sd) age of + . years, and the mean difference in date of diagnosis between cases and control was . days. in one matched case-control pair, one intern was matched with a final year undergraduate student which was the closest possible match for the case, the remaining case-control pairs' profession was perfectly matched. (table ) out of cases, ( . %) cases were admitted in a hospital while ( . %) cases opted for home isolation. majority of the participants ( . %) were below years of age. nearly two-thirds of participants ( . %) were male. more than half of the participants ( . %) had their duties in covid wards or covid- screening opd in last one month. most participants ( . %) were not doing any physical activity during the study period. among the various modes of physical activity jogging and yoga were opted by participants each ( . %), gymnasium by ( . %), and sports by ( . %) participants. among participants, ( . %) were nursing officers, ( . %) were supporting staffs, ( . %) were resident doctors, ( . %) were interns, ( . %) were students and ( . %) were faculty members. half of the participants ( . %) were staying in an extended family, one-third participants ( . %) were staying with friends, and others were either staying in a nuclear family ( . %) or living along ( . %). (table ) out of participants, participants ( from cases and from controls) have taken any is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint ( . %) participants had a history of two-dose ivermectin prophylaxis ( µg/kg at day and day ) however ( . %) participants took only one dose ( µg/kg), and ( . %) participants continued the same dose for three or more days. out of participants, who took vitamin-c prophylaxis, participants took at a dose of mg once daily, and participants took vitamin-c mg twice daily. majority of participants took vitamin-c for less than one month; however, participants were continuing vitamin-c prophylaxis for more than one month. hcq prophylaxis was practised mg once a week. out participants, who took hcq prophylaxis, ten participants took for three or more weeks, five participants took for two weeks, and four participants took for a week. in the matched pair analysis, ivermectin prophylaxis ( . , % ci, . - . ) was associated with the reduction of covid- infection however vitamin-c prophylaxis ( . , % ci, . - . ) and hcq prophylaxis ( . , % ci, . - . ) had no significant association with covid- infection. (table ) in multivariate conditional logistic regression model , those who did any physical activity for more than one hour ( . % ci, . - . ) compared to who did not do any physical activity had an increased odds of contracting covid- infection. in the multivariate conditional logistic regression model , ivermectin prophylaxis ( . , % ci, . - . ) was associated with a reduction of covid- infection after adjusting for covid duties, type of household, physical activity, vitamin-c prophylaxis and hcq prophylaxis. however, physical activity for more than one hour was an independent risk factor ( . % ci, . - . ) for covid- infection. (table ) our study has shown that two doses of ivermectin prophylaxis at a dose of μg/kg given hours apart was associated with a % reduction of covid- infection among hcws for the is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint following month. our results are similar to the randomised trial conducted by waheed shouman from the zagazig university of egypt in which out of the hcws in the intervention arm, only . % developed symptoms versus the . % of the hcws in the control arm after days of enrolment. the study also reported no mortality or serious adverse events due to ivermectin in the intervention arm. mostly, ivermectin prophylaxis will benefit the hcws who are vulnerable to the infection because of their profession. our study findings throw light in the same direction that ivermectin may play a vital role in the prevention strategy of covid- infection. our study also documented that single-dose ivermectin prophylaxis at a dose of µg/kg, hcq prophylaxis, and vitamin-c prophylaxis is not associated with preventing covid- infection. engaging in physical activity for more than one hour daily, which is taken for lack of physical distancing was an independent risk factor for covid- infection in our study. study participants practised outdoor physical activity like walking or jogging, many also worked out at the gymnasium, and few were involved in playing sports. the possible explanations may be that physical activity poses a greater risk of exposure to infection due to is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . however, at hour-duration, there was no further reduction observed in the viral rna levels. the study also determined the ic (half maximal inhibitory concentration) of ivermectin treatment to be . µm under the test conditions. our study also estimated that single-dose prophylaxis has no association with a reduction of covid- and two-dose of ivermectin ( µg/kg) was associated with a reduction of covid- . our study finding is supported by another study conducted by chang et al., which found ivermectin to be useful as prophylaxis among healthcare personnel. their study aimed to investigate specifically post-exposure prophylaxis in contacts who tested negative for sars-cov- with ivermectin dose of . mg/kg body weight on day one with an additional second dose of ivermectin on day or for men aged more than years. in contrast, our study investigated the healthcare workers who had tested for covid- with positive cases and negative controls. we also matched the cases and controls according to age, gender, designation, and date of testing. also, our sample size was larger than the former study. a randomised controlled study conducted by boulware et al. tested hydroxychloroquine as postexposure prophylaxis among high-risk contacts of confirmed covid- cases. similar to our study findings, their study reported no significant difference between the hcq arm and the placebo arm. there were also more side effects reported with hydroxychloroquine in their study. in literature, the proposed hypothesis is that vitamin c may have a role to prevent covid- infection due to its strong antioxidant and immunomodulatory effects. [ ] [ ] [ ] however, no is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint research or hardcore evidence supports these findings. in our study, we did not find any association between vitamin-c prophylaxis and prevention of covid- infection. the strengths of our study are the adequate sample size, completeness of the data collection and verification from subjects. all the hcws received ivermectin procured from a single manufacturer and belonged to the same batch for each strength. we adjusted for confounders by matching and multivariate analysis. though recall bias is inherent in case-control studies, our data regarding the drug intake within the last one month is less likely to be forgotten by hcws. due to its observational nature, our study's findings need further confirmation using longitudinal studies or interventional studies to strengthen the evidence before its large-scale use among hcws and the implementation of public health programs. we conclude that two-dose ivermectin prophylaxis at a dose of μg/kg body weight with a gap of hours was associated with a % reduction of covid- infection among hcws in the following one month. this is an intervention worth replicating at other centres until a vaccine is available. based on the long history of clinical use, favourable safety profile, and the reportedly promising effect of ivermectin as a prophylactic agent in covid- , the expert committee group proposes the following consensus statement: suggested prophylaxis for doctors/nurses/staff/students of aiims bhubaneswar with ivermectin* first dose: ivermectin μg/kg body weight on day (directly observed) and ( hours apart). for - kg: mg, - kg: mg, > kg: mg subsequent dose: once a month dose (as above/kg body weight) on every th day after the last dose. ivermectin should be taken on an empty stomach with water. *the above schedule will be followed till further guideline/new evidence is available. *pregnant women will not be given this drug. women of childbearing age will be warned not to conceive while on this drug, in case they decide to take the drug. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint world health organisation ministry of health and family welfare government of india home page infection and death in healthcare workers due to covid- : a systematic review covid- has infected some , health workers and killed , in the americas, paho director says -paho/who protective measures for covid- for healthcare providers and laboratory personnel infection control guidance for healthcare professionals about coronavirus (covid- ) coronavirus disease (covid- ) outbreak: rights, roles and responsibilities of health workers, including key considerations for occupational safety and health: interim guidance hydroxychloroquine as prophylaxis for coronavirus sars-cov- infection: review of the ongoing clinical trials revised advisory on the use of hydroxychloroquine (hcq) as prophylaxis for covid- infection (in suppression of previous advisory date world health organization. coronavirus disease (covid- ): hydroxychloroquine a phase iii trial to promote recovery from covid with combined doxycycline and ivermectin along standard care use of ivermectin as a prophylactic option in asymptomatic family close contact for patient with covid- covid- : ivermectin prophylaxis in adult contacts. first report on health personnel and post-exposure prophylaxis icon (ivermectin in covid nineteen) study: use of ivermectin is associated with lower mortality in hospitalized patients with covid world health organization. health workers exposure risk assessment and management in the context of covid- virus: interim guidance covid- and social distancing covid- preventive measures showing an unintended decline in infectious diseases in taiwan impact of self-imposed prevention measures and short-term government-imposed social distancing on mitigating and delaying a covid- epidemic: a modelling study effectiveness of preventive measures against covid- : a systematic review of in silico modeling studies in indian context impact of social distancing measures on coronavirus disease healthcare demand the fda-approved drug ivermectin inhibits the replication of sars-cov- in vitro a randomized trial of hydroxychloroquine as postexposure prophylaxis for covid- quercetin and vitamin c: an experimental, synergistic therapy for the prevention and treatment of sars-cov- related disease (covid- ) ascorbate as prophylaxis and therapy for covid- -update from shanghai and u.s. medical institutions vitamin c as prophylaxis and adjunctive medical treatment for covid- ? we are thankful to all the participants for their involvement in the study. key: cord- - okrjbfn authors: wang, ya‐ling; wang, shih‐han; yang, ai‐yu title: pharmacist's perspective on hcq treatment of covid‐ date: - - journal: kaohsiung j med sci doi: . /kjm . sha: doc_id: cord_uid: okrjbfn nan covid- have been published. since hcq is widely used for the treatment of systemic lupus erythematosus (sle) and rheumatoid arthritis (ra), we will use the pharmacodynamic/pharmacokinetic (pd/pk) data of hcq to discuss the role of hcq in covid- treatment. after oral absorption, hcq is widely distributed in various tissues in the body. according to the results of animal experiments, the tissues with the most hcq distribution were the lungs, with a concentration of up to times that of plasma, followed by the spleen, liver, and kidney. this finding is similar to another in-vitro study on although hcq is safer and more effective than chloroquine, special attention should be paid to the interaction and side effects when it is used with azithromycin. especially in terms of cardiovascular, both hcq and azithromycin are likely to cause arrhythmias (such as qt prolongation and torsades de points), and combined use will increase the incidence, which was also observed by molina. there's no guidance for the treatment of covid- has indicated that the dose of hcq can be calculated according to body weight and adjusted according to different races. further studies are needed in the future to provide more comprehensive data. all authors declare no conflict of interest. tissue distribution of chloroquine, hydroxychloroquine, and desethylchloroquine in the rat in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) hydroxychloroquine and azithromycin as a treatment of covid- : 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- infection observational study of hydroxychloroquine in hospitalized patients with covid- key: cord- - sors bg authors: clementi, nicola; criscuolo, elena; diotti, roberta antonia; ferrarese, roberto; castelli, matteo; dagna, lorenzo; burioni, roberto; clementi, massimo; mancini, nicasio title: combined prophylactic and therapeutic use maximizes hydroxychloroquine anti-sars-cov- effects in vitro date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: sors bg while the sars-cov- pandemic is heavily hitting the world, it is of extreme importance that significant in vitro observations guide the quick set up of clinical trials. in this study, we evidence that the anti-sars-cov activity of a clinically achievable hydroxychloroquine concentration is maximized only when administered before and after the infection of vero e and caco- cells. this suggests that only a combined prophylactic and therapeutic use of hydroxychloroquine may be effective in limiting viral replication in patients. on th march , who director-general characterized covid- as a pandemic. as of th june , , , total cases were confirmed, accounting for , deaths (dong et al., ) . to date, the clinical management of covid- subjects almost exclusively consists of supportive therapy and, in particular, of ventilatory support for the most severe cases. several drugs, some already in clinical trials, are currently being used in order to limit the inflammatory response or to hamper sars-cov- replication (tu et al., ) . regarding the latter, no sars-cov- -specific drugs are currently available and, as a consequence, there is the need for repurposing drugs used in other settings, such as chloroquine (cq) and hydroxychloroquine (hcq) (gao et al., ; gautret et al., ; zhou et al., ) . several studies already demonstrated the in vitro efficacy of cq and hcq, evidencing the latter's higher activity on sars-cov- (colson et al., ; wang et al., ) . importantly, a recent study also modeled the main pharmacokinetic features of hcq trying to infer its lung concentration after different dosing regimens (yao et al., ) . it is therefore important to use this data in order to set up possible scenarios related to the real clinical use of this drug. in this study, we tested hcq against a sars-cov- italian clinical isolate, by using different protocols of drug administration corresponding to its possible prophylactic, therapeutic, and prophylactic/therapeutic use in patients. a single hcq concentration easily reachable in the lung and characterized by high anti-sars-cov- activity was used for the three protocols wang et al., ; yao et al., ) . vero e (vero c , clone e -crl- ; atcc) cells were cultured in dulbecco's modified eagle medium (dmem) supplemented with non-essential amino acids (neaa, x), penicillin/streptomycin (p/s, u/ml), hepes buffer ( mm) and % (v/v) fetal bovine serum (fbs). caco- (human epithelial colorectal adenocarcinoma cells, atcc htb- ) cells were cultured in minimum essential medium (mem) supplemented with neaa ( x), p/s ( u/ml), hepes buffer ( mm), sodium pyruvate ( mm), and % (v/v) fbs. calu- (human lung cancer cell line, atcc htb- ) were cultured in mem supplemented with neaa ( x), p/s ( u/ml), hepes buffer ( mm), and % (v/v) fbs. a clinical isolate hcov- /italy/unisr / (gisaid accession id: epi_isl_ ) was isolated and propagated in vero e cells, and viral titer was determined by % tissue culture infective dose (tcid ) and plaque assay for confirming the obtained titer. all the infection experiments were performed in a biosafety level- (bls- ) laboratory of microbiology and virology at vita-salute san raffaele university, milan, italy. bafilomycin a (bfla ) and hydroxychloroquine (hcq) were obtained from merck. an aliquot ( . ml) of the transport medium of the nasopharyngeal swab (copan's kit utm r universal viral transport medium-copan) of a mildly symptomatic sars-cov- infected patient was mixed with an equal volume of dmem without fbs and supplemented with double concentration of p/s and amphotericin b. the mixture was added to % confluent vero e cells monolayer seeded into a cm tissue culture flask. after h adsorption at • c, ml of dmem supplemented with % fbs and amphotericin b were added. twenty-four hours post-infection (hpi) another ml of dmem supplemented with % fbs and amphotericin b were added. live images were acquired (olympus ckx inverted phase-contrast microscopy) daily for evidence of cytopathic effects (cpe), and aliquots were collected for viral rna extraction and in-house one-step real-time rt-pcr assay ( . / s - ( ) - ). five days post-infection (dpi) cells and supernatant were collected, aliquoted, and stored at − • c (p ). for secondary (p ) virus stock, vero e cells seeded into cm tissue culture flasks were infected with . ml of p stored aliquot, and infected cells and supernatant were collected hpi and stored at − • c. for tertiary (p ) virus stock, vero e cells seeded into cm tissue culture flasks were infected with . ml of p stored aliquot and prepared as above described. p virus stocks were titrated using both plaque reduction assay (pra, pfu/ml) and endpoint dilutions assay (eda, tcid /ml). for pra, confluent monolayers of vero e cells were infected with -fold-dilutions of virus stock. after h of adsorption at • c, the cell-free virus was removed. cells were then incubated for h in dmem containing % fbs and . % agarose. cells were fixed and stained, and viral plaques were counted. for eda, vero e cells ( × cells/ml) were seeded into wells plates and infected with base dilutions of virus stock. after h of adsorption at • c, the cell-free virus was removed, and complete medium was added to cells. after h, cells were observed to evaluate cpe. tcid /ml was calculated according to the reed-muench method. viral genome from supernatant infected cells was extracted using qiaamp viral rna mini kit following manufacturers' instructions. reverse transcription and subsequent amplification were performed using random hexamer primers. the amplicons were sequenced on the illumina miseq ngs platform (illumina, san diego, ca, usa). amplicon purification and quantification were performed by agencourt ampure xp (beckman coulter, villepinte, france) and qubit dsdna assay kit (thermofisher scientific, waltham, ma, usa), respectively. library preparation was performed by using the nextera xt dna library prep kit (illumina, san diego, ca, usa). the library generated was then diluted and sequenced with miseq reagent kit v ( -cycles) (illumina, san diego, ca, usa) on the miseq platform. the quality of raw sequences obtained from miseq run was first checked using fastqc (v . . ) (babraham bioinformatics). the reads were aligned on reference sequence (gisaid accession id: epi_isl_ ) using bwa-mem and rescued using samtools alignment/map (v . ) and bamtofastq. finally, the contigs were generated using spades (v . . ). vero e cells ( × cells/ml) were seeded into wells plates and treated with hcq at different concentrations ( : serial dilutions, spanning from to . µm). cells were pretreated with the drug for h prior to virus infection at • c, followed by virus adsorption ( tcid /ml, pfu/ml, sars-cov- ) for h in the presence of the molecule. then, the cells were washed with pbs and further cultured at • c with the molecule-containing medium for h. then, cytopathic effect (cpe) was assessed using a scoring system ( = uninfected; . to . = increasing number/area of plaques; = all cells infected) to evaluate treated and untreated cells. infection control (score ) was set as % infection inhibition, uninfected cells (score ) as % infection inhibition. the whole surface of the wells was considered for the analysis ( x magnification). cell supernatants were collected for real-time pcr (rt pcr) analysis. all conditions were tested in quadruplicate. vero e cells ( × cells/ml) were seeded into wells plates and treated with µm hcq. in detail, cells were pretreated with the drug for h prior to virus infection at • c, followed by virus adsorption at different concentrations ( . - moi, sars-cov- ) for h in the presence of the molecule. then, the cells were washed with pbs and further cultured at • c with the molecule-containing medium for h. then, the cytopathic effect (cpe) was assessed using the scoring system above described, and results were normalized on virus infection control. cell supernatants were collected for rt-pcr analysis. all conditions were tested in triplicate. cell viability assay was performed using the cell proliferation kit ii (xtt) (roche diagnostics, merck). briefly, the tetrazolium salt , -bis-( -methoxy- -nitro- -sulfophenyl)- h-tetrazolium- -carboxanilide (xtt) is cleaved by viable cells to form an orange formazan dye that can be quantified photometrically at nm. before the assay, vero e , caco- , and calu- cells ( × cells/ml) were cultured in -well plates for h. the culture medium was replaced by medium containing µm hcq following full-time experimental setting, and cells were incubated for h. xtt was added to each well and the plates were incubated for an additional h. the optical density was measured at nm (reference wavelength− nm) using a multiskan go plate reader (thermo scientific instruments). for quantifications, the background levels of media without cultured cells were subtracted. vero e cells ( × cells/ml) were seeded into wells plates and treated with hcq ( µm) at different stages of virus infection. for full-time treatment, cells were pre-treated with the drug for h prior to virus infection at • c, followed by virus adsorption for h in the presence of the molecule. then, cells were washed with pbs, and further cultured at • c with the molecule-containing medium until the end of the experiment. for pre-adsorption treatment, the agent was added to the cells for h at • c before virus infection and maintained during virus adsorption. then, the mixture was replaced with fresh medium without molecule till the end of the experiment. for post-adsorption assays, the drug-containing medium was added to cells only after virus adsorption and maintained until the end of the experiment. bfla ( nm) was tested as control of inhibition of viral infectivity at a phagolysosome level only in a pre-adsorption treatment, alone or in combination with pre-adsorption hcq treatment. uninfected cells were included in all experimental settings to exclude possible drug-toxicity cpe. for all the experimental groups, cells were infected with tcid /ml sars-cov- , and absorption was performed for h at or • c. the full-time experimental setting was figure | hcq dose-response assessment. hcq ec against sars-cov- was obtained by both cpe and rt-pcr analysis on results from full-time experimental setting on vero e cells. mean values and sd (for rt-pcr values) and sem (for cpe values) are reported for all experimental replicates. all conditions were tested in quadruplicate and tested in duplicate in rt-pcr. figure | hcq antiviral effect using different multiplicities of infection (moi). cpe analysis resulted in a statistical difference between treated and untreated cells (****p < . ) when using . moi or less, rt-pcr only from . moi to decrease (****p < . ). performed also using caco- and calu- cells. live images were acquired (olympus ckx inverted phase-contrast microscopy), cpe was assessed as described above and cell supernatants were collected for rt-pcr analysis at hpi. all conditions were tested in quadruplicate. viral rna extraction and real-time rt-pcr viral rna was purified from µl of cell culture supernatant using the qiaamp viral rna mini kit (qiagen), following the manufacturer's instructions. subsequently, the purified rna was used to perform the synthesis of the first-strand cdna, using the superscript tm first-strand synthesis system for rt-pcr (thermo fisher scientific), following the manufacturer's instruction. real-time pcr, using sybr r green dye-based pcr amplification and detection method, was performed in order to detect the cdna. we used the sybr tm green pcr master mix (thermo fisher scientific) the forward primer n f: tta caa aca ttg gcc gca aa, the reverse primer n r: gcg cga cat tcc gaa gaa, and the following pcr conditions: • c for min, cycles of • c for s, annealing at • c for s and elongation at • c for s, followed by a final elongation at • c for min (hirotsu et al., ) . rt-pcr was performed using the abi-prism ht fast real-time instruments (applied biosystems) by using optical-grade -well plates. samples were run in duplicate in a total volume of µl. cpe observed for different experimental settings using hcq and bfla , alone or in combination, were normalized to figure | hcq cpe reduction on veroe cells infected with sars-cov- . cpe was assessed using a scoring system ( = uninfected; . to . = increasing number/area of plaques; = all cells infected) to evaluate treated and untreated cells. infection control (score ) was set as % infection inhibition, uninfected cells (score ) as % infection inhibition. the whole surface of the wells was considered for the analysis ( x magnification). blue gradient indicates the reciprocal of cpe detected in treated cells compared to virus infection control (white color corresponds to % cpe). protection levels are indicated by color gradient: white corresponds to no protection, dark blue shows full protection. bfla treatments are reported as experimental control of virus fusion process inhibition. virus adsorption was performed at and • c. frontiers in microbiology | www.frontiersin.org corresponding virus infection control. rt-pcr results were analyzed calculating delta ( ) ct as the difference between ct values obtained for experimental settings and infection control. then, two-way anova and tukey's multiple comparisons (for caco- and calu- experiments and virus curve for testing hcq susceptibility) or sidak's multiple comparisons (for hcq dose-response curve, time of addiction experiments and xtt cell viability evaluation) test were performed for the evaluation of cpe scoring and ct differences (graphpad prism ). ec was calculated using non-linear regression with least squares regression as a fitting model (graphpad prism ). virus isolation was achieved after < h. at hpi the cytopathic effect (cpe) was already evident on vero e cells. ngs analysis was performed by illumina miseq obtaining the whole genome sequence of the cultured isolate hcov- /italy/unisr / (gisaid accession id: epi_isl_ ). compared to the first italian isolate sequenced (hcov- /italy/cdg / ), that diverged from the hcov- /wuhan/wiv / reference strain at position , , , , and , , two more nucleotide mismatches were identified, g a and c a. g a is located in the ′ utr, while c a is a missense mutation causing the i l variation in the orf a polyprotein, specifically in the region that is part of the nsp membrane domain. no obvious consequence of the two polymorphisms can be drawn based solely on the sequence. different concentrations of hcq were tested on vero e to determine the effective concentration of the drug against sars-cov- in vitro infection (figure ) . ec resulted from both cpe and rt-pcr analysis were . and . µm, respectively. as ec analysis resulted in a low hcq effective dose, it was tested using different viral loads to better define the optimal conditions to complete the preliminary setting for subsequent experiments (figure ) . cpe analysis resulted in a statistical difference between treated and untreated cells (p < . ) when using . moi or less, rt-pcr only from . to decrease (p < . ). xtt assay was performed to determine cell tolerability to hcq treatment, and µm of the molecule tested in full-time treatment resulted in no drug-related toxicity on all three cell lines tested in the study (figure ) . median values for all experimental replicates, tested each one in duplicate in rt-pcr, and % ic range reported with error bars (*p < . ; **p < . ; ****p < . ). all conditions were tested in quadruplicate. frontiers in microbiology | www.frontiersin.org the two molecules were tested using different experimental protocols, and virus adsorption was performed at both and • c. cpe was assessed at hpi (figures , ) . virus infection positive control showed marked effects on cell morphology at • c as well as • c adsorption conditions. hcq was effective in full-time treatment at both adsorption temperatures, and in post-adsorption treatment only when the virus was added to cells for h at • c. the molecules did not show the same degree of protection from cpe in pre-adsorption treatment at both adsorption temperatures, as well as in post-adsorption treatment at • c adsorption. bfla was tested as control of inhibition of viral infectivity at a phagolysosome level in a preadsorption treatment, showing full cpe protection at • c virus adsorption, while only partial protection was observed when the virus was added to cells at • c (figures , ) . no drugrelated cytotoxic effect was observed on uninfected cells, in all experimental settings. cell supernatants of different experimental settings were collected and analyzed by rt-pcr, and results confirmed cpe data analysis (figure ) . in detail, a significant statistical difference of ct was observed with hcq full-time treatment compared to infection control, both at • c (p < . ) and • c (p < . ) virus adsorption, while hcq post-adsorption treatment was effective (p < . ) only when the virus was added to cells at • c. interestingly, bfla addition to hcq preadsorption treatment resulted in a significant statistical difference of ct when compared to infection control at • c virus adsorption. when tested on different cell lines, hcq resulted extremely effective on caco- cells (p < . for the higher tested concentrations, p < . for the minor one), while a non-statistical difference was observed when tested on calu- cells (figure ) . in the lack of sars-cov- -specific drugs, it is extremely important to evaluate the clinical potential of drug-repurposing to face the current pandemic (yao et al., ) . hcq has gained the attention of the scientific and medical community based on previous in vitro data on similar viruses (sars and mers) and on preliminary reports discussing its possible clinical effectiveness in therapy or prophylaxis based on observations performed on vero e -based infection models (colson et al., ; gao et al., ; gautret et al., ) . in this atypical context, in which on-field medicine often anticipates experimental laboratory pre-clinic, there is an urgent need for prompt experiments addressing specific clinical questions. for example, it is not clear what is the best administration regimen to maximize possible hcq anti-viral effectiveness in covid- patients. in this regard, a recent study evaluated the direct anti-sars-cov- effect in vitro and, importantly, modeled its bioavailability at the lung level where it could maximally exert its antiviral activity. based on a dosing regimen of mg given twice daily for day, followed by mg twice daily for more days, it also suggested the more useful drug concentrations to be used in clinically-oriented phenotypic laboratory assays wang et al., ; yao et al., ) . on that basis, we evaluated the hcq antiviral activity when administered before (pre-adsorption), after (postadsorption), or before and after (full-time) virus adsorption to simulate, on vero e cells, its possible prophylactic, therapeutic and prophylactic/therapeutic clinical use. moreover, we first focused our attention on a single concentration ( µm) easily achieved, well-tolerated and endowed with a strong antiviral activity (yao et al., ) . moreover, to speculate on its possible mechanism of action, we also evaluated hcq activity performing virus adsorption at and • c. in fact, at • c the virus enters the cell in a more physiological context while, conversely, at • c virus it can dock to the cell receptor, but its internalization is much more limited. in the prophylactic setting (pre-adsorption), µm hcq did not interfere effectively with the viral replicative cycle neither at • c nor at • c, as evidenced in the cpe and the rt-pcr analysis. limited antiviral activity was also observed in the therapeutic setting (post-adsorption) but, interestingly, a higher hcq antiviral activity was observed at • c, suggesting its predominant sars-cov- interference at the endosomal level (vincent et al., ; hu et al., ) . overall, and most importantly, these results suggest a limited activity of hcq when administered only prophylactically or therapeutically. on the contrary, significant antiviral activity was observed in the prophylactic/therapeutic (full-time) experimental setting both at and • c, as evidenced both by cpe and rt-pcr analyses. this observation allows us to speculate on the need for a combined prophylactic and therapeutic clinical use of hcq to maximize its antiviral effects. however, a possible bias of in vitro studies aimed at evaluating the antiviral activity of putative drugs can be represented by the moi used for testing the inhibitory activity of drugs. that is why we used different moi for testing the inhibitory activity of hcq. our data suggest how using moi higher than . to perform in vitro testing can impact on the hcq inhibitory capability. we also determined, by cpe evaluation and rt-pcr analysis, the hcq ec ( . and . µm, respectively) at h post-infection. moreover, to better translate to the clinic what observed on vero e , hcq activity was assessed also on cells of human origin: the caco- (colorectal adenocarcinoma epithelial-like cells) and the calu- (lung adenocarcinoma epithelial cells). as observed for vero e cells, no hcq direct toxicity was observed for both caco- and calu- . interestingly, a dose-response effect of hcq was appreciated only on caco- cells. on the contrary, no significant reduction of viral rna amount was appreciated on calu- cells. this observation will deserve certainly further investigations focused on both mechanistic aspects of hcq-mediated inhibition of sars-cov- and on the possible interpretation of clinical trial outcomes. in the atypical scenario of an ongoing pandemic, preclinical medical research should be focused on simple and fast observations potentially useful for the prompt set up of clinical trials. however, all possible hcq side effects already known (chatre et al., ; zhou et al., ) should be considered and evaluated also under the light of concerns regarding two clinical trials recently described and retracted (mehra et al., a,b; science aaftao, ) . as an example, our observation could be translated in a clinical study on extremely high-risk categories, such as health care workers, based on the prophylactic administration of hcq followed by its therapeutic use in case of positivity to sars-cov- . whole genome sequence data of hcov- /italy/unisr / have been uploaded on gisaid database (https://www.gisaid. org/) with the following accession id: epi_isl_ . the study involving human participants was reviewed and approved by ospedale san raffaele irb in the covid- biobanking project. the patient from whose samples the virus was isolated provided written informed consent. this manuscript has been released as a pre-print at biorxiv preprint. doi: . / . . . , hu et al. ( ) . nc and nm conceived the study. ec, rd, and rf performed the experiments. nc, nm, ec, mca, and mcl analyzed the data. nc, nm, ec, and rd wrote the manuscript. nc, nm, ld, rb, and mcl revised the manuscript. all authors contributed to the article and approved the submitted version. cardiac complications attributed to chloroquine and hydroxychloroquine: a systematic review of the literature chloroquine and hydroxychloroquine as available weapons to fight covid- an interactive web-based dashboard to track covid- in real time breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial double-quencher probes improved the detection sensitivity of severe acute respiratory syndrome coronavirus (sars-cov- ) by one-step rt-pcr. medrxiv insights from nanomedicine into chloroquine efficacy against covid- hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro retraction: cardiovascular disease, drug therapy, and mortality in covid- retracted: hydroxychloroquine or chloroquine with or without a macrolide for treatment of covid- : a multinational registry analysis the pandemic's first major research scandal erupts a review of sars-cov- and the ongoing clinical trials chloroquine is a potent inhibitor of sars coronavirus infection and spread remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) covid- : a recommendation to examine the effect of hydroxychloroquine in preventing infection and progression the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © clementi, criscuolo, diotti, ferrarese, castelli, dagna, burioni, clementi and mancini. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -l l pco authors: roldan, eugenia quiros; biasiotto, giorgio; magro, paola; zanella, isabella title: the possible mechanisms of action of -aminoquinolines (chloroquine/hydroxychloroquine) against sars-cov- infection (covid- ): a role for iron homeostasis? date: - - journal: pharmacol res doi: . /j.phrs. . sha: doc_id: cord_uid: l l pco the anti-malarial drugs chloroquine (cq) and primarily the less toxic hydroxychloroquine (hcq) are currently used to treat autoimmune diseases for their immunomodulatory and anti-thrombotic properties. they have also been proposed for the treatment of several viral infections, due to their anti-viral effects in cell cultures and animal models, and, currently, for the treatment of coronavirus disease (covid- ), the pandemic severe acute respiratory syndrome caused by coronavirus (sars-cov- ) infection that is spreading all over the world. although in some recent studies a clinical improvement in covid- patients has been observed, the clinical efficacy of cq and hcq in covid- has yet to be proven with randomized controlled studies, many of which are currently ongoing, also considering pharmacokinetics, optimal dosing regimen, therapeutic level and duration of treatment and taking into account patients with different severity degrees of disease. here we review what is currently known on the mechanisms of action of cq and hcq as anti-viral, anti-inflammatory and anti-thrombotic drugs and discuss the up-to-date experimental evidence on the potential mechanisms of action of cq/hcq in sars-cov infection and the current clinical knowledge on their efficacy in the treatment of covid- patients. given the role of iron in several human viral infections, we also propose a different insight into a number of cq and hcq pharmacological effects, suggesting a potential involvement of iron homeostasis in sars-cov- infection and covid- clinical course. chloroquine (cq) and its hydroxy-analogue hydroxychloroquine (hcq), both -aminoquinolines, have been extensively used in the treatment of malaria in the last century, but the continuous emergence of drugresistant strains of plasmodium falciparum has accelerated the development of new antimalarial drugs. nevertheless, these compounds, particularly the less toxic hcq, are currently widely used to treat autoimmune diseases like rheumatoid arthritis (ra), systemic lupus erythematosus (sle) and antiphospholipid syndrome (aps), due to their immunomodulatory and anti-thrombotic properties. cq and hcq have also been proposed for the treatment of viral infections, since they have been demonstrated to directly inhibit viral entry and spread in several in vitro and in vivo models. treatment with these drugs has then been proposed for several viruses in humans, including coronaviruses like sars-cov and sars-cov- . nonetheless, a clear evidence of their utility in human viral infectious diseases still lacks [ ] . translation from laboratory to clinic should be based on detailed analysis of results from randomized controlled studies and observational outcome registries focused on the efficacy, duration and toxicities of treatments with these drugs that could be useful to understand their real effectiveness. here we review the current knowledge on the mechanisms of action of cq and hcq as anti-viral, antiinflammatory and anti-thrombotic drugs and discuss the current experimental evidence on the potential mechanisms of action of cq/hcq on sars-cov . we also propose a different insight into some of cq and hcq effects, suggesting a potential role of iron homeostasis in sars-cov- disease (covid- ), similarly to several other human viral infections [ ] [ ] [ ] . finally, we briefly review and discuss the current knowledge on their efficacy in the treatment of patients with covid- . we conducted a literature search using different database (pubmed, science direct and web of science) up to april th . the search strategy was to use different search terms alone and in any combination, such as "sars-cov- disease", "covid- ", "sars-cov- ", "coronavirus", "clinical trial", "treatment", "drug", "chloroquine", "hydroxychloroquine", "iron", "virus", "viral entry", "viral spread", "anti-viral activity", "infection", "inflammation", "immunity", "innate immunity", "cytokine", "il- ", "tnf-", "il- ", "adaptive immunity", "thrombosis", "in vitro". only english articles with available data were included. then, cq/hcq could have inhibitory effects on virus attachment and entry in the host cell, possibly resulting in blocking the viruses in endocytic vesicles. cq/hcq have also been shown to display anti-viral activity even when administered after viral infection. this effect has been observed in vitro also in sars-cov and sars-cov infections [ , , ] . further mechanisms could then be involved in antiviral drug action. through the alkalization of endosomes, cq/hcq might also act inhibiting or preventing endosomelysosome membrane fusion that leads to membrane viral receptor recycling, viral uncoating and viral genome release into the cytosol, as observed for sars-cov [ ] . [ ] . cleavage sites other than those recognized by cathepsin and tmprss have been identified in the s protein of sars-cov- that may be cleaved by furin-like proteases. furin is localized in the tgn, is highly expressed in the lung, its cleavage of s protein could be implicated in virus egress and spread [ ] and might be inhibited by cq, as observed in chikungunya virus infection [ ] . due to their basic properties and consequent disruption of cellular vesicle compartments, cq/hcq may also inhibit virion budding, occurring when encapsidated viral genomes bud into the ergic membranes in which viral envelope proteins are inserted, forming mature virions [ ] . within infected airway epithelium, through their binding to toll-like receptors (tlrs), several respiratory viruses (among them some coronaviruses) activate the mitogen-activated protein kinase (mapk) pathways, particularly the p mapk. respiratory viruses harness mapk activation to their own advantage, exploiting the triggering of their downstream targets for trafficking their own proteins and for viral assembly and spread. these effects are particularly important in people suffering for underlying airway diseases, like asthma or chronic obstructive pulmonary disease (copd), who undergo a respiratory viral infection, since these pathways are already turned on and aberrantly activated [ ] . cq has been shown to hinder viral infections through the inhibition of p mapk activation [ , ] . this inhibitory action could then be of particular importance in covid- since people with copd are among the worst affected by sars-cov- infection. j o u r n a l p r e -p r o o f therefore, besides viral attachment and entry, also viral uncoating, genome release, protein maturation process and assembly of new virions for budding and spread may be inhibited by the basic drugs, resulting in reduced infectivity. little is currently known about the mechanism(s) of action of cq/hcq in the treatment of sars-cov- infection. the first published experimental evidence of the potential efficacy of cq/hcq in this disease comes from in vitro experiments. wang and colleagues [ ] results suggested that cq could be more efficacious than hcq. the authors also confirmed that cq acts at both entry and post-entry stages and this double action was also observed for hcq. in order to shed light on potential mechanisms of action of the drugs, the authors studied the cellular localization of viral particles and found virions partly in early endosomes (ees) and more in late endosome-lysosomes (lels) in control conditions. when cells were treated with cq and hcq, more viral particles were observed in ees, suggesting that the drugs block endocytotic vesicle maturation at intermediate stages and probably stall the virus transport from ees to lels, a crucial step for the release of viral genome. the authors also observed that both cq and hcq treatments resulted in abnormally enlarged ees, but while hcq increased lel size and number, cq treatment induced no changes in number and size of lels but the vesicle structure was disrupted, suggesting partially distinct mechanisms of action of the two drugs. the anti-sars-cov- in vitro action of cq and hcq was confirmed in the same cellular model by [ ] . these authors found ec values of . and . for cq and . and . mm at and h for cq and hcq respectively, that, in contrast with the previous report, was a better performance for hcq. they also confirmed that the drugs have anti-viral activity also when administered prior to viral infection, with ec values of > , and . for cq and . and . mm for hcq respectively at and h. cq/hcq are also used as anti-inflammatory and immunomodulatory drugs in autoimmune diseases, like ra and sle. these properties derive from their multiple effects on the immune system cells and their modulation of crucial pro-inflammatory cytokines [ , ] . see table for a schematic list of cq/hcq biological activities as anti-inflammatory drugs. cq/hcq may impair the correct maturation and recognition of viral antigens by antigen-presenting cells (apcs) that require endosomal acidification for antigen processing. by phagocytosis and macropynocytosis, dendritic cells (dcs) capture pathogens, process and present their antigens to activate t cells. through their antigen-specific b cell receptor and cme and clathrin-independent endocytosis, b cells recognize specific antigens and present their peptides to specific t cells. through phagocytosis, cme, caveolin-mediated endocytosis and macropinocytosis, macrophages internalize pathogens, process their proteins and present antigens to t cells. t cells, through their antigen specific receptors, interact with apcs, recognizing antigenic peptides immobilized on their surface by major histocompatibility complex (mhc) class i (for cd + t cytotoxic cells) and mhc class ii (for cd + t helper cells) molecules. mhc class i molecules are loaded with those antigens mainly derived from proteasome degradation of cytosolic proteins (like viral proteins) in the er, but also from phagocytosed/endocytosed material, processed through endosomes and lysosomes. mhc class ii molecules bind antigens obtained by endosomal and lysosomal processing and, as mhc class i, require the low ph of these organelles to activate protease and other enzymes and form the complexed antigen [ , ] . cq downregulates picalm affecting cme and possibly antigen-processing [ ] . cq/hcq also alkalinize vesicles of the endosome-lysosome-autophagy pathway, possibly further interfering with antigen presentation by apcs, both acting on antigen degradation and mhc molecules processing. the inhibition of antigen presentation may reduce t cell activation and differentiation and in turn decrease the production of pro-inflammatory cytokines. tlrs are involved in innate immunity against bacteria and viruses. tlr and tlr are located in the er and move to endosomes and lysosomes where they recognize bacterial and viral nucleic acids. by this binding, their activation results in the production of pro-inflammatory cytokines and chemokine like interleukin- (il- ), interleukin- (il- ), type i interferons (ifn s), interferon- (ifn-) and cytokines promoting the stimulation of th cells and disrupting the th /th balance. the activation of tlr and tlr by their ligands requires the low ph of endosomes and lysosomes and an active autophagic pathway, then cq/hcq, through vesicle alkalinization, may inhibit this interaction and tlr signaling, in turn inhibiting the inflammatory response [ ] . matrix metalloproteinases (mmps) are a family of zinc endopeptidases that regulate inflammation and tissue repair at several levels. mmps regulate pro-inflammatory and antiinflammatory cytokine and chemokine production by proteolytic processing and may in turn be induced by cytokines and chemokines. cq has been shown to downregulate mmp- expression through the inhibition of tlr signaling [ ] . cq has also been demonstrated to inhibit phospholipase a and block the arachidonic acid cascade that leads to the production of prostaglandins and tromboxanes. in this context, cq may have both antiinflammatory and anti-thrombotic effects [ ] . this is an important point, since sars-cov- can induce pulmonary microthrombi and coagulopathy, being characterized in its severe forms by d-dimer level elevation [ , ] . interestingly, phospholipase a has recently been shown to be critically involved in coronavirus replication due to its role in the production of lipids required to form the membranous structures that are essential for virus replication and transcription [ ] . then, inhibiting phospholipase a , cq may be a triple arm against sars-cov- , acting through the anti-inflammatory, anti-thrombotic and antiviral mechanisms. as stated above, respiratory viruses can activate the p mapk pathway in infected airway epithelium and harness the trafficking machinery of infected cells to assist viral assembly and spread. p mapk activation also leads to the release of cytokines like tumor necrosis factor- (tnf-α), interleukin-  (il- β), il- and il- that turn on the inflammatory response, but this reaction can be inhibited by the cq [ , ] . as previously mentioned, covid- is characterized by the release of high levels of il- , il-  and tnf-that arekey modulators of inflammation [ , ] . cq/hcq have been shown to inhibit their release by immune cells through several mechanisms like transcription and post-transcriptional regulation, p mapk signaling block as stated above or disruption of cellular iron metabolism among others [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . tnf- is then, the challenge for anti-il- therapy in covid- is to identify when il- is detrimental or beneficial that is when clinicians would act on this pathway [ ] . a possible role of hcq in the prevention of thrombosis in hip replacement surgery has been proposed since the late s [ - ]. in this pathology, a decrease of pulmonary embolism has been reported using hcq doses between and mg per day [ , ] . hcq is now commonly used as therapeutic in sle and j o u r n a l p r e -p r o o f aps, also due to its ability to decrease the incidence of thrombosis at the dose of mg to mg per day [ ] [ ] [ ] [ ] [ ] [ ] . some authors associated this anti-thrombotic effect to the decrease of the anti-phospholipid (apl) antibody titers [ ] [ ] [ ] , while others to a more general and transversal contribute to immunomodulatory, anti-inflammatory, metabolic and anti-thrombotic effects [ , ] . chinese cardiologists have reported diffuse micro-vascular thrombosis in various organs on autopsy examination in patients died for covid- , particularly in the lung. taking into consideration this diffuse thrombosis, chinese physicians have recommended anti-coagulant therapy in these patients, but no pharmacological protocols have currently been published [ ] . the anti-thrombotic effect of cq/hcq may derive from different mechanisms. see table for a schematic list of cq/hcq biological activities as anti-thrombotic drugs. cq/hcq could act by interfering with platelet aggregation, decreasing the activation of collagen and alpha granule discharge [ , ] . platelet activation induces degranulation with the release of adenine diphosphate (adp) and other molecules in order to amplify the platelet activation process. hcq has been shown to decrease adp effects on platelet activation and in vitro platelet aggregation induced by the antibiotic ristocetin [ , ] . as mentioned above, cq/hcq could inhibit phospholipase a and decrease arachidonic acid, prostaglandin and tromboxane release [ , ] . when administered within h before surgery, hcq induces the increase of fibrinogen causing a decrease of plasmatic and blood viscosity [ ] . a possible role in rheological property decrease of red blood cells (rbcs) has been proposed, but this effect is not perfectly clarified [ ] . neutrophil extracellular traps (nets) are released by activated neutrophils that unleash intracellular molecules such as granules, proteins, dna and histones in tissues or circulation to capture and kill pathogens during infections [ ] . nets have been shown to increase thrombosis in autoimmune diseases and in inflammation [ , ] . golonka and colleagues [ ] have recently hypothesized that neutrophil abundance in covid- could be associated to the increase of nets. in a murine model of pancreatic adenocarcinoma, nets have been shown to stimulate the discharge of tissue factors and promote platelet activation and aggregation. in this model, cq/hcq showed, besides their proper anti-thrombotic characteristics, the ability to inhibit nets, in turn further decreasing hypercoagulability [ ] . j o u r n a l p r e -p r o o f hcq has been shown to inhibit the binding of the apl antibody- -glicoprotein i (gpi) complex to the phospholipid bilayer, decreasing the pro-thrombotic effect [ ] . annexina (anxa ) is an anticoagulant protein with great affinity for negatively charged phospholipids. this protein forms a -dimensional crystal over membrane phospholipids and exerts its potent anticoagulant activity shielding the phospholipid bilayer from the interaction with coagulation enzymes [ ] . in vitro experiments on human umbilical vein endothelial cells (huvec) have evidenced the protective effect of hcq, which acts restoring the anxa anticoagulant shield and therefore prevents the beginning of coagulation cascade [ , ] . hcq is used in the treatment of aps. its ability to reduce endothelial dysfunction has been studied in animal models. vascular reactivity has been studied in mice injected for three weeks with apl antibodies directed against  gpi to induce aps, with or without hcq co-treatment. hcq improved endotheliumdependent dilatation by the amelioration of no bioavailability and by the decrease of oxidative stress [ ] . in another study, apl antibodies were administered in mice and in human aortic endotelial cells (haec), in the presence or absence of hcq, to evaluate endothelial function and endothelial nitric oxide synthetase (enos) modulation. in both models, thrombosis was evaluated by thrombin generating time (tgt) and tissue factor (tf) production. apl antibodies increased vascular cellular adhesion molecule (vcam- ) expression and decreased endothelial relaxation induced by acetylcholine in haec cells. in mice, apl antibodies increased thrombus size and shortened the time needed to generate arterial occlusion. tgt and tf were increased in both models. hcq confirmed its anti-thrombotic activity decreasing clot formation, ameliorating tgt and improving endothelial relaxation. in addition, hcq ameliorated enos activation, increasing the p-enos/enos ratio with consequent improvement of nitric oxide (no) production [ ] . cytokines like tnf-, il-  and apl antibodies induce endosomal nadph oxidase (enox) that is implicated in the pro-inflammatory signal transduction. hcq inhibits these up-regulation in human monocytes, preventing the translocation of the catalytic subunit gp phox in the endosome and so interfering with enox functions. further, hcq decreases the thrombotic effect induced by apl antibodies by inhibiting ndaph oxidase (nox ), the nadph oxidase platelet isoform that is involved in platelet activation [ ] . an additional indirect effect of hcq on thrombosis is its ability to improve blood lipid profile. studies in sle and ra patients treated with steroids have evidenced lower ldl cholesterol and triglyceride levels [ ] . these data were confirmed in further sle cohorts treated with hcq in association with steroids: the treatment decreased total cholesterol and vldl level and increased hdl cholesterol [ , ] . iron is an essential element for all organisms. this is due to its redox potential which makes it an essential cofactor for several proteins and enzymes involved in vital cellular functions like energy production, dna replication and transcription. even most viruses need iron, since they require the host metabolic apparatus to replicate their genome and produce mrnas for their translation in functional viral proteins [ ] . therefore, while cellular iron repletion may boost viral replication and spread, iron deficiency may interfere with viral life cycle. during infections and inflammation, anemia is frequently observed and caused by pro-inflammatory cytokines. some of them directly affect iron homeostasis, like il- , tnf-and il- . the release of these cytokines, mainly il- , results in the upregulation of the iron-regulatory hormone hepcidin (hamp), primarily produced by hepatocytes and released in the blood flow to regulate systemic iron homeostasis. systemic hamp blocks cellular iron export through ferroportin (fpn ), resulting in reduced intestinal iron absorption, increased iron retention in hepatocytes and macrophages and ultimately anemia of infection/inflammation [ , ] . several cells other than hepatocytes have been demonstrated to produce and release hamp that can act as autocrine and paracrine molecule, modulating local iron homeostasis [ , ] . not only cells of the immune system like lymphocytes, monocytes and macrophages (including alveolar macrophages) but also airway epithelial cells have been demonstrated to produce hamp during infection and inflammation and potentially contribute to lung injury [ ] [ ] [ ] . hamp is also a peptide involved in innate antimicrobial immunity and an acute phase protein [ ] . further acute phase iron-related proteins like transferrin (tf), lactoferrin (lf), ferritin (ft), haptoglobin (hp) and hemopexin (hpx) are modulated by viral infections, further underlining the crucial role of iron in anti-viral host defense. the role of iron metabolism has been thoroughly investigated in several human viral infections. for extensive reviews on this topic and for details on systemic and cellular iron metabolism, please refer to [ ] [ ] [ ] ] . cq/hcq have been shown to modulate iron metabolism, impairing its homeostasis at different levels [ , ] , and to decrease inflammatory cytokines like il- , il-  and tnf-. here we summarize the more striking evidences on cq/hcq action on cellular iron trafficking and their impact on cellular and systemic iron metabolism. we further propose alternative modes of action for these drugs, currently used in the treatment of covid- , that might also work through the interference with local and/or systemic iron metabolism, restricting this essential element in infected cells and/or immune cells involved in virus clearance and/or acting on virus cell cycle (figure ). in several experimental models, cq has been shown to restrict iron entry into the cells. in the eukaryotic model saccharomyces cerevisiae, cq interferes with iron uptake, acting as a competitive inhibitor of iron entry and inducing iron starvation. iron-deprived yeast, by means of knocking-out genes involved in iron uptake or by using iron chelators, shows increased sensitivity to cq [ ] . in mammalian cells, cq has similar effects acting through the inhibition of the tf/transferrin receptor (tfr ) complex endocytosis. the first evidence of cq inhibition of tf uptake by cells was obtained in cultured rat embryo fibroblasts [ ] . tf is the main plasma iron carrier which, maintaining this cofactor in a redox inert state, distributes it to most cells of the human body. tf tightly binds two fe + . tfr , located on the plasma membrane of most types of cell, binds and internalizes tf into endocytic vesicles through cme [ ] . as stated above, cq has been demonstrated to reduce picalm expression in macrophages of treated mice [ ] . this ubiquitously expressed protein is involved in cme and its deficiency has been demonstrated to result in anemia and abnormal iron metabolism in mice [ , ] and iron starvation, with increased surface tfr expression and decreased intracellular iron levels, in murine embryonic fibroblasts [ ] . then, cq/hcq treatment may result in the inhibition of tf/tfr complex uptake and cellular iron starvation. the release of iron from tf after iron-loaded tf/tfr complex cme and its translocation into the cytosol is a fundamental step for iron cellular acquisition and further usage. the acidic milieau of endosomes weakens the binding of tf to fe + , enabling its release within these vesicles [ ] . cq/hcq, by virtue of their basic properties, raise ph of endocytic vesicles, possibly inhibiting iron removal from tf within endocytic vesicles. within endocytic vesicles, released fe + is reduced to fe + by the metalloreductase six-transmembrane epithelial antigen of the prostate (steap ) and exported from the lysosomes to the cytosol through the divalent metal-ion transporter (dmt ), mucolipin (trpml /mcoln ) and other transporters [ ] . iron transport through dmt is ph dependent, being stimulated at low ph. dmt is indeed a h + /fe + symporter that needs a proton electrochemical potential gradient as driving force to transport iron from endosomes into the cytoplasm [ ] . trpml /mcoln is a non-selective channel permeable to various cations such as ca + , na + and k + that can also transport fe + . it mainly localizes in late endosomes and lysosomes and the acidic environment of these vesicles activates this channel and the release of cations from the lumen into the cytosol [ ] . then cq/hcq could also inhibit iron release from endosomes into the cytosol. the alkalizing properties of cq/hcq have been widely used to impair the endosome/lysosome fusion and inhibit autophagic flux [ ] . ft is the main iron storage cellular protein that compartmentalizes iron in a nonreactive form within the cell, until use. iron release from ft mainly occurs through protein degradation by a selective lysosome-autophagy pathway named ferritinophagy [ , ] that is inhibited by cq [ ] . further, dmt and trpml have been implicated in the release of iron derived from ferritinophagy and entrapped in autophagic vesicles and, as stated above, both require an acidic environment for their channel functions. all the steps described above result in cellular iron starvation. this condition might possibly influence sars-cov- life cycle, although currently there is no experimental evidence in this new pandemic infection, but we suggest research in this direction. sars-cov- enter cells through ace receptor that is expressed broadly in the human body, mainly in the vascular endothelia, gastrointestinal system, heart, kidney, muscle, skin and bronchial and lung alveolar epithelial cells [ ] . in all human cells, then also sars-cov- target cells, iron is a cofactor of several crucial proteins, involved in bioenergetics, cellular growth and a second important point raised by the iron starvation conditions induced by cq/hcq is their effect on immune cells, involved in the innate and adaptive immune responses against the virus. as all cells in the body, immune cells require iron for their proper functions and for their activation and proliferation. iron excess often results in impaired immune response to infections, as observed in patients suffering from hemochromatosis (hh) or thalassemia [ ] . excessive or dysregulated immune response is of particular importance in the pathogenesis of covid- [ ] and diseases caused by other coronaviruses [ ] [ ] [ ] . direct infection of innate and adaptive immune cells has been described in some coronavirus infections [ , ] , then iron starvation could also possibly inhibit these infections. resident macrophages can polarize under the stimuli of cytokines in classically activated pro-inflammatory macrophages (m ), induced by interferon- (ifn-) and tnf- or, under interleukin- (il- ) and (il- ) stimuli, alternatively activated macrophages (m ), involved in pathogen clearance, tissue repair and inflammation reduction. while m macrophages have low iron levels, m macrophages are characterized by iron retention, secrete high levels of pro-inflammatory cytokines, produce high amounts of radicals to kill pathogens and produce hamp that acts in an autocrine manner to minimize iron exit. increased iron deposition in macrophages has been shown to induce m polarization and the persistence of a pro-inflammatory state due to an incomplete switch to m state [ ] . iron retention in macrophages could then favor intracellular virus life cycle in the case of their infection and further promote the process of inflammation, while iron starvation could result in the opposite effects. further, iron excess in macrophages favors secondary infections with other j o u r n a l p r e -p r o o f microbes. chronic administration of cq has been shown to decrease iron content in a rat model. legssyer and colleagues [ , ] showed that cq decreased iron content in the liver of control, iron-loaded and iron-deficient rats, in the spleen of control and iron-loaded animals and, importantly, in alveolar macrophages of iron-loaded rats. they also interestingly observed that cq treatment of primary cultures of alveolar macrophages obtained from all three groups of rats and treated with lipopolysaccharide (lps) significatively reduced the oxidative response, measured by no release, suggesting that cq might prevent infections, particularly those associated with diseases characterized by iron overload, through limiting iron availability not only in infected cells but also in macrophages, in turn reducing inflammation. however, heterozygous and homozygous mutations in the hemochromatosis gene (hfe) reduce the cq effect of iron removal in porphyria cutanea tarda [ ] . also in murine models, cq has been shown to decrease macrophage activation syndrome (hemophagocytic syndrome) induced by pristane, by reducing macrophage infiltration, phagocytic functions, cytokine production and reducing ft, lactate dehydrogenase and triglyceride levels [ ] . although immune cells need iron for their proliferation and activation, excess iron has also been reported to impair antigen processing and presentation by apcs, suppress cd + cells and alter cd + /cd + lymphocyte ratio, increase circulating immunoglobulin-producing b cells, reduce natural killer (nk) cell lysing efficiency and perturb complement activation [ ] , while iron deficiency has an immunosuppressive effect on t cells [ ] . then, cq/hcq treatment, through their action on iron homeostasis, could possibly have also a broad role in the modulation of the adaptive response to sars-cov- . a third important point to highlight derives from the inhibitory action of cq/hcq on il- , tnf- and il- release. first, tnf-α acts on iron homeostasis, reducing intestinal iron absorption and blocking iron recycling from macrophages, then inducing iron retention in these cells and their polarization to m proinflammatory phenotype [ ] . conversely, cq inhibits tnf- acting also through the induction of cellular iron starvation [ ] . then, the inhibition of the cytokine by cq/hcq, also through their effect on iron homeostasis, could alleviate inflammation, restoring a balanced systemic iron homeostasis and rescuing erythropoiesis. secondly, il- , tnf- and il- as stated above, induce the systemic and local release of hamp. iron is essential for life, but excess iron is toxic, due to its redox potential that can hamper oxidative stress damaging crucial cellular components. iron availability is then tightly regulated both at the cellular and systemic levels [ - , , ] . the main systemic regulator is hamp. this hormone peptide is produced mainly in the liver and is regulated by circulating and tissue iron levels through the bone j o u r n a l p r e -p r o o f morphogenetic/small mother against decapentaplegic (bmp/smad) pathway in such a way that iron load induces while iron deficiency inhibits its expression, mainly acting on intestinal iron absorption and iron retention/release by macrophages and hepatocytes. hamp expression is also downregulated by hypoxia and erythropoietin (epo) through erythroferrone (erfe) to allow iron mobilization for erythropoiesis, while it is upregulated by inflammation. il- is the main signal that induce hamp during inflammation, through the jak/stat pathway in association with the bmp/smad pathway, but also il-  and tnf- have a direct role on hamp regulation [ , , ] . as stated above, cq/hcq not only interfere with cellular iron inducing its starvation in alveolar macrophages [ , ] , then possibly resulting in a switch to m antiinflammatory state, but also inhibit il- , il-  and tnf- release, possibly reducing local hamp release by macrophages. this reduction can result in further decreased iron retention in these cells allowing direction towards inflammation resolution. further, cytokine decrease could result in systemic hamp decrease that, through increased intestinal iron absorption, may ameliorate anemia of infection. interestingly epo treatment has recently been found to attenuate covid- severe symptoms [ ] while, through in silico modeling, sars-cov- has been found to possibly bind heme dissociating iron from porphyrin [ ] . numerous studies correlated thrombocytosis induced by iron deficiency anemia (ida) with thrombotic events [ ] [ ] [ ] [ ] . a recent study has evidenced that ida patients manifesting thrombocytosis had -fold augmented risk of thrombosis when compared with ida patient with normal platelet count [ ] . interestingly, covid- patients with severe pneumonia seem to have high platelet counts compared with patients with severe non-covid- pneumonia [ ] and this increase is more evident among the nonsurvivor compared with survivor covid- patients [ ] . to clarify the effect of iron deficiency on thrombotic propensity in animal models, thrombosis was induced in sprague-dawley rat fed with irondeficient diet or normal diet as control. iron deficiency induced thrombocytosis and platelet number resulted proportional with thrombus size. in addition, platelet adhesion and aggregation were impaired. taking into account data obtained in this model, the authors concluded that anemia of inflammation, caused by hamp-mediated iron sequestration in the liver, spleen and macrophages, as possibly occurs in covid- patients, may be considered a functional iron deficiency (id) and patients affected by this condition should be treated as patients with high risk of thrombosis [ ] . in conclusion, the multiple action of cq and hcq as anti-viral, anti-inflammatory and anti-thrombotic drug may be also strictly linked to their effects on iron homeostasis, both at the local and systemic level. interestingly, another common drug, frequently used in the treatment of covid- patients with markedly j o u r n a l p r e -p r o o f elevated d-dimer levels is heparin. like cq and hcq, heparin is a versatile drug, as defined by jecko thachil [ ] , due to its possible actions as anti-coagulant, anti-inflammatory and anti-viral drug. it is interesting to note that, like cq/hcq, heparin too has been demonstrated to modulate iron metabolism: this antithrombotic drug has indeed been demonstrated to inhibit hamp expression in human macrophages, to increase fpn plasma-membrane expression and promote iron export, resulting in cellular iron starvation [ ] . all this evidence suggest a possible role of iron in sars-cov- infection that would be explored in future basic and clinical research, also considering it as a potential target for covid- therapy as proposed for other human infectious diseases [ ] [ ] [ ] . molecular mechanism and theoretical mode of action of cq and hcq on multiple steps of the viral pathway and the demonstrated in vitro activity against covid- [ ] [ ] [ ] have prompted their off-label use in clinical setting during this pandemic emergency. several clinical trials have been launched to evaluate the effectiveness of these drugs [ , ] . until now, published clinical data on cq and hcq are limited and regard only small, poorly controlled or uncontrolled clinical studies. we found only four published reports of these trials in pubmed till april th, . the first clinical study to evaluate the efficacy and safety of cq/hcq in patients with covid- has been reported by huang and colleagues [ ] from china. the authors screened patients tested positive for , % of controls. treatment was more effective in patients with symptoms than in asymptomatic patients. the same researchers [ ] further conducted an uncontrolled non-comparative observational study with enrolled patients and the same treatment protocol for a maximum of days. six of these patients were from the previous study. most patients ( , , %) were discharged from the infectious disease unit (idu) with a mean length stay of . days, while were still hospitalized, died and stopped treatment for the potential interaction with other drugs. on the total, % required oxygen therapy, were transferred in icu and two of them improved and returned to idu, the third remained in icu. overall, for almost all enrolled patients the clinicians obtained a better clinical improvement with this treatment when compared with other treatments. further, viral load rapidly decreased, with % negative patients at day and % at day of treatment. only patients had still detectable viral load at day . molina and colleagues [ ] studied covid- patients ( / with fever and nasal oxygen therapy, with significant comorbidities) treated with hcq plus azithromycin, using the same dosing regimen reported by gautret and colleagues [ , ] . one patient died and were transferred to icu within days after treatment initiation, one therapy was discontinued after days for severe adverse event (prolongation of the qt interval), suggesting the poor clinical outcome of the combined treatment. in contrast with results obtained by [ , ] , nasopharyngeal swabs were still positive for viral rna in / patients at days to . researchers concluded by saying that, in their experience, they found no evidence of anti-viral activity and clinical benefit by using the combined therapy in severe covid- . not only clinical efficacy but also optimal dosing regimen, therapeutic level, duration of treatment and pharmacokinetics in patients with different severity degrees of the disease are uncertain and currently there are no standard dosages or duration of treatment around the world for covid- patient treatment [ ] [ ] [ ] [ ] [ ] . numerous studies are under way to evaluate their efficacy in treating and prevention of covid- and to establish benefit versus harm of cq/hqc treatment [ ] . notwithstanding, in the emergency phase of covid- pandemic many old drugs have been off-label used for the treatment of the infection, based only on theoretical or in vitro efficacy and without enough clinical evidence based on randomized clinical trial. j o u r n a l p r e -p r o o f as we have described, cq/hcq are likely to have: ( ) a direct antiviral action stopping viral infection in several steps; ( ) a hypothetical ability to attenuate the progression of covid- to severe disease exploiting its inflammatory mechanisms, but also ( ) through its potential anti-thrombotic effect. based on our review and because of their minimal toxicity profile and complex action, we suggest to use cq/hcq cq beyond - days of treatment in patients with covid- according to the hypothesis that their utility can extend also after ending sars-cov- high replication phase and considering also the possibility of a reactivation of the infection [ ] . randomized controlled studies and observational outcome registries focused on efficacy, duration and toxicities of treatment with these drugs could be useful to understand their real effectiveness while more specific anti-covid- drugs are available. this study was partly supported by the university of brescia (fondi ex % to eugenia quiros-roldan, giorgio biasiotto and isabella zanella). the authors declare the following financial interests/personal relationships which may be considered as of chloroquine and covid- 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metabolism and the inflammatory response. iubmb life the role of iron and iron binding proteins in lymphocyte physiology and pathology anti-tnf-α monoclonal antibody therapy improves anemia through downregulating hepatocyte hepcidin expression in inflammatory bowel disease does recombinant human erythropoietin administration in critically ill covid- patients have miraculous therapeutic effects covid- : attacks the -beta chain of hemoglobin and captures the porphyrin to inhibit human heme metabolism iron deficiency and thrombosis: literature review iron deficiency anemia as a risk factor for cerebrovascular events in early childhood: a case-control study association between ischemic stroke and irondeficiency anemia: a population-based study association between venous thromboembolism and iron-deficiency anemia: a population-based study characterization of the rate, predictors, and thrombotic complications of thrombocytosis in iron deficiency anemia difference of coagulation features between severe pneumonia induced by sars-cov and non-sars-cov anticoagulant treatment is associated with decreased mortality in severe coronavirus disease patients with coagulopathy iron deficiency induced thrombocytosis increases thrombotic tendency in rats heparin inhibits intracellular mycobacterium tuberculosis bacterial replication by reducing iron levels in human macrophages treating covid- with chloroquine hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label nonrandomized clinical trial clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in covid- patients with at least a six-day follow up: a pilot observational study no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe covid- infection towards optimization of hydroxychloroquine dosing in intensive care unit covid- patients of the n-glycosylation of the cell surface viral receptor ace  inhibition of the n-glycosylation of the viral spike (s) proteins  inhibition of the synthesis of cell membrane sialic acids  inhibition of picalm expression, cme and pathogen internalization  vesicle alkalinization and inhibition of endosomal/lysosomal antigen processing  vesicle alkalinization and inhibition of mhc processing and mhc-antigen we would like to thank andrea zanella for helpful language editing. isabella zanella wishes to dedicate this work to her father, antonio zanella, who recently died for covid- .due to space constraints, we were unable to provide a comprehensive citation of all the relevant primary literature. we apologize to those whom we have even unintentionally omitted.j o u r n a l p r e -p r o o f key: cord- - m bc authors: aquino, yves s j; cabrera, nicolo title: hydroxychloroquine and covid- : critiquing the impact of disease public profile on policy and clinical decision-making date: - - journal: j med ethics doi: . /medethics- - sha: doc_id: cord_uid: m bc the controversy surrounding the use of hydroxychloroquine (hcq), an antimalarial drug, for covid- has raised numerous ethical and policy problems. since the suggestion that hcq has potential for covid- , there have been varying responses from clinicians and healthcare institutions, ranging from adoption of protocols using hcq for routine care to the conduct of randomised controlled trials to an effective system-wide prohibition on its use for covid- . in this article, we argue that the concept of ‘disease public profile’ has become a prominent, if not the sole, determinant in decision-making across various healthcare responses to the pandemic. in the case of covid- , the disease’s public profile is based on clinical and non-clinical factors that include contagiousness, clinical presentation and media coverage. in particular, we briefly examine the dangers of a heightened public profile in magnifying the inequality of diseases and undermining three key ethical concepts, namely ( ) evidence-based practice, ( ) sustainable allocation and ( ) meaningful consent. the controversy surrounding the off-label use of hydroxychloroquine (hcq) for covid- is just one among numerous ethical problems raised by the current pandemic. in this article, we examine hcq for covid- as a case study in highlighting emerging ethical dilemmas. in particular, we document the role of disease public profile as the most prominent, if not the sole, determinant of decisionmaking that potentially undermines ( ) evidencebased practice, ( ) sustainable allocation and ( ) meaningful consent. we acknowledge that although there are justifiable variations among national policies, practice guidelines and institutional protocols as a response to the outbreak, it remains important to identify both tacit and implicit values that underlie the decision-making in healthcare settings. hcq, along with chloroquine, belongs to a group of antimalarial agents known as -aminoquinolines. as more was learnt about hcq's immunomodulatory effects, its use expanded from the prevention and treatment of malaria to the treatment of various rheumatological and autoimmune conditions. hcq putatively increases intracellular vacuolar ph to modify the processing of antigens within macrophages and other antigen-presenting cells. this mechanism likely pre-empts inflammatory responses that could be maladaptive and lead to the symptoms of autoimmune disease. data from randomised controlled clinical trials (rcts) have established the efficacy of hcq for the treatment of specific rheumatological conditions such as systemic lupus erythematosus and rheumatoid arthritis. a potential role of hcq in the treatment of covid- has been suggested. in vitro data have demonstrated activity against severe acute respiratory syndrome coronavirus (sars-cov- ), likely through the inhibition of nucleic acid synthesis, viral protein glycosylation, virus assembly, virus particle transport and viral release. clinical data on its use in covid- are sorely limited. a nonrandomised open-label clinical study conducted in france included patients treated with hcq and controls. researchers reported a greater decline in viral load on day of treatment compared with untreated patients, particularly when hcq was combined with azithromycin. the conclusions of the study have been thoroughly scrutinised owing to its methodological limitations. [ ] [ ] [ ] in contrast, a small randomised controlled study in china that included patients and compared hcq with standard care did not demonstrate a difference in viral clearance on day of treatment. systematically collected data on clinical outcomes over a reasonable follow-up period have yet to be reported but may have emerged by the time of this publication. in the face of limited data, the us food and drug administration (fda) has opted to grant limited emergency use authorisation, though not approval, to the use of hcq in the treatment of covid- . hcq as well as many other drugs being repurposed to treat covid- are not without risk of serious adverse events, including potentially fatal cardiac abnormalities (eg, arrhythmias). these risks may be heightened in critically ill patients with comorbidities that place them at high cardiovascular risk. in the wake of widespread use in healthcare facilities across the world, the infectious disease society of america has issued recommendations to use hcq only in the context of a clinical trial. the american thoracic society-led international task force issued guidelines of their own that suggested considering the use of hcq for hospitalised patients with covid- pneumonia on a case-by-case basis, taking into account shared decision-making, collection of valid data and the severity of the patient's condition. regulatory boards, healthcare institutions and clinicians are now involved in a contest on whether hcq is appropriate for one medical condition but not for another. the pandemic demonstrates the role of covid- 's public profile in magnifying the inequality of diseases. here, we discuss clinical and non-clinical factors that underpin disease public profile that potentially privileges one medical condition over others. first, contagiousness is a key factor that causes one disease to be deemed or perceived as far more important than others. contagiousness, including ease and mode of transmission, of a disease determines the imperatives to intervene. in the case of covid- , the transmission is through the transfer of respiratory droplets between persons who are in close contact (within m). other modes of covid- transmission include direct contact with infected individuals, as well as contact with surfaces used or touched by the infected individual. worse, studies have shown that sars-cov- , the novel coronavirus causing covid- , has a higher transmissibility rates than other viruses such as middle east respiratory syndrome coronavirus (mers-cov) and h n . concerns have also been raised regarding transmissions from asymptomatic and presymptomatic persons that are expected to challenge containment efforts. thus, unlike in malaria and lupus, contagiousness in covid- entails a sense of larger scale, more sense of urgency and greater threat. second, the clinical presentation (ie, acuity, prognosis and case-fatality rate) determines the perceived importance of some diseases over others. accumulating clinical experience documented in case reports [ ] [ ] [ ] and relayed in anecdotes among treating clinicians contribute to the sense that patients may deteriorate rapidly and dramatically, making covid- distinct from most viral types of pneumonia. the case-fatality rate for covid- has varied widely, reflecting differences in multiple factors such as testing practices, containment efforts or demographic structure. estimates have ranged from as low as . % in germany and . % in china to as high as % in spain and . % in italy. in contrast, the united states centers for disease control and prevention (cdc) has estimated that from october to april , there have been - million cases of illness and - deaths from influenza, which translates to a case-fatality rate of only up to . %. third, media coverage is another factor that informs the inequality of disease conditions. although media coverage of a disease is a non-clinical factor, it is correlated with the contagiousness and clinical presentation of the disease in question. public profile due to media coverage is best exemplified by conditions in two extremes of perceived worthiness as a cause: breast cancer and (any type of) orphan disease. breast cancer has a high public profile mainly due to effective awareness campaigns across the globe that often involve celebrities who encourage regular examination and support for research. on the other extreme are orphan diseases, so-called because they are so rare that drug companies are not inclined to adopt them to develop treatments. in the current scenario, lupus (and to some extent malaria) are closer to the orphan disease side of the spectrum and covid- in the other extreme. unlike breast cancer, the worthiness of covid- as a cause is not due to any corporatised awareness campaign. rather it is due to the / coverage, the grim picture in countries like italy and iran, the daily updates on incidence and fatality rates and the extreme social interventions (eg, lockdowns) among others. increased public profile in itself is not problematic, as some rare or unknown diseases have received increased research funding through this route. a famed example is the 'ice bucket challenge', involving celebrities dumping ice water on their heads to promote awareness of and research on amyotrophic lateral sclerosis, a rare motor neuron disease. the danger of increased public profile of disease is demonstrated by the tendency of public officials with no medical expertise, such as us president donald trump and french president emmanuel macron, to explicitly recommend drug treatments and prophylaxis against covid- . one harmful result of president trump's pronouncements is the death of a man from arizona who ingested a cleaning cocktail that contains the same active ingredient as hcq. furthermore, increased public profile heightened by unfounded treatment recommendations have negatively impacted the academic and medical community, with scientific publications accelerating dramatically at the expense of academic rigour that validates the integrity of the research. as a result, several published articles have been retracted by elite medical journals the lancet and the new england journal of medicine following revelations of questionable data sources. in the next three sections, we elaborate the ways in which one disease's public profile, such as covid- 's, can become so extraordinary that it undermines evidence-based clinical practice, drives unsustainable resource allocation and authorises structural forms of coercive consent. the heightened public profile of disease appears to increase the imperative to resort to off-label treatments, both for therapeutic and prophylactic purposes. in the case of covid- , various drugs are proposed or currently under investigation as potential off-label treatments. however, at the time of this writing, there was still no standard of care. off-label use refers to the use of treatment (medication or device) for regimens not stated in the approved labelling or package insert. off-label use is considered a common practice and sometimes clinically appropriate in cases when there are no alternatives and potential benefits outweigh potential risks. however, off-label use has raised longstanding debates on whether it is morally justified. by its very definition, off-label use entails treatments that are not yet fully supported by evidence. in response to the potential harms of unproven treatments, national medical associations have stated some guidelines on off-label treatments. for example, the american medical association's code of ethics states that off-label and other innovative treatments must be based on 'sound scientific evidence and appropriate clinical expertise'. the australian medical association similarly recommends that off-label use should be based on 'sufficient evidence to support its efficacious and safe use and overall favourable harm:benefit ratio'. although there are several morally justifiable reasons for off-label use in general clinical practice, our analysis focusses on the influence of disease public profile on the unapproved or emergency use of hcq for covid- without sufficient scientific evidence. but what counts as scientific and/or sufficient evidence? despite the good intentions of these practice guidelines, it seems that off-label use will remain in a 'catch- ' situation given the contradicting regulations. on one hand, a common regulation states that drugs can only be used for indications of which they are approved by authorities such as the us fda. on the other hand, drug authorities do acknowledge and to some extent permit the use of drugs for unapproved or unproven indications granted the best available evidence supports this unapproved use. if there were evidence deemed by authorities to be sufficient to support use for the indication in question, this labelling would be approved and the use no longer off-label. it is widely accepted that the strongest evidence for adopting a treatment comes from well-designed randomised controlled trials, or a meta-analysis of such well-designed trials if study designs are sufficiently similar. anecdotes are powerful, but they have a limited ability to make fair comparisons between any two treatment options. outside a controlled setting, observed differences in outcomes may be due to the differences in the treatments. but they are just as likely due to pre-existing differences in the risks of patients themselves for developing a poor outcome, and these are misattributed to the treatments received. variables that are readily or quickly measured (eg, laboratory test results such as persistent detectability of viral particles) may be used to compare the two treatments, but they do not necessarily translate to the clinical outcomes that are directly experienced by patients (eg, resolution of symptoms, discharge and death). thus, good clinical trial design, ideally including the randomisation of patients to different treatment arms, is the key to producing interpretable data that best inform the care of patients. however, when such data do not yet exist, clinicians nevertheless must make patient care decisions using the best available evidence. any evidence that falls short of an rct can be classified into supported, suppositional and investigational. the classification relies on the availability of non-rct evidence and expert opinion. among the three, the investigational off-label use has the lowest level of certainty. at the time of this writing, a search in clinicaltrials. gov yielded studies on the use of hcq to prevent or treat covid- in various stages ranging from prerecruitment to completed. though there is much disagreement on the merits of hcq as a treatment, most researchers and clinicians likely at least agree that equipoise exists. though we are uncertain, the probability that hcq works is at least enough to merit investigation through rcts that will either affirm or debunk its role in care. while awaiting the results of these rcts, treating clinicians have taken a wide range of positions on the off-label use of hcq in treating covid- . for the sake of this discussion, we can consider two: the treating clinician should consider the off-label use of hcq in a closely monitored inpatient setting for patients at increased risk of an adverse outcome from covid- versus the treating clinician should provide supportive care alone and not consider any targeted off-label therapy outside the context of an rct. the position to consider off-label use has been described as the compulsion to 'just do it', or the drive to do something rather than nothing. in contrast, the position to refrain from off-label use outside the context of an rct has been presented as a necessary measure to protect patients from adverse consequences of treatment that might later prove to lack efficacy anyway. in this light, the position to offer and use hcq off-label (ie, to 'just do it') may be perceived as instinctual or irrational. nevertheless, it must be recognised that the position to refrain from offlabel hcq use to avoid serious adverse events from unproven therapy is based on an alternate instinct as well. the latter position instinctively presupposes that death from a serious adverse event from hcq that is later proven ineffective for covid- must be a worse outcome than death from covid- if hcq is later proven effective but was not tried. the former position presupposes the opposite. given the heightened public profile of covid- , it is conceivable that some next of kin of deceased patients would find greater comfort or less regret in thinking, 'we tried', or, 'we did everything we could', whereas others by, 'we did not know it could have worked'. however, when experts determine that off-label use outside of an rct (of an agent that has enough potential to be investigated in an rct) should be discouraged when managing an individual patient with their own preferences, they do make an inadvertent claim that one value or preference is objectively superior to another. however, these preference hierarchies are by nature subjective and, thus, requiring continued ethical scrutiny. the impact of covid- 's public profile on evidentiary concerns trickles down to issues regarding resource allocation based on government policies, hospital protocols and norms of clinical practice. in the case of hcq, one issue is regarding the fair distribution of the drug among its indicated uses (eg, malaria and lupus) and off-label use for covid- . as a consequence of the interest in hcq for covid- , no doubt fuelled by president trump's endorsement, demand for the drug has skyrocketed. the precarity of supply has prompted diverging responses at the level of the state and healthcare organisations or at least varying opinions from different stakeholders on how to manage the scarcity. a patient with lupus has reportedly been issued a letter by her healthcare network explaining that the hcq supply was being conserved in favour of those critically ill with covid- . the covid- global rheumatology alliance highlighted the major risk of disease flare and organ damage in patients with lupus who might be unable to refill their hcq prescriptions. in their statement, they discouraged off-label use until further efficacy data emerge in favour of its use in covid- and the supply is bolstered. at least until before the us fda's emergency authorisation, certain states had opted to restrict dispensing hcq for conditions outside the proven indications. as various expert panels and bodies have highlighted the threat to the hcq supply for lupus patients generated by hcq use in the treatment of covid- , media reports of unusual prescribing patterns on the part of healthcare providers have emerged. there is suspicion that physicians, dentists and other licensed providers are issuing themselves or family members prescriptions of hcq to stockpile them, ostensibly to take them prophylactically or to use them should they develop symptoms. the magnitude of this practice is unclear, but it does pose an ethical concern that treatment practices surrounding patients hospitalised with covid- are being influenced by an allocation problem generated by some prescribers hoarding for themselves or significant others who are not or only mildly symptomatic. bioethical debates regarding medical resource allocation generally involve two main competing positions: the maximising utility view and the equity view. the former follows the utilitarian precept of promoting the greatest good for the greatest number of persons, whereas the latter highlights the need to redress social injustice and socioeconomic inequalities that result in unfair distribution of goods. using the utility view, the competition among diseases means that governments and health institutions should focus on the disease where treatment will result in the greatest good for the greatest number. the key criticism against utility view is that the greatest good is often at the expense of a vulnerable minority, whether in terms of persons or diseases. in contrast, the equity view recognises that certain groups of people are already at a disadvantage. the equity view can be useful in demonstrating that in a public health crisis, some disease conditions are vulnerable to neglect as resources are diverted disproportionately to the disease that receives high public attention. in the example of breast cancer, there has been a growing criticism against the 'pink ribbon' campaign as receiving excessive attention at the expense of other types of cancer. the case of hcq for covid- demonstrates a problematic allocation of resources that cuts across various domains of healthcare. intensive care unit beds, mechanical ventilators and human resources are now largely diverted toward covid- . arguably, diverting resources toward a highly contagious disease during a pandemic is justifiable. however, such a strategy can have unintended harmful consequences. for example, health experts warn against the surge of polio, measles and other infectious diseases as covid- efforts trigger the suspension of worldwide vaccination campaigns. a patient with lupus who is unable to fill their hcq prescription could end up requiring hospital care from a life-threatening lupus flare, but be unable to receive optimal care from a system now primed to focus on covid- . hence, the trend of allocating resources, including hcq, toward covid- is not only disproportionate but may also prove to be unsustainable or counterproductive in the long run. the public profile of a disease similarly impacts the creation of undue influence on patient consent to participate in research trials. when the us fda granted emergency authorisation for hcq, there was concern that offering off-label therapy outside rcts leads to a squandered opportunity to enrol these patients in them. if off-label therapy is offered outside the context of an rct, there is reduced motivation or incentive to participate in one. however, if they could be persuaded to participate, their outcomes would provide clarity to the treating clinician in their care of future patients. in bioethics, consent is deemed meaningful and informed if the person giving it is 'competent to act, receives a thorough disclosure, comprehends the disclosure, chooses voluntarily and consents to the intervention'. competence, comprehension and voluntariness can be compromised by coercion and undue influence, which are two overlapping concepts. although coercion is generally understood as direct use of threat of violence, undue influence occurs when there is an excessive and inappropriate reward to obtain compliance. for some authors, coercion can occur when there is a substantially asymmetric power dynamic between the physician/researcher and the patient/participant. patients and potential research participants may be coerced to consent if they fear they have something to lose by not complying with the request. in a less pessimistic light, coercion may occur when patients overly trust their physicians so that a physician's invitation to participate may be viewed as a recommendation rather than a request. concerns regarding potential coercion of participants have been demonstrated in clinical trials for cancer treatments, hiv cure and treatment for psychiatric disorders, among others. authors argue that therapeutic clinical trials tend to collapse the two roles of a clinician as providing clinical care and performing research, and this collapse can contribute to patients' misconception that participation in the trial is a matter of necessity if not an obligation. clinicians and future patients with covid- would be best served by the participation of current patients with covid- in these rcts. nevertheless, one of the cornerstones of modern ethical codes governing clinical research is preserving the participant's ability to provide meaningful consent. withholding an ff-label agent (with sufficient potential to warrant rct investigation) in the course of care functions as an inducement to consent to join the study. study participation might entail being randomised to placebo (ie, supportive care alone) when the patient would otherwise not have agreed to be. understandably, in practice, many rcts often involve incentives, but these are closely scrutinised precisely because of the risk of compromising meaningful consent. a conflict, therefore, arises between future patients on one side and the current patient whose only access to off-label therapy is through an rct. although the informative findings of an rct might serve the 'greater good,' this must be carefully weighed against protecting the meaningful consent of patients being recruited. the controversy surrounding hcq further demonstrates that undue influence and coercion can be structural. wider social, economic and political contexts can undermine understanding and voluntariness as components of consent. structural and indirect forms of coercion can be shaped by social disparities that perpetuate unequal access to resources that help people make healthier and more informed choices. for example, us reports show that the pandemic has highlighted the disparity in access to technology and online information from reputable websites such as the us centers for disease control and prevention, leading to hoax and conspiracy sites that promote unfounded and harmful covid- treatments. moreover, structural forms of coercion are demonstrated by the ways in which the public profile of covid- magnifies the impact of political, social and institutional influence on participants consenting to ongoing hcq trials. on a political level, some state leaders, such as president trump and president macron, have been vocal in their confidence with hcq, putting demands on researchers and clinicians who are already under intense pressure to develop treatments for covid- . consequently, political faith on a treatment that is not supported by evidence has spurred greater society's demand to have access to hcq, either as a prophylaxis or treatment against covid- . the hype may play a role in influencing patients' decision to participate in trials or to seek treatments that are at best not proven to be effective or worst associated with potentially fatal side effects. in this article, we argued that the case of hcq being touted as potential covid- treatment demonstrates the dangers of a disease's public profile becoming the overarching influence in healthcare decision-making. the public profile of a disease, which is based on a combination of clinical features and media coverage, not only magnifies the inequality of diseases but also potentially undermines the ethical concepts of evidence-based medicine, sustainable allocation of resources and meaningful consent. given the rapid progression of the pandemic, we acknowledge the need for quick clinical and policy responses. although we do not advocate for a particular outcome of decision-making, we wish to highlight the need for ethical deliberation to make explicit the values that are informing widespread and immediate responses to the pandemic. we need to strike a balance between matters of urgency and matters of equity, with some medical conditions becoming vulnerable to neglect and ethical concepts being dismissed, as resources and attention are diverted towards covid- . the key claims during the development stage; responsible for the ethical discussion raising specific ethical concepts in the manuscript. nc is responsible for the technical details of the drugs and the diseases discussed. funding the authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors. competing interests none declared. provenance and peer review not commissioned; externally peer reviewed. data availability statement there are no data in this work. this article is made freely available for use in accordance with bmj's website terms and conditions for the duration of the covid- pandemic or until otherwise determined by bmj. you may use, download and print the article for any lawful, non-commercial purpose (including text and data mining) provided that all copyright notices and trade marks are retained. yves s j aquino http:// orcid. org/ - - - hydroxychloroquine: from malaria to autoimmunity mechanism of action of hydroxychloroquine as an antirheumatic drug hydroxychloroquine in systemic lupus erythematosus: results of a french multicentre controlled trial (plus study) a long-term study of hydroxychloroquine withdrawal on exacerbations in systemic lupus erythematosus. the canadian hydroxychloroquine study group hydroxychloroquine compared with placebo in rheumatoid arthritis. a randomized controlled trial in vitro antiviral activity and projection of 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doctors stockpile trial coronavirus drugs for themselves, states say the new york times ethics of resource allocation equity and population health: toward a broader bioethics agenda backlash against "pinkwashing" of breast cancer awareness campaigns allocation of scarce resources during the covid- pandemic: a jewish ethical perspective polio, measles, other diseases set to surge as covid- forces suspension of vaccination campaigns science the ethics of consent: theory and practice expanding the frame of "voluntariness" in informed consent: structural coercion and the power of social and economic context consent to clinical research--adequately voluntary or substantially influenced? seeking informed consent to cancer clinical trials: describing current practice informed consent to hiv cure research the ethics of clinical trials research in severe mood disorders the routledge companion to bioethics. abingdon-on-thames ethical issues surrounding forced, mandated, or coerced treatment how health disparities are shaping the impact of covid- pandemic response lays bare america's digital divide. the guardian coronavirus: the spread of misinformation the authors would like to thank the journal of medical key: cord- -tbtmqmpr authors: acharya, yogesh; sayed, abida title: chloroquine and hydroxychloroquine as a repurposed agent against covid- : a narrative review date: - - journal: ther adv infect dis doi: . / sha: doc_id: cord_uid: tbtmqmpr the predicament arising from the coronavirus disease (covid- ) pandemic has become one of the most significant modern public health challenges. despite uncertainties in the viral determinants and pathogenesis, it is crucial to accurately inspect all available evidence to construct accurate clinical guidelines for optimised patient care. this study aims to discuss the available evidence for the use of chloroquine (cq) and hydroxychloroquine (hcq) against covid- . early in vitro studies of cq/hcq against severe acute respiratory syndrome coronavirus (sars-cov- ) are convincing. but contradictory evidence exists on the clinical use of cq/hcq, either alone or in combination with azithromycin. as of now, there is no compelling clinical evidence on cq, hcq, and azithromycin in covid- and the available evidence is limited to methodologically inferior non-randomised studies. studies have also shown detrimental drug reactions to cq and ‘hcq plus azithromycin’, mainly cardiac side effects in hospitalised patients with coexisting cardiovascular comorbidities. therefore, we recommend that physicians avoid high doses and exercise extreme caution in the compassionate use of cq/hcq, either alone or in combination with other antiviral drugs. the sudden outbreak of coronavirus disease (covid- ) , stemming from a novel coronavirus originating from wuhan, china, has grown into a global pandemic as the third major outbreak of the virulent coronavirus family after severe acute respiratory syndrome (sars) and middle eastern respiratory syndrome (mers). despite radical containment efforts, the sars coronavirus (cov ) continues to spread globally. currently, therapeutic tactics are only supportive as there are no proven pharmacological agents active against the virus. international efforts are focussed on searching for effective therapies to counter the disease's effects. one strategy is to search for nobel agents by repurposing older drugs with known antiviral activity that have been studied in the past. in this regard, chloroquine (cq) and hydroxychloroquine (hcq) have garnered much attention as they were well studied during previous coronavirus epidemics with sars and mers. this study aims to discuss the general properties and antiviral history of cq and hcq, and to analyse the available evidence against covid- , either alone or in combination with other drugs. a literature review was performed using pubmed and google scholar to identify all relevant english language scientific studies based on our study objectives. non-specific combinations of the search strings included (coronavirus or severe acute respiratory syndrome or sars or chloroquine and hydroxychloroquine as a repurposed agent against covid- : a narrative review cq and hcq share similar pharmacokinetics, with rapid absorption from the gastro-intestinal surfaces, and are renally and hepatically eliminated. as they are weak bases, they increase the ph of acidic vesicles. notably, hcq increases intracellular ph levels by inhibiting lysosomal activity in immune cells to prevent downstream immune cell interaction, antigen processing, and cytokine responses. furthermore, the increment in the ph interferes with viral entry into endosomes and blocks viral-endosome fusion. although cq and hcq are relatively well-tolerated, both drugs are associated with systemic adverse effects, including qt syndrome, hypoglycemia, hepatitis, pancreatitis, neutropenia, retinopathy, anaphylaxis, and cardiac toxicities. the united states food and drug administration (fda) has issued warnings on cq/hcq, particularly based on the associated cardiac side-effects. the cq/hcq, either alone or in combination with other drugs like azithromycin, can cause possible cardiac complications, including conduction defects, such as bundle branch blocks, atrioventricular blocks, qt prolongation, torsades de point (tdp), and even dangerous ventricular arrhythmias. although the cq and hcq use during pregnancy and in lactating mothers are believed to be safe, [ ] [ ] [ ] they should be avoided in children due to narrow therapeutic and toxic windows. children can suffer from apnea, seizures, and arrhythmias if they exceed the recommended therapeutic dose. antiviral activity: sars, mers and others clark, in , conducted studies depicting the potential anti-microbial activity of cq by effective inhibition of deoxyribonucleic acid (dna) synthesis on a protozoan parasite, plasmodium gallinaceum. this led to further investigations by schellenberg and coatney in , who demonstrated cq-induced inhibition of the incorporation of radioactively labelled nucleic acid substituents in dna and ribonucleic acid (rna) of plasmodium gallinaceum. these studies showed that cq could inhibit the nucleic acid, and this nucleic-acid inhibitory potential could be utilised in viruses. mallucci conducted one of the first animal studies in to demonstrate the antiviral activity of cq on lysosomes of hepatitis virus-infected mouse cells. the mechanism of inhibition was unknown before the animal studies, and cq was believed to lower virus yield, hypothesised by the prevention of new viral synthesis or viral uncoating. in , shimizu et al. investigated the antiviral effects of cq on non-oncogenic viruses in animal cell culture. , these inhibitory effects were noted with cq treatment both before and after exposure to the sars virus, suggesting its prophylactic as well as therapeutic utility. vincent et al. found that cq interferes with the terminal glycosylation process of cellular receptors, particularly angiotensinconverting enzyme (ace ), with the potential to prevent virus-receptor interaction, and interferes with viral spreading by increasing vesicular ph levels. in , savarino et al. pointed out the importance of the cq as a broad-spectrum antiviral agent. they stated, 'the broad-spectrum antiviral effects of chloroquine deserve particular attention in a time in which the world is threatened by the possibility of a new influenza pandemic, and the availability of effective drugs would be fundamental during evaluation of an effective vaccine.' in , keyaerts et al. further investigated both the in vitro and in vivo antiviral activities of cq against human cov strain oc (hcov-oc ) in newborn mice. they concluded that cq interferes with the in vitro replication of hcov-oc , and showed % survival in newborn mice treated pre-partum with cq. their result showed that cq could be immensely useful against hcov-oc . although there were some promising results concerning cq/hcq in sars/mers, most of these credible pieces of evidence were based on in vitro studies. only a few clinical studies were available, which were methodologically inferior with small sample size, high dropout rates, variable baseline viral loads compared with monotherapy, and combination therapy of cq/hcq and differences in toxicities, which impacted the quality and validity of the results obtained. as the sars/ mers outbreaks were limited within a particular region and lasted for a short time, there were no follow ups, more extensive observational studies, or controlled clinical trials to support this evidence. since the beginning of the covid- the cq and hcq duo was one of the very first contenders in the race against covid- . although there was no high-quality clinical evidence of cq/hcq against sars and/or mers, the in vitro and in vivo results of cq/hcq were promising. therefore, as sars-cov shared genetics and pathological similarities with sars/ mers within the coronavirus families, cq/hcq were considered to be initial candidates for drug repurposing in covid- . [ ] [ ] [ ] [ ] we have summarised these in vitro and in vivo studies in table , and each of these studies is described in brief based on the sequential unfolding of the evidence. on february , an editorial in springer nature ® conducted by wang et al. was one of the first to describe the effects of cq in conjunction with other five antiviral drugs in vero e cells infected with ncov betacov. they performed a standard in vitro assay in -ncov clinical specimens to elucidate the antiviral activity in terms of cytotoxicity, virus yield, and infection rate. notably, their time-of-addition assay showed that cq blocked the virus at low concentrations during both entry and post-entry phases of cellular infection. they concluded that cq has a high prospect in -ncov as it is a standard drug with known safety profile, is relatively cheap, achieves a wide volume of distribution after oral intake, and can easily attain the ec seen in vero e ( . μm) with standard mg dose. on february, a chinese briefing reported cq as a successful treatment regimen in greater than patients infected with covid- . the patients showed improvements in lung function demonstrated by radiological evidence, viral clearance, and slowing of disease progression. they recommended cq to be listed in the standard guidelines for management of covid- associated pneumonia in the national health commission, china, based on the successful outcomes in terms of safety and efficacy within the country. another in vitro study conducted by yao et al. on april , saleh et al. conducted a prospective observational safety study to evaluate the effect of cq, hcq, and azithromycin in association with qt interval and risk of tdp and sudden cardiac death in covid- patients. in this extensive cohort study to date with cq/hcq/ azithromycin, no tdp or sudden death due to arrhythmia was noted. these medications did prolong the qt interval but did not lead to the discontinuation of therapy. they concluded that further research is needed to establish drug safety before a definitive recommendation can be made. in addition, a retrospective observational cohort safety study conducted by van den broek et al. on april , investigated cq-induced qt prolongation in covid- patients. they concluded that cq leads to qt prolongation and recommended ecg monitoring of all patients taking cq. similarly, on may , mercuro et al. in boston, massachusetts, investigated the risk of qt prolongation with the use of hcq with or without azithromycin used in combination in covid- confirmed-patients. they observed that patients receiving hcq as a treatment for covid- pneumonia were at significant risk for qt prolongation and that parallel treatment with azithromycin further elevated the risk. they emphasised extreme caution and careful monitoring of the patients with a careful weighing of both risk and benefits before initiation of the treatment with hcq/azithromycin. on may , a rapid systematic review consisting of clinical trials carried out by chowdhury, rathod and gernsheimer showed inadequate evidence to encourage the use of cq/hcq in covid- . they concluded that healthcare professionals should dissuade from the clinical use of cq/hcq until the ongoing studies provide more evidence on efficacy and safety profiles. similarly, on may , jain et al. conducted a safety study to establish an improved ecg monitor system of covid- patients undergoing pharmaceutical treatment associated with a risk of qt prolongation. they created a tool called situation background assessment recommendation (sbar) that identifies patients requiring ecg monitoring and tags qt prolongation within the ecgs. they showed that sbar efficiently identified qt prolongation, with . % being related to qt-prolonging medications. furthermore, on may , geleris et al. conducted an observational study to determine an association between hcq and the need for intubation or risk of death. based on the observations of consecutive patients with covid- who received hcq ( mg twice daily on initial admission followed by mg daily for days), they conclude that there was no significant association of hcq with lowering the risk of intubation or death (hr . ; % ci . - . ). geleris et al. concluded that hcq should only be used within clinical trial settings unless its efficacy can be thoroughly tested and established. this study had many potential limitations, including missing data and inaccurate health record-keeping, incomplete documentation on smoking, and comorbidity status on patients as well as a single-centre design, limiting the generalisability of the study data. an open-labeled randomised controlled trial was published on may , by tang apart from a general warning, the fda has cautioned against the indiscriminate use of cq/ hcq, either alone or in combination with azithromycin, in covid- patients due to the potential linkage to cardiac toxicities, including severe complications like rhythm disturbances. while the fda continues to explore these adverse events and will communicate their findings with the public once more information becomes available, the latest covid- nih treatment guidelines panel recommends 'against using high-dose chloroquine ( mg twice daily for days) for the treatment of covid- (ai), because the high dose carries a higher risk of toxicities than the lower dose'. similarly, the ema has advised the close monitoring of the covid- patients receiving cq/hcq. current clinical recommendations on cq/hcq in covid- are not backed by substantial evidence, and most of the studies are methodologically inferior. in this unique circumstance, we are made to decide between providing medical care or producing reliable and scientifically valid data. this dilemma may lead to compromising the generation of evidence-based and clinically reliable results. in many cases, drugs like cq and hcq are being given compassionately due to the severe nature of the disease, despite the discrepancies and safety warnings. as there is insufficient clinical evidence to either refute or accept the use of cq/hcq in covid- , we would like to advise physicians across the globe to avoid high dose and exercise extreme caution in the compassionate use of cq/hcq, particularly in patients with cardiac comorbidities. understandably, there is an overwhelming need to identify plausible treatment options against a devastating and deadly disease like covid- . in their quality analysis of the existing studies of cq/hcq in covid- , alexander et al., reported that the majority of the existing studies on covid- are general, biased, and methodologically non-rigorous. they concluded that the available clinical studies have many limitations, including sample size, unclear reporting of study methodology, no blinding and/or randomisation, missing clinical data, inconsistencies in treatment versus control groups such as groups taken from different healthcare centers, lack of control groups, lack of matching and stratification, inconsistent and low event rates, and an unadjusted analysis. observational studies can lead to biases, like selection, collider, and confounding bias, that can significantly influence studies outcomes, making them challenging to interpret and/or replicate. , this is mostly due to the high demand for evidence on ongoing pandemics and uncertainty regarding the virus. although we did not perform the quality assessment of the available studies, the majority of the available studies were case-based small studies that are often not controlled with randomisation and/or standardisation. there is no denial that high-quality research, such as rcts or extensive registry-based observational studies, are still warranted to have conclusive evidence before reaching any consensus. despite these methodological challenges, it is still up to the research community and clinicians to accurately appraise studies and prioritise the publication of credible evidence. given the promising early in vitro results of cq/ hcq in sars-cov and existing therapeutic dilemmas, the compassionate use of cq/hcq can be an option in the ongoing crisis despite the absence of convincing clinical evidence in covid- . however, physicians should avoid high doses of cq/hcq and exercise caution, particularly in patients with existing cardiovascular disease. we suggest that clinicians and researchers regularly update and adhere to the available credible evidence and findings of the ongoing clinical trials. • given the promising early in vitro results and therapeutic dilemma, the compassionate use of cq/hcq in covid- can be an option. • however, there is no convincing clinical evidence to support the use of cq and hcq, either alone or in combination with azithromycin, in covid- . • misuse of cq/hcq or use beyond the prescription can result in serious health problems, including cardiac toxicity and even death. • it is recommended to avoid high dose cq/ hcq and exercise extreme caution while using cq/hcq even in hospitalised patients. • clinicians and researchers should regularly update and adhere to the available credible evidence and findings of the ongoing clinical trials. ya and as made substantial contributions to the conception and study design, data acquisition and interpretation. all authors were involved in drafting the manuscript as well as the revision process. all authors have approved the final version of the manuscript. the reproductive number of covid- is higher compared to sars coronavirus evaluation and treatment coronavirus drug repurposing against covid- : focus on anticancer agents chloroquine for covid- : rationale, facts, hopes pharmacology of chloroquine and hydroxychloroquine covid- : a recommendation to examine the effect of hydroxychloroquine in preventing infection and progression treating covid- -off-label drug use, compassionate use, and randomized clinical trials during pandemics aralen® chloroquine phosphate, usp: description transfer of chloroquine and 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association of treatment with hydroxychloroquine or azithromycin with in-hospital mortality in patients with covid- in new york state. jama. epub ahead of print a randomized trial of hydroxychloroquine as postexposure prophylaxis for covid- recoverytrial.net breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies a systematic review on the efficacy and safety of chloroquine for the treatment of covid- epub ahead of print a systematic review of the prophylactic role of chloroquine and hydroxychloroquine in coronavirus disease- (covid- ) virological and clinical cure in covid- patients treated with hydroxychloroquine: a systematic review and meta-analysis a rapid systematic review of clinical trials utilizing chloroquine and hydroxychloroquine as a treatment for covid- hydroxychloroquine in patients with covid- : a systematic review and meta-analysis fda cautions use of hydroxychloroquine/chloroquine for covid- coronavirus disease (covid- ) treatment guidelines covid- coronavirus research has overall low methodological quality thus far: case in point for chloroquine/hydroxychloroquine collider bias undermines our understanding of covid- disease risk and severity robins-i: a tool for assessing risk of bias in non-randomised studies of interventions methodological challenges in studying the covid- pandemic crisis the authors declare that there is no conflict of interest. journals.sagepub.com/home/tai the authors received no financial support for the research, authorship, and/or publication of this article. yogesh acharya https://orcid.org/ - - - key: cord- - vlc rx authors: stricker, raphael b; fesler, melissa c title: flattening the risk: pre-exposure prophylaxis for covid- date: - - journal: infect drug resist doi: . /idr.s sha: doc_id: cord_uid: vlc rx to date, more than million people worldwide have been infected with severe acute respiratory syndrome coronavirus (sars-cov- ), the agent of coronavirus disease (covid- ), and more than one million have died in the covid- pandemic. international economies are stalled and social isolation based on palpable fear of death remains the order of the day. the united states and other countries are moving toward resuming work activities and social interaction to boost economic recovery. while this makes financial sense, from a medical perspective our population has already suffered and will continue to suffer severe losses in the absence of a viable aggressive prophylaxis strategy for sars-cov- . herein, we present a plan to address this problem. the medical approach to sars-cov- is based on three principles: prevention, early treatment and late treatment. we previously presented a novel plan to implement early treatment for covid- on a large scale as needed. although this plan may salvage patients with early disease, it is less practical than prevention for people who are resuming work activities and who may be exposed to sars-cov- via contact, aerosol and/or droplet transmission. as for late treatment, the high mortality rate of % or more for patients on respiratory support makes implementation of prevention and early treatment an urgent priority. furthermore, the emergence of persistent debilitating symptoms in survivors indicates that covid- is a complex illness that is best avoided. residual risk of covid- will likely persist in the future, and this risk is associated with additional global mental health problems such as stress, anxiety, depressive symptoms, insomnia, denial, anger and fear. , the medical costs of a poorly controlled covid- pandemic are projected to reach hundreds of billions of dollars in the united states. prophylaxis for viral infections traditionally relies on vaccines. although a vaccine for covid- would be an optimal solution, the prospects for a safe and effective vaccine are murky at best and probably not a viable option in the next two years due to mutational variation of the virus and potential vaccine enhancement of viral infection. , herd immunity to sars-cov- also may be difficult to achieve because the infection rate is too low. , therefore, an alternate prevention strategy is needed when social distancing is phased out during economic recovery. drawing on lessons from the hiv/aids pandemic, we know that pre-exposure prophylaxis (prep) with antiviral agents is a viable strategy to prevent infection. in the case of hiv/aids, however, this treatment-intensive and expensive strategy only works because of the relatively small number of subjects at risk. for the covid- pandemic, we need a more practical approach to prevention. based on worldwide experience with sars-cov- , we now know that certain groups carry a higher risk of developing severe complications of covid- . in addition, medical personnel have more than three times the risk of acquiring the disease from repeated exposure to infected patients and household contacts. , as schools reopen and asymptomatic children increase the risk of viral transmission, more teachers and parents will need protection against viral infection. other high-risk groups include first responders, factory workers and sailors confined to close quarters, and prisoners. individuals with certain genotypes may also have increased susceptibility to severe covid- . these subjects would benefit from a simple prep-type treatment for sars-cov- . where can we find a prophylactic treatment that is easy to use, inexpensive and already available? one answer lies in a medication that has generated significant controversy so far in the pandemic: hydroxychloroquine (hcq). this medication has been used in combination with azithromycin for treatment of established sars-cov- infection, with variable results. , there has been concern about cardiac and retinal toxicity with high-dose hcq, especially because azithromycin increases blood levels of the medication. , furthermore, shortages of the two antimicrobials have occurred due to overprescribing in the face of the pandemic. nevertheless, the combination appears to be effective in patients with early covid- , although it appears to be less effective in hospitalized patients with advanced disease. , hcq treatment was shown to protect multiple organs from inflammation in hospitalized covid- patients. in one uncontrolled study, hcq prophylaxis in a hospital setting with a known sars-cov- exposure prevented dissemination of viral infection. an overlooked property of hcq is its extremely long half-life, estimated at up to days with oral dosing. , this extraordinary property has allowed the medication to be used for decades as weekly prophylaxis for malaria. , although hcq-resistant malaria has eventually developed in parts of the world, the benefit of the medication is still recognized in terms of low cost, ease of use and limited side effects in areas with susceptible disease. [ ] [ ] [ ] malaria prophylaxis with hcq may contribute in part to the reportedly low rate of covid- infection seen so far in sub-saharan africa. , data from three randomized clinical trials using hcq for the prevention and treatment of covid- did not suggest significant safety concerns, and there were no sudden deaths in any trial. hcq treatment is also considered safe for use in pregnancy and childhood diseases. , unfortunately, extrapolation of inpatient hcq use to outpatient settings has muddied both the risks and benefits of this readily available preventive treatment for covid- . based on these observations, we propose instituting a prophylactic regimen for sars-cov- in the high-risk patients listed above. the dose limit should be mg weekly based on malaria prophylaxis recommendations and continuing for one month or longer depending on the degree of social interaction and risk of viral exposure. the use of this prophylactic regimen will allow individuals to resume work with some modicum of protection against covid- . prep would also prevent high-risk individuals from acquiring infection from close contacts infected with the virus, and it would hopefully attenuate sars-cov- infection if it did occur. although a higher-dose regimen might be more effective, higher dosing would also increase the risk of adverse events without clear benefit at this point in the pandemic. , if symptoms of covid- develop despite the use of prep, further treatment should be instituted immediately, as outlined in our previous publication. recently three studies of hcq prophylaxis in healthcare workers (hcws) from india reported encouraging results. one cohort study found that % of untreated hcws developed covid- infection compared to % of hcws treated with weekly hcq prep (p< . ). the second case-control study of hcws found that four or more weekly doses of hcq resulted in significantly less infection with sars-cov- (adjusted odds ratio . , p< . ). the third study was a questionnaire-based analysis showing that hcws who had taken a full course of hcq prep (seven or more weekly doses) had significantly less infection with sars-cov- compared to those who had taken either an incomplete course or no hcq at all (p = . ). additional clinical observations suggest that hcq prep may be effective for covid- . in a multicenter retrospective study of , rheumatic disease patients from china, patients who were taking hcq had a lower risk of sars-cov- infection compared to patients taking other disease-modifying anti-rheumatic drugs (odds ratio . , p= . ). another population-based analysis of over , subjects from portugal found that chronic hcq treatment was associated with a significant decrease in sars-cov- infection (adjusted odds ratio . , p= . ). an observational study of , early outpatient covid- cases in new jersey found that a prescription of hydroxychloroquine reduced the risk of hospitalization by % (odds ratio . ). the benefit appeared to be lost if treatment was delayed more than two days after the onset of symptoms. in a retrospective cohort study of , rheumatic disease patients from the us veterans health administration, the incidence of sars-cov- infection was equivalent regardless of chronic hcq use ( . % in users versus . % in non-users), but mortality was significantly decreased in patients taking hcq (odds ratio . , p= . ). hcq prep failed to prevent experimental viral infection in syrian hamsters and macaque monkeys, but all of the animals cleared their infections and none died. thus, the hamster and monkey prep models have questionable significance for humans. , larger randomized controlled trials (rcts) of hcq prep are ongoing in the united states and elsewhere. [ ] [ ] [ ] [ ] the results of two rcts that enrolled , hcws were recently released, and among , subjects who received hcq prep there were no hospitalizations, no deaths and no cardiac complications. in addition, once-weekly dosing appeared to be as effective as twice-weekly or daily dosing for hcq prep. because the studies were terminated prematurely they were underpowered to show a treatment benefit, however. , since rcts require an average of . years for completion at an average cost of over a million dollars, it may take a long time to obtain conclusive results from these studies. [ ] [ ] [ ] in the face of a public health crisis, it is important to consider life-saving approaches based on scientific logic and clinical availability even if definitive results are pending. , unfortunately, hcq has generated significant controversy resulting in the public perception that the medication is dangerous and ineffective. in light of this problem, weekly prophylaxis with mefloquine mg could be used instead. this oral medication has a half-life of about three weeks and appears to be active against sars-cov- in vitro, which makes it suitable for weekly antiviral prophylaxis. , the troubling neuropsychiatric side effects of mefloquine make the drug a less attractive option for prep. another option is the antiparasitic drug ivermectin. this antimicrobial agent with antiviral properties appears to be effective against sars-cov- in vitro and in vivo. [ ] [ ] [ ] [ ] the half-life of ivermectin is - hours in humans, and its metabolites may persist for up to days due to high liposolubility. it can be dosed daily or weekly at . - . mg/kg with minimal side effects, but clinical prep trials for covid- using ivermectin have not been organized to date, and appropriate antiviral dosing remains questionable. individuals with specific gene mutations may have adverse reactions to ivermectin. tafenoquine, a newer antimalarial agent that can be dosed weekly with a half-life of - weeks, has been suggested for covid- prep, but the side effects and cost of this medication make it a less attractive candidate for widespread prophylaxis. , in patients with hiv/aids, treatment with tenofovir disoproxil fumarate (tdf)/emtricitabine (ftc) lowered the risk of covid- , but these medications are expensive and must be dosed daily. therapy with convalescent plasma has been administered to severely ill covid- patients and prophylaxis with plasma or neutralizing monoclonal antibodies has been suggested, but use of these nascent non-standardized treatments for prep has not been evaluated. [ ] [ ] [ ] [ ] [ ] the efficacy and safety of barrier protection (primarily facemasks) in the covid- pandemic continues to be debated, and formal studies in various settings have been recommended. , for hcws with high-risk patient for the general population, facemasks appear to be less effective due to technical limitations, compliance issues and variable transmission risks. , the theory of "variolation", which states that population-wide masking may limit viral load exposure and thereby attenuate the severity of covid- , remains to be proven. although not a perfect solution, medication-based prep should largely eliminate the drawbacks of barrier protection by providing consistent prophylaxis against viral infection for as long as the medication is used and the virus remains clinically susceptible. as economies reopen, schools resume and passive social distancing becomes more difficult, there is an urgent need for active prophylaxis against covid- . prep modeled on malaria and 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long-term hydroxychloroquine use in patients with rheumatic conditions and development of sars-cov- infection: a retrospective cohort study hydroxychloroquine proves ineffective in hamsters and macaques infected with sars-cov- hydroxychloroquine use against sars-cov- infection in non-human primates will hydroxychloroquine impede or prevent covid- (whip covid- ) protecting frontline health care workers from covid- with hydroxychloroquine pre-exposure prophylaxis: a structured summary of a study protocol for a randomised placebo-controlled multisite trial in toronto vitamin c, vitamin d, and zinc for the prevention of covid- infection (helpcovid- ) hydroxychloroquine in the covid- pandemic era: in pursuit of a rational use for prophylaxis of sars-cov- infection efficacy and safety of hydroxychloroquine vs placebo for preexposure sars-cov- prophylaxis among health care workers: a randomized clinical trial hydroxychloroquine as pre-exposure prophylaxis for covid- in healthcare workers: a randomized trial beyond the randomized controlled trial: a review of alternatives in mhealth clinical trial methods effect of a us national institutes of health programme of clinical trials on public health and costs characteristics and strength of evidence of covid- studies registered on clinicaltrials.gov covid- : real-time dissemination of scientific information to fight a public health emergency of international concern mefloquine at the crossroads? implications for malaria chemoprophylaxis in europe repurposing of clinically approved drugs for treatment of coronavirus disease in a -novel coronavirus ( -ncov) related coronavirus model the broad spectrum antiviral ivermectin targets the host nuclear transport importin α/β heterodimer the fdaapproved drug ivermectin inhibits the replication of sars-cov- in vitro ivermectin, a new candidate therapeutic against sars-cov- /covid- icon (ivermectin in covid nineteen) study: use of ivermectin is associated with lower mortality in hospitalized patients with covid ivermectin and covid- : a report in antiviral research, widespread interest, an fda warning serious ivermectin toxicity and human abcb nonsense mutations a recommendation for the use of chloroquine, hydroxychloroquine, primaquine, or tafenoquine for prophylaxis against the novel coronavirus (covid- ) with note to the ophthalmic considerations tafenoquine inhibits replication of sars-cov- at pharmacologically relevant concentrations in vitro incidence and severity of covid- in hiv-positive persons receiving antiretroviral therapy -a cohort study the convalescent sera option for containing covid- effect of convalescent plasma therapy on time to clinical improvement in patients with severe and life-threatening covid- . a randomized clinical trial lessons learned from early compassionate use of convalescent plasma on critically ill patients with covid- potent neutralizing antibodies directed to multiple epitopes on sars-cov- spike antibody therapies could be a bridge to a coronavirus vaccine -but will the world benefit? universal use of face masks for success against covid- : evidence and implications for prevention policies use of facemasks during the covid- pandemic . doi: . /nejmp and the mechanisms of resistance development and diffusion in both hospitals and the community. the manuscript management system is completely online and includes a very quick and fair peerreview system, which is all easy to use key: cord- - t ce hb authors: baroutjian, amanda; sanchez, carol; boneva, dessy; mckenney, mark; elkbuli, adel title: sars-cov- pharmacologic therapies and their safety/effectiveness according to level of evidence date: - - journal: am j emerg med doi: . /j.ajem. . . sha: doc_id: cord_uid: t ce hb introduction: there is a pressing need for covid- transmission control and effective treatments. we aim to evaluate the safety and effectiveness of sars-cov- pharmacologic therapies as of august , according to study level of evidence. methods: pubmed, sciencedirect, cochrane library, jama network and pnas were searched. the following keywords were used: ((covid- ) or (sars-cov- )) and ((((((therapeutics) or (treatment)) or (vaccine)) or (hydroxychloroquine)) or (antiviral)) or (prognosis)). results included peer-reviewed studies published in english. results: peer-reviewed articles met study inclusion criteria, of which were rcts and one was a systematic review with meta-analysis. the following pharmacologic therapies were evaluated: chloroquine (cq), hydroxychloroquine (hcq), antivirals therapies, plasma therapy, anti-inflammatories, and a vaccine. conclusion: according to level evidence reviewed here, the most effective sars-co-v- pharmacologic treatments include remdesivir for mild to severe disease, and a triple regimen therapy consisting of lopinavir-ritonavir, ribavirin and interferon beta- b for mild to moderate disease. also, dexamethasone significantly reduced mortality in those requiring respiratory support. however, there is still a great need for detailed level evidence on pharmacologic therapies. introduction also showed that the high-dose group had a higher incidence of qt prolongation greater than ms. following an unplanned interim analysis of study findings due to cq dosages related safety concerns, the study independent data safety and monitoring board (dsmb) recommended the immediate interruption of the trial for patients on high dose cq from all age groups, unmasking, and converting all to low dose cq. they concluded that patients with severe covid- should not be given a high dose of cq especially with azithromycin and oseltamivir due to risk of qt prolongation and associated lethality. however, findings from patients with prolonged qt showed no clear association between the first day of prolonged qt and day of death, and that cumulative dosages were not higher among prolonged qt associated fatalities. in addition, it is important to be aware that this study had a small sample size, lacked of a placebo control group and used a historical control group. instead, findings were only adjusted by age, and pre-protocol analysis was not conducted due to inability to register daily untaken or mistaken cq doses because or renal or liver failures. an open label rct conducted on patients years and older with mild or moderate ongoing sars cov- investigated the effects of hydroxychloroquine (hcq) on negative conversion by days. patients were administered mg of hcq daily for three days followed by a maintenance dose of mg daily ( days if mild, days if moderate). results showed that those treated with standard care plus hcq had an . % probability of negative conversion by day ( % ci . - . ) , whereas those treated with standard care alone had an . % chance ( % ci . - . ). however, this difference was reportedly not significant. due to the trial ending early and only two patients (out of ) with severe disease being enrolled, results on clinical improvement were not presented. this study was limited by its underpowered sample size, non-computerized randomization protocol, and open label design. a more recent, multicenter, open-labeled controlled trial was conducted to assess the efficacy of hcq with and without azithromycin compared to the standard of care. the study was performed on patients with suspected or confirmed mild to moderate covid- with or fewer days since symptom onset. patients in the hcq group received a dose of mg twice daily for seven days. patients in the hcq plus j o u r n a l p r e -p r o o f azithromycin additionally received a dose of mg of azithromycin once daily for seven days. clinical status at days was evaluated using a -level ordinal scale. results showed no significant difference in the -level ordinal scale at days between those treated with hcq and standard care (or . , % ci . - . , p= . ), or between those treated with hcq + azithromycin and standard care (or . , % ci . - . , p= . ). there were also no significant differences in the number of days free from respiratory support, use of high-flow nasal cannula or non-invasive ventilation, use of mechanical ventilation, duration of hospital stay, in-hospital death, thromboembolic complications, or acute kidney injury between the groups. they also found that prolongation of qt interval was more frequent in the experimental groups (especially the hcq plus azithromycin group), and elevation of liver enzymes was more frequent in the hcq plus azithromycin group than the control group. limitations of this study include lack of blinding, concomitant treatment of patients with other pharmacologic agents, and the fact that some patients were previously treated with hcq ± azithromycin at other hospitals prior to enrollment in this trial. another rct was conducted to assess the efficacy of hcq as a post-exposure prophylaxis. participants were adults with household or occupational exposure to someone with laboratory confirmed covid- at a distance of less than ft for more than minutes without a face mask and/or eye shield. time from exposure to enrollment varied between - days in all participants. patients in the hcq group were administered mg hcq once, followed by mg in to hours, then mg daily for additional days for a total course of days. results showed that the incidence of new illness compatible with covid- did not significantly differ between participants receiving hcq ( . %) and placebo ( . %) (p= . ). also, there was no meaningful difference in the effectiveness according to the time of starting post-exposure prophylaxis. side effects were significantly more frequent in the hcq group by day (p< . ), with nausea, loose stools and abdominal discomfort being the most commonly reported side effects. no serious adverse reactions or cardiac arrhythmias were reported. this study is limited by its use of an a priori symptomatic case definition in some patients as opposed to diagnostic testing. j o u r n a l p r e -p r o o f ii. preliminary results of a double-blind randomized controlled trial by beigel et al. suggest that a -day course of remdesivir ( mg loading dose on day , followed by mg daily for up to additional days) is superior to placebo. this study, which was conducted in sites throughout the world, analyzed , patients and aimed to assess the effect of remdesivir on time to recovery, clinical improvement, and mortality in patients with varying baseline severity. those who received remdesivir had a statistically significant different median recovery time than placebo, days vs days (rate ratio for recovery, . ; % ci, . to . ; p< . ). the authors additionally stratified these results by disease severity, where the beneficial effects of remdesivir appeared to be more pronounced in the severe disease stratum. also, the remdesivir group had higher odds of improvement in the -level ordinal scale score at day compared to placebo (or . , % ci . - . ). although mortality was numerically lower in the remdesivir group, this difference was not statistically significant. patients on remdesivir who were receiving high-flow oxygen, mechanical ventilation, or extracorporeal membrane oxygenation did not achieve significant differences compared to placebo. another double blind, placebo-controlled, multicenter rct was conducted on the effectiveness of remdesivir in confirmed sars-cov- positive patients with severe covid- . patients were either assigned to receive intravenous remdesivir or placebo infusions. remdesivir was administered at mg on day followed by mg on days - . their primary outcome was time to clinical improvement within days after randomization. some patients were concomitantly treated with corticosteroids, lopinavir-ritonavir or interferons. intention to treat analysis revealed a non-significant decrease in the time to clinical improvement for the remdesivir group compared to placebo. survival at days and clinical improvement at and days were also not statistically significantly different, although numerically higher in the remdesivir group. serious adverse events occurred in % and % of the remdesivir and placebo groups respectively. intravenous remdesivir did not provide significant improvements in j o u r n a l p r e -p r o o f patients with severe covid- . this study was limited by insufficient power, the late initiation of therapy and absence of data on viral recovery. another rct evaluated the efficacy of remdesivir therapy after a -or -day regimen in patients with varying baseline clinical status. clinical status on day as measured by a -point ordinal scale was the primary endpoint. patients were administered mg of remdesivir on day , followed by mg once daily for the next or days. results showed that clinical improvement of points or more occurred in % of patients in the -day group and % of patients in the -day group. after correction of imbalance of baseline clinical status, clinical status at day was similar between the -day and -day groups (p= . ). it was concluded that there was no significant difference in efficacy between a -day or -day course. this study is limited by the fact that the patients in the -day group had a significantly worse clinical status than those in the -day group (p= . ), however the authors state that results were adjusted for this discrepancy. other limitations include lack of placebo and the open-label design. one clinical trial was conducted on -day triple medication protocols compared to -day lopinavirritonavir therapy alone. this open-label, randomized trial tested a triple medication regimen including interferon beta- b, lopinavir-ritonavir, and ribavirin. patients enrolled had mild to moderate covid- . the dosage for the experimental group was lopinavir mg and ritonavir mg every h, ribavirin mg every h, and three doses of million iu of interferon beta- b on alternate days. the control group received days of lopinavir mg and ritonavir mg every h. patients who were admitted to the clinical trial after the th day of experiencing symptoms were not treated with interferon beta- b due to its proinflammatory properties. their primary outcome measure was time to a negative rt-pcr assay by nasopharyngeal swab. the combination group had a significantly shorter median time to a negative rt-pcr than the control group. a negative sars-cov- was achieved in a median time of days in the experimental group vs in the control. additionally, clinical improvement was significantly better in the experimental group than the control with a median time to alleviation of symptoms of vs days. this study had an open-label design, absence of placebo group, and was also confounded by j o u r n a l p r e -p r o o f subgroup omitting of interferon beta- b within the combination group, depending on time from symptom onset. another randomized controlled open-label trial in hospitalized patients with confirmed sars-cov- with severe covid- was done to compare the clinical effectiveness of lopinavir-ritonavir to standard care alone. severe covid- was defined as sars-cov- positivity, pneumonia confirmed by chest imaging, and an oxygen saturation of % or less while breathing ambient air or a ratio of the partial pressure of oxygen to the fraction of inspired oxygen at or below mg hg. patients in the experimental group were treated with mg/ mg of lopinavir-ritonavir for days. the time to clinical improvement, mortality at day , and detectable viral load were not significantly different between groups. limitations of this study include a non-blinded protocol, higher baseline throat viral loads in the lopinavir-ritonavir group, and absence of data on lopinavir exposure levels in severe and critically ill patients. a more recent rct done on patients with mild to moderate covid- aimed to compare the difference in rate of positive-to-negative conversion of sars-cov- nucleic acid between lopinavir/ritonavir and arbidol (umifenovir). patients were administered either mg/ mg lopinavir/ritonavir po twice daily for - days, or mg of umifenovir po three times daily for - days. results showed no significant difference in the rate of positive-to-negative conversion between the lopinavir/ritonavir, arbidol, and control groups (p> . ). there was also no significant differences between the groups for the rates of antipyresis, cough alleviation, or improvement of ct findings at day or (p> . ). the lopinavir/ritonavir and arbidol groups experienced adverse effects; whereas the control group did not. limitations include small sample size, single center design, and lack of blinding to clinicians who recruited patients and research staff. iii. anti-inflammatory agents j o u r n a l p r e -p r o o f a multicenter, single-blind rct was conducted to assess the time to clinical improvement in patients with severe covid- treated with ruxolitinib, a jak inhibitor. time to clinical improvement was measured as time from randomization to an improvement of points on a -category ordinal scale, or live discharge from the hospital. patients in the experimental group received mg twice daily of ruxolitinib. results showed that ruxolitinib plus standard-of-care was associated with a non-statistically significant decrease in median time clinical improvement ( [iqr, [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] days vs. [iqr, - ] days). however, % of ruxolitinib patients had significant ct improvement at day compared to . % of control patients (p= . ), and levels of cytokines (including il- , il- and vegf) were significantly decreased in the experimental group, demonstrating the anti-inflammatory effects of ruxolitinib. also, the -day overall mortality was % in the experimental group and . % in the control group. this study is limited by its small sample size, use of an ordinal scale to assess primary end points, concomitantly treatment of some patients with other pharmacologic agents, and lack of inclusion of critically ill patients and patients with invasive ventilator dependence. a preliminary, open-label rct was conducted to assess the effect of dexamethasone on -day mortality in hospitalized patients with clinically suspected or laboratory confirmed sars-cov- infection. a randomized, double-blind, placebo-controlled trial was conducted to assess the effectiveness of an ad -vectored covid- vaccine. there were two experimental groups, one of which received a higher dose of viral particles ( x particles) and another that received a lower dose of viral particles ( x particles). participants who received either a low or high dose of viral particles had a significant increase in rbd-specific elisa antibodies, seroconversion rates, and neutralizing antibody responses compared to the placebo group. the placebo group showed no increase in antibody from baseline, and no ifnγ-elispot responses. severe adverse reactions occurred in % of the high dose patients and % of the low dose patients, although no serious adverse reactions were documented. it is important to note that % of participants had high pre-existing immunity, and % of the participants had low pre-existing immunity. the authors also did not calculate sample size based on study power in advance, and only reported data within days of vaccination. another rct sought to evaluate the effects of convalescent plasma therapy on the time to clinical improvement within days in patients with severe or life threatening covid- . according to the level evidence reviewed here, the most effective treatments against sars-cov- , measured by time to negative rt-pcr and time to clinical improvement, are remdesivir therapy and a triple medication regimen (lopinavir-ritonavir, ribavirin, and interferon beta- b). [ ] [ ] remdesivir showed beneficial effects in patients with varying baseline severity. it resulted in a decrease in mean recovery time, higher odds of improvement on an -level ordinal scale at day , and a non-statistically significant decrease in mortality in patients with mild to severe covid- . it also resulted in a nonstatistically significant reduction in time to clinical improvement in patients with severe covid- with no effect on mortality. one reason for not finding a significant effect of remdesivir in severe covid- patients could be insufficient power. remdesivir also appears to have some beneficial effects in severe j o u r n a l p r e -p r o o f covid- patients irrespective of the time to initiation of therapy. however, there was no difference in clinical improvement between a -day and -day course of remdesivir in patients with varying baseline clinical status. in patients with mild to moderate covid- , the triple medication regimen appeared to be most beneficial, as it resulted in a significantly shorter median time to negative rt-pcr compared to therapy with just lopinavir-ritonavir. evidence gathered from other rcts show several additional findings. first, in patients with severe covid- , treatment with lopinavir-ritonavir showed no significant difference in time to clinical improvement, mortality at day , or detectable viral load compared to standard care alone. also, treatment with lopinavir/ritonavir did not significantly affect the rate of positive-to-negative conversion when compared to arbidol in patients with mild to moderate covid- . second, mortality and qt prolongation was worse in severely ill patients taking high doses of cq compared to low doses. qt prolongation was also significantly higher in patients with mild to moderate covid- treated with hcq and hcq plus azithromycin. additionally, hcq showed no significant effect on the probability of negative conversion by day or virologic cure compared to standard care alone in patients with mild to moderate covid- . , it also did not reduce the prevalence of unfavorable secondary outcomes such as need for respiratory support, mechanical ventilation, or thromboembolic complications in patients with mild to moderate covid- . moreover, hcq did not reduce the incidence of new illness when used as a post-exposure prophylaxis. however, meta-analysis did reveal that hcq treatment resulted in fewer cases of radiological progression of lung damage. furthermore, treatment of severe covid- patients with ruxolitinib resulted in a non-statistically significant decrease in median time to clinical improvement, and a statistically significant decrease in levels of seven cytokines including il- , il- and vegf, indicating that it may be useful in treating cytokine storm. convalescent plasma was also efficacious in reducing the time to clinical improvement in severe and life threatening covid- . oral or intravenous dexamethasone was shown to significantly reduce mortality among hospitalized covid- patients j o u r n a l p r e -p r o o f receiving mechanical ventilation or oxygen without mechanical ventilation. finally, vaccination of healthy individuals using an ad -vectored covid- vaccine showed significant increase in immunity to sars-cov- by days. while we await higher quality evidence from randomized control trials and meta-analyses, these results provide some context on the efficacy of pharmacologic therapy in covid- patients. as of may , , the fda has granted emergency use authorization for intravenous remdesivir for severe covid- . however, they have revoked emergency use authorization for use of hydroxychloroquine and chloroquine due to their high risk to benefit ratio. covid- has undoubtedly posed a detrimental health burden worldwide. there is still a great need for detailed evidence on individual pharmacologic therapies. the findings from our review suggest that there is currently inconclusive evidence for one therapy. it is difficult to conduct studies on one category of pharmacologic treatment due to the lack of a universal systematic approach to treating covid- . in the absence of a vaccine available to the public, there is a great need for level evidence from randomized controlled trials and meta-analyses to support the development of evidence-based guidelines to treat covid- patients. aside from being novel, part of what makes treatment of sars-cov- difficult is its ability to affect multiple organ systems. the disease is characterized as an acute respiratory failure but may have systemic outcomes such as gastrointestinal, cardiovascular, and nervous system symptoms in addition to multi-organ failure. there has been evidence of high incidence of pulmonary embolism and thrombotic events. these severe cases often present with thrombocytopenia, elevated d-dimer levels, and pt prolongation. the hypercoagulable state often seen in covid- patients can be explained by the j o u r n a l p r e -p r o o f overwhelming production of inflammatory cytokines. this increase in inflammatory markers leads to an activation of the coagulation cascade and inhibits the fibrinolytic pathway. being over years old had a significantly larger impact on mortality than sex at birth and preexisting comorbidities. another important point to be discussed is the increase of non-evidence-based treatment and the unintended morbidity and mortality that results from it. there has been a large increase in the spread of false information and non-evidence-based remedies, such as consumption of cow urine and high proof alcohol that have resulted in illness and even death. furthermore, there has been a recent concern for patients who are using ace-inhibitors. a casepopulation study in spain on the admission rate of covid- patients on ace-inhibitors compared to other antihypertensive medications revealed that there was no increased risk of covid- related admission to a hospital, and concluded that ace-inhibitors not be discontinued. however, there remains a concern, especially among uninformed providers and patients, on whether use of ace-inhibitors pose a risk to patients during this pandemic. a similar review done by sanders et al. on pharmacologic treatment for covid- report similar findings regarding the available pharmacologic options and the inconclusive nature of the available data on these drugs. they additionally offer useful resources for clinical treatment guidance. in contrast, we have tailored our review to provide a more up to date, in-depth and systematic analysis using only level evidence. additionally, our discussion touches on the multisystem effects of sars-cov- . undoubtedly, it is of utmost importance to discuss the safety profile of all the medications included. many of these pharmacologic agents result in side effects ranging from mild to severe. first, hcq and cq have both been shown to cause cardiac electrical disturbances and cardiomyopathy. one clinical trial using a dose of mg twice daily for ten days was terminated early due to the death of patients as a result of arrhythmias by the th day. other adverse effects associated with hcq include retinopathy, gastrointestinal disturbances, and suicidal behavior. additionally, agents like hcq and cq can cause qt prolongation and their toxicity may be exacerbated when combined with other agents that also prolong the qt interval, such as azithromycin. patients who develop qt prolongation without torsades de pointes should be treated immediately by correcting oxygen, potassium, calcium, and magnesium concentrations. magnesium sulphate is recommended as the first-line therapy for torsades de pointes. cardiotoxicity has not been reported with remdesivir use. however, side effects of remdesivir include allergic reactions and increased liver enzymes. adverse effects associated with triple therapy j o u r n a l p r e -p r o o f using interferon beta b, lopinavir-ritonavir and ribavirin include diarrhea, nausea, and increased alanine transaminase levels, all of which stopped in one trial upon discontinuation of the drugs. additional side effect concerns with lopinavir-ritonavir include haptic injury, pancreatitis, acute gastritis, and qt prolongation. use of ruxolitinib in covid- patients showed a favorable side-effect profile of the drug according to the rct reviewed in this study. some of the adverse reactions included mild anemias, neutrocytopenia, thrombocytopenia, elevated liver enzyme levels, dizziness, rash, and nausea. there were no serious adverse events such as acute heart failure, shock, and sepsis. adverse reactions of dexamethasone were not evaluated in the rct included in this study; however, clinicians treating covid- patients with dexamethasone should monitor their patients for hyperglycemia, secondary infections, psychiatric effects and avascular necrosis. the ad -vectored vaccine also showed a favorable side-effect profile with most side effects being a result of the injection itself such as skin induration, redness, and swelling. the systemic side effects reported were headache, vomiting, diarrhea, joint pain, muscle pain, fatigue, headache, and cough. lastly, adverse effects associated with the convalescent plasma trial included dyspnea, fever, and an allergic reaction caused by transfusion. these findings lead us to recommend that physicians follow updated guidelines from reputable sources. the society of surgical oncology also offers frequently updated resources to assist physicians in treating particularly vulnerable patients with cancer. currently, there is also a great deal of randomized clinical trials that are ongoing and should provide the medical community with more conclusive evidence in the near future. according to the nih, there are , active studies on covid- as of august , . the studies included in this review had several limitations. first, there was an issue of small sample size for several studies. , [ ] [ ] another limitation to the findings is the inability to generalize them to all patients as a result of specific exclusion criteria such as individuals with mild or severe disease. [ ] [ ] [ ] , [ ] [ ] [ ] [ ] also, several studies reviewed above aimed to focus on the efficacy and safety of one drug, but employed multiple drugs in the treatment of patients. , , these limitations make it difficult to compare j o u r n a l p r e -p r o o f journal pre-proof the efficacy and safety profiles of the drugs being used. the findings listed are dependent on the accuracy and validity of data used to assess sars-cov- pharmacological therapies. lastly, given the rapidly evolving nature of this pandemic, it is difficult to ensure that all the existing evidence has been included up until this article's publication date and new information and trials will arriving. there remains uncertainty regarding the safest and most effective pharmacologic therapy for covid- disease. however, the findings from this review conclude that, according to level evidence, remdesivir therapy in mild to severe disease, and the triple medication regimen (lopinavir-ritonavir, ribavirin and interferon beta- b) in mild to moderate disease are the most efficacious against sars-cov- in terms of symptom improvement and time to a negative rt-pcr. also, dexamethasone was significantly able to reduce mortality in patients receiving respiratory support. we recommend that physicians remain informed on up to date evidence such as preliminary data from rcts, and work with their institution and scientific societies in developing evidence-based systematic guidelines in the treatment of covid- patients. j o u r n a l p r e -p r o o f hospitalized patients with clinical suspicion of covid- , aged years or older, with respiratory rate higher than rpm and/or heart rate higher than bpm (in the absence of fever) and/or peripheral oxygen saturation lower than % in ambient air and/or shock. participants who received either a low or high viral particles dose had a significant increase in rbd-specific elisa antibodies, seroconversion rates and neutralizing antibody responses compared to the placebo group. placebo group showed no antibody increase from baseline. no ifnγ-elispot responses in placebo group. points on a -point disease severity scale, or discharge. clinical improvement occurred at a higher rate in those with severe disease compared to those with life threatening disease ( . % vs . %). there was no significant difference in day mortality (or . , % ci . - 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- journal: cureus doi: . /cureus. sha: doc_id: cord_uid: lb jcl m the severe acute respiratory syndrome coronavirus (sars-cov- ) pandemic and its high virulence along with its variable presentation have generated a significant amount of interest within the medical community. the heterogeneous nature of the symptoms of the disease caused by sars-cov- , coronavirus disease (covid- ), ranging from being asymptomatic to severe acute respiratory distress syndrome (ards), has created significant interest in potential therapeutics. given the lack of randomized controlled trials, most medications are experimental, and only anecdotal evidence is available so far regarding their efficacy. one medication that emerged as an early frontrunner as a promising therapeutic was hydroxychloroquine (hcq), a common antimalarial and lupus drug. the adverse side effects that could result from its use did not gain much attention initially. we present the cases of two covid- -positive patients treated with hcq at our institution, which showed adverse effects of the medication. while hcq may have some therapeutic effect, it should be borne in mind that patients may experience more harm than benefit from its use. the ongoing coronavirus disease (covid- ) pandemic has led to global panic regarding its highly infectious process. in the race to find novel treatment strategies for covid- , numerous pharmacological agents are being touted as the silver bullet. the unfortunate reality of the covid- pandemic is that, as providers, we are dealing with a highly virulent, variable, and potentially aggressive pathogen as evidenced by a reported r-naught (r ) of . [ ] . hydroxychloroquine (hcq), a common antimalarial and lupus drug, has been shown to potentially reduce viral carriage and the number of symptomatic days in covid- patients according to an open-label non-randomized french case study of patients [ ] . however, the findings of a subsequent randomized controlled trial have led to the fda revoking hcq's emergency use authorization [ ] . the purpose of this case series was to highlight some of the cardiovascular complications related to hcq and to engage in a risk-benefit analysis of its use in mild/moderate presentations of covid- . a -year-old female with a past medical history of coronary artery disease (cad), chronic obstructive pulmonary disease, and autoimmune hepatitis on tacrolimus presented with a sixday history of fatigue, dry cough, shortness of breath, chest tightness, nausea, vomiting, and diarrhea. her vitals were stable and the exam demonstrated bilateral decreased breath sounds and wheezing on admission. she was admitted initially for possible community-acquired pneumonia and started on empiric coverage of ceftriaxone and azithromycin (azm). ondansetron was also started due to her nausea. on day two of hospitalization, the patient's condition improved symptomatically, but she was subsequently found to be covid- -and influenza-b positive. hcq and tamiflu® (roche pharmaceuticals, basel, switzerland) were started while azm was continued. labs demonstrated troponin of . ng/ml (three sets), potassium (k) level of . meq/l, magnesium (mg) level of . meq/l, and white blood cell (wbc) count of . thousand/mm at the time of initiation. on the morning of admission, the patient was found to have a qtc of ms ( figure a) . a chest x-ray demonstrated a left perihilar infiltrate ( figure b) . echocardiogram demonstrated left ventricular ejection fraction of % and no diastolic dysfunction. clostridium difficile testing was negative. a repeat ekg on day five of hospitalization demonstrated prolongation of qtc to ms, and several premature ventricular contractions with r-on-t waves were noted ( figure c ). she was asymptomatic at this time. hcq, ondansetron, and azm were subsequently discontinued. furthermore, her electrolytes were optimized, specifically with mg of > . meq/l and k of > . meq/l. by hospital day nine, the patient stated that her symptoms had improved. her qtc was noted to be ms on ekg on the same day ( figure d ). she made an uneventful recovery and was discharged on hospital day . this patient had a moderate presentation of covid- infection. her case was complicated by co-infection with influenza b, radiographic evidence of perihilar infiltrate, and leukopenia. given these combined findings, the initial benefit of hcq seemed superior to the risk given the fear of rapid progression to severe acute respiratory distress syndrome (ards). however, after a day of supportive therapy, the patient's condition dramatically improved. it is important to note that her tisdale score was after hcq, ondansetron, and azm administration (high risk for qt prolongation) [ ] . she was also having premature ventricular contractions (pvcs) throughout her hospitalization. especially worrisome were r-on-t waves noted on her telemetry, which could be a forewarning for impending ventricular fibrillation, especially in the setting of acquired long qt and her history of cad [ ] . in this case, the patient's hospital course was clearly complicated by hcq in addition to the many qtc-prolonging agents she was prescribed; hence, the overall benefit seemed marginal. a -year-old female with no significant medical history presented with a one-day history of nausea, vomiting, and diarrhea. she stated that she had recently returned from scotland and had begun experiencing symptoms of rhinorrhea starting in late january . she was not tested immediately for covid- due to a lack of fever. she was given a steroid dose-pack and a five-day course of azm at that time. she improved initially but later relapsed one week prior to admission when she developed symptoms of shortness of breath and mild wheezing. figure ) noted marked sinus bradycardia with a ventricular rate of bpm. the patient had no prior history of arrhythmias. it was believed that her symptoms of sudden diarrhea, nausea, and vomiting were secondary to hcq as was the bradycardia. the medication was discontinued, and her symptoms resolved; her heart rate improved to bpm, and she was uneventfully discharged on no antimicrobial medication. this case showed a mild incidence of covid- in a patient who was at low risk for qt prolongation given a tisdale score of . given the low risk of adverse effects, it initially appeared reasonable that the patient's primary care provider had started her on hcq treatment. the marked sinus bradycardia was a worrisome finding that thankfully resolved with discontinuation of the medication. it should be noted that the patient's baseline ekg was unknown. providers should consider obtaining a baseline ekg, renal panel, and hepatic panel prior to the administration of hcq in these patients. this case illustrates the fact that hcq provided little benefit for such a mild presentation of covid- as in this patient. while hcq was demonstrated to be effective in the french study by gautret et al., it is not a proven treatment modality and it should be used with caution. the above two cases add to the ongoing discussion as to whether hcq therapy in covid- patients is beneficial for all, especially given how it was paired with another arrhythmogenic agent azm. we believe these are among the first few cases illustrating adverse cardiovascular effects of the experimental five-day hcq therapy in mild/moderate presentations of covid- . while the safety profile on hcq is relatively favorable, the drug is a well-known arrhythmogenic medication that can lead to life-threatening ventricular arrhythmias, most commonly recognized as torsades de pointes (tdp) [ ] . admittedly, the relationship between qtc prolongation and tdp is not linear; nevertheless, clinicians are well aware of the risks of prolonged qtc. the tisdale risk stratification scoring system was invented for predicting the risk of qt prolongation [ ] . as shown in table with respect to our patients, the tisdale score characterizes patients as low risk (score< ), moderate risk (score - ), and high risk (score > ). case , which was considered as high risk, involved a patient with potentially lethal asymptomatic prolongation of the qtc segment. additionally, the r-on-t phenomenon noted on telemetry was worrisome as this could have potentiated a polymorphic ventricular tachycardia (pvt) or even ventricular fibrillation [ ] . these were clear complications of the hcq and, subsequently, prolonged the patient's hospital stay. case , considered as low risk, demonstrated how hcq therapy initiated in an outpatient resulted in an adverse outcome that led to hospital admission. the bradycardia was thankfully recognized early as this could have progressed to potential atrioventricular block had the patient continued the medication [ ] . both of these cases throw light on hcq-related complications resulting in prolonged hospital stay/hospitalization that exposed these patients to potential hospital-acquired infections or even re-infection with covid- , which has only been sporadically reported [ ] . age ≥ years the american college of cardiology (acc) has issued guidance on how to proceed in patients who could potentially receive hcq for covid- therapy. in the outpatient setting, a baseline assessment with ekg, renal function panel, and hepatic function panel should be completed and, if possible, the ekg should be assessed by an electrophysiologist [ ] . furthermore, other qtc-prolonging agents should be discontinued if possible [ ] . relative contraindications in these patients would include: ) history of long qt syndrome, ) qtc of > ms, and ) tisdale score of ≥ [ ] . for inpatients, the recommendations include the same as above plus: ) placing patients on telemetry ) obtaining serum potassium on a daily basis, and ) obtaining ekg two to three hours after the second dose of hcq. guidance on qtc increases of > ms or overall qtc of > ms seems to point towards the discontinuation of azm and subsequent dose decrease of hcq. if qtc does not improve, the acc recommends complete discontinuation of hcq [ ] . the emergence of new data regarding hcq's application in the early disease course of covid- has called its use into question. in the outcomes related to covid- treated with hydroxychloroquine among in-patients with symptomatic disease (orchid) trial conducted by the petal network, patients were enrolled in a double-blinded, placebo-controlled randomized trial to determine if five-day therapy with hcq led to more favorable outcomes [ ] . the dosing regimen included hcq sulfate mg po twice daily on day one followed by mg twice daily doses on days two to five [ ] . the initial results from the trial suggested that there was no benefit or harm from hcq use, and the trial was subsequently halted in mid-june [ ] . the data is still awaiting full analysis for submission to peer review [ ] . in june , the fda officially revoked emergency use authorization for hcq as there was no benefit demonstrated from the aforementioned trial [ ] . moreover, the agency has cautioned against its use in the outpatient setting given its potential cardiovascular complications and inability to closely monitor patients [ ] . the trial data and fda recommendations in their totality demonstrate that hcq use likely is not beneficial in the early disease course of these patients. regarding the lack of harm shown by the orchid trial, more subgroup analysis is warranted in these patients to fully determine if patients with cardiovascular disease burden suffered harm, despite the absence of mortality among patients. the potential for arrhythmogenic effects of these medications, especially in patients with cardiovascular disease, should be seriously weighed against their benefit before administration. based on the recent preliminary analysis of data from the orchid trial, there is no benefit from hcq use. if hcq is commenced by providers for the treatment of covid- in patients with a cardiac history, it should be done at the guidance of an infectious disease physician in conjunction with a cardiologist. while the prospect of potential lifesaving therapeutics seems tempting, the long-standing principle of "do no harm" is of importance now more than ever. experimental therapy with hcq in mild/moderate presentations of covid- should be balanced with considerations of the risk of potential cardiac complications in both inpatient and outpatient settings. this is especially the case as there have been no double-blinded randomized control trials with results demonstrating that the benefits of the therapy outweigh the risks. disclosures high contagiousness and rapid spread of severe acute respiratory syndrome coronavirus . emerg infect dis hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial (epub ahead of print) fda cautions against use of hydroxychloroquine or chloroquine for covid- outside of the hospital setting or a clinical trial due to risk of heart rhythm problems development and validation of a risk score to predict qt interval prolongation in hospitalized patients r-from-t as a common mechanism of arrhythmia who malaria policy advisory committee meeting: the cardiotoxicity of antimalarials heart conduction disorders related to antimalarials toxicity: an analysis of electrocardiograms in patients treated with hydroxychloroquine for connective tissue diseases recurrence of positive sars-cov- rna in covid- : a case report ventricular arrhythmia risk due to hydroxychloroquineazithromycin treatment for covid- outcomes related to covid- treated with hydroxychloroquine among in-patients with symptomatic disease (orchid) orchid: outcomes related to covid- treated with hydroxychloroquine among in-patients with symptomatic disease human subjects: consent was obtained by all participants in this study. hca institutional review board issued approval - . this research activity was determined to be exempt or excluded from the institutional review board (irb) oversight in accordance with current regulations and institutional policy. the internal reference number for this determination is - . conflicts of interest: in compliance with the icmje uniform disclosure form, all authors declare the following: payment/services info: all authors have declared that no financial support was received from any organization for the submitted work. financial relationships: all authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. other relationships: all authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work. key: cord- - jpdvy n authors: annangi, srinadh title: chloroquine and hydroxychloroquine for covid‐ : a word of caution date: - - journal: respirology doi: . /resp. sha: doc_id: cord_uid: jpdvy n nan the novel coronavirus, severe acute respiratory syndrome coronavirus- (sars-cov- ), is the causative pathogen of the coronavirus disease (covid- ) pandemic. there is interest in recommending chloroquine (cq) or hydroxychloroquine (hcq) for treatment and prophylaxis, as they are readily available and affordable. shah et al. have recently reviewed the current literature on the use of cq and hcq as prophylaxis against covid- . prior research including with human immunodeficiency virus (hiv) and sars-cov- has shown that in vitro antiviral activity of cq and hcq does not necessarily translate to in vivo clinical efficacy. however, the % genomic similarity between sars-cov- and - , along with their similar receptorbinding domain structures, has stimulated interest in cq and hcq. yao et al., in their antiviral pre-treatment activity analysis, reported ec ( % maximal effective concentration) for cq and hcq at h as > and . μm, respectively, much higher compared to values from their treatment analysis arm. it is unclear how the differences in the ec values will impact the in vivo prophylactic antiviral efficacy at their recommended doses of hcq mg twice daily (bd) on day and then mg bd for days. ongoing prophylaxis clinical trials in the united states are using doses ranging from to mg daily or weekly, with or without a loading dose for a total duration of days to months. the results of these studies will only answer the effectiveness, if any, of these dosing regimens. analysis of cytotoxic effects is based on cck assays, an indirect colorimetric assay evaluating the number of viable cells by measuring their metabolic activity. however, some of the side effects of cq and hcq are based on their inherent pharmacological properties rather than their direct cytotoxic effect. in a recent unpublished study by chorin et al. from new york evaluating hospitalized sars-cov- -infected patients receiving hcq + azithromycin, qtc (corrected qt interval) prolongation by > ms was noted in % of subjects and % had a prolongation to > ms. baseline qtc did not predict the risk for qtc prolongation. cardiac injury, renal impairment, concomitant use of drugs that prolong qtc and electrolyte imbalance will further increase the risk of fatal ventricular arrhythmias. anecdotal reports of death from self-administered cq phosphate were reported. although retinal toxicity is rare at currently recommended doses, risk increases among patients with renal or hepatic impairment with decreased drug elimination. in a recent mice study, using a combination of cq and hcq ( mg/kg daily for weeks) with metformin ( mg/kg daily for weeks) resulted in - % lethality. the safety profile of this combination at the much lesser doses currently being recommended for prophylaxis is yet to be established, and caution should be excised by prescribers until then. potential overdose can also happen in obese patients if the dose is calculated using total body weight instead of ideal body weight as these drugs are not retained in the fatty tissue. published studies to date have not reported prophylactic efficacy of these compounds. even though gautret et al. supported the treatment efficacy, it was extensively criticized for study design limitations. some subjects attained virologic cure as early as day ; however, these patients might have attained virologic cure irrespective of treatment and attributing this to the antiviral efficacy of hcq is not appropriate. chen et al., in their analysis, did not report virologic cure. the hippocratic oath of 'first do no harm' should drive our instincts in these uncertain times. authorities should make recommendations based on clinical evidence and not speculations. there is no conclusive evidence to support these drugs either for treatment or prophylaxis. even if the results from ongoing trials are more conclusive, ideal dosing regimens should be evaluated to avoid unwarranted overdosing. until then, cq and hcq should only be used in the setting of clinical trials with a clear understanding among physicians and patients of uncertain efficacy and potentially harmful side effects. a systematic review of the prophylactic role of chloroquine and hydroxychloroquine in coronavirus disease- (covid- ) genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus dis. . . pii: ciaa the qt interval in patients with sars-cov- infection treated with hydroxychloroquine/ azithromycin fatal toxicity of chloroquine or hydroxychloroquine with metformin in mice dosage of antimalarial drugs for children with juvenile rheumatoid arthritis and systemic lupus erythematosus: a clinical study with determination of serum concentrations of chloroquine and hydroxychloroquine hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial efficacy of hydroxychloroquine in patients with covid- : results of a randomized clinical trial key: cord- -m ywt authors: leung, alexander kc; mcmillan, tara; human, andrea; lam, joseph m title: hydroxychloroquine-induced hyperpigmentation in a -year-old female with systemic lupus erythematosus date: - - journal: drugs context doi: . /dic. - - sha: doc_id: cord_uid: m ywt hydroxychloroquine (hcq)-induced hyperpigmentation is uncommon but is increasingly recognized. to our knowledge, hcq-induced hyperpigmentation has not been reported in the pediatric age group. herein, we present the case of a -year-old girl with systemic lupus erythematosus, who developed hyperpigmentation on her shins and dorsum of the left foot, approximately years after initiating treatment with hcq. physicians who treat children with hcq for reasons such as rheumatologic disorders, dermatologic disorders and, more recently, coronavirus disease- should be aware of this less-known side effect of hcq. chloroquine-induced hyperpigmentation is well known. on the other hand, hydroxychloroquine (hcq)-induced hyperpigmentation is an uncommon but increasingly recognized side effect of hcq therapy. the hyperpigmentation may develop from a few months to a few years following the initiation of hcq therapy. , to our knowledge, hcq-induced hyperpigmentation has not been reported in the pediatric age group. herein, we report on the case of a -year-old girl with systemic lupus erythematosus (sle), who developed hyperpigmentation on her shins and dorsum of the left foot after treatment with hcq for years. no review board approval was necessary and was therefore not obtained. signed consent was obtained. a - year-old indigenous canadian female with sle presented with a -year history of asymptomatic hyperpigmentation on the bilateral shins and the dorsum of the left foot. the hyperpigmented patches first appeared over the bilateral shins and the dorsum of the left foot in areas of previous bruising. there was no history of trauma. the patient's symptoms began at the age of years, when she presented with polyarthritis, low serum c and c levels, positive antinuclear antibodies, anti-double-stranded dna antibodies, and anti-smith antibodies. she was seen by a pediatric rheumatologist who diagnosed her with sle based on the clinical and laboratory findings. the patient was treated with oral methotrexate, mg, weekly and oral hcq at a dose of mg per week ( mg daily on weekdays and mg daily on weekends, averaging . mg/kg/day). during the course of her illness, she was also treated with cyclophosphamide, corticosteroids, rituximab, and mycophenolate mofetil at various points in time. she did not have a known coagulopathy and was not on anticoagulation therapy. physical examination revealed fitzpatrick type iv skin with symmetrical, bluish-grey patches bilaterally on the pretibial areas and dorsum of the left foot ( figure ). there was no pigmentation at other body sites including the oral mucosa and the nails. a clinical diagnosis of hcq-induced hyperpigmentation was made based on the typical appearance of symmetrical, bluish-grey patches bilaterally on both shins. the hcq was discontinued. however, the patient expired from complications of lupus prior to the observance of any effect. issn: - case report -hydroxychloroquine-induced hyperpigmentation drugsincontext.com hcq was first developed as an antimalarial agent, and is now also used in the treatment of sle, juvenile idiopathic arthritis, rheumatoid arthritis, sjogren syndrome, dermatomyositis, actinic lichen planus, oral lichen planus, and sarcoidosis owing to its anti-inflammatory and immune-modulating properties. , [ ] [ ] [ ] [ ] recently, hcq has also been used in the treatment of coronavirus disease- (covid- ). - hcq has also been suggested as a candidate prophylactic agent for covid- in populations at high risk for covid- , such as high-risk groups in new york and italy. hcq is preferred to chloroquine because it is less toxic. for the treatment of covid- , hcq works by blocking severe acute respiratory syndrome-related coronavirus (the causative virus for covid- ) viral entry into host cells through inhibition of angiotensin-converting enzyme receptor glycosylation, reducing viral replication, and blocking the export of newly constructed virions. [ ] [ ] [ ] hcq has a relatively favorable safety profile, is generally well tolerated, and is readily available at relatively low cost. hcq-induced hyperpigmentation of the skin is increasingly recognized as a side effect of hcq therapy. although infrequently reported in the literature, a recent cross-sectional study found that % of patients on hcq developed hcq-induced hyperpigmentation. in this regard, hyperpigmentation associated with chloroquine therapy is more common than that of hcq. , typically, hcq-induced hyperpigmentation presents as bluish, blue-grey macules/ patches most commonly on the shins, but can also been seen on the arms, forearms, face, oral mucosa, trunk, nails, and axilla. , , , there is no apparent predilection for sunexposed areas and oral mucocutaneous hyperpigmentation also occurs. , the involvement is typically bilateral, although unilateral involvement has rarely been reported. the differential diagnosis includes erythema nodosum, ecchymosis, minocycline dyspigmentation, and erysipelas. the color, chronicity, and associated hcq use help to distinguish hcq-induced hyperpigmentation from these other disorders. there are no formal criteria for hcq-induced hyperpigmentation as the diagnosis is usually clinically apparent. a skin biopsy is often not required but can help confirm the diagnosis. our patient presented with symmetrical, poorly defined, grey patches bilaterally on the shins and the dorsum of the left foot. cutaneous pigmentation occurs in - % of patients after a few months to a few years of treatment with hcq. , , , in one study of patients treated with hcq, pigmentation appeared after a median duration of hcq treatment of months (range - months). there is no clear association with duration of treatment or cumulative dose of hcq. in the present case, the child developed hyperpigmentation on the shins and dorsum of the right foot approximately years after initiating hcq therapy. to our knowledge, hcq-induced hyperpigmentation of the skin has not been reported in children. as such, our patient represents the first case reported in the pediatric age group. hcq-induced hyperpigmentation usually resolves within months after hcq has been discontinued , , , but, occasionally, the hyperpigmentation may persist. if possible, hcq should be substituted with another medication that has similar therapeutic effects. the use of a q-switched -nm alexandrite laser may be considered for the treatment of persistent hyperpigmentation. the exact pathogenesis for the development of hcq-induced hyperpigmentation is not known. a strong association has been found with preceding ecchymosis or bruising, which suggests that the mechanism may be, at least in part, due to localized trauma. other predisposing factors include the use of oral anticoagulants or antiplatelet agents, long-term use of corticosteroids, and antiphospholipid syndrome. , , , , hcqinduced damage of dermal vessels with leakage of erythrocytes is another possibility. histopathological examination of the hyperpigmented lesion shows yellow-brown granules within macrophages in the dermis, melanin in reticular dermis, and perivascular iron/hemosiderin on special staining. , hcq can accumulate within the skin and has a strong binding affinity to melanin. , , it has been suggested that the hyperproduction of melanin by epidermal melanocytes can be under the direct influence of hcq. hcq-induced hyperpigmentation is more other hcq-induced cutaneous manifestations include xerosis, pruritis, nail hyperpigmentation, hair discoloration, alopecia, urticarial and lichenoid skin rash, toxic epidermal necrolysis, stevens-johnson syndrome, and exacerbation of pre-existing psoriasis. , , non-cutaneous adverse effects of hcq include nausea, diarrhea, prolongation of qt-interval, corneal lens opacity, retinopathy, ciliary body dysfunction, posterior subcapsular lens opacity, myopathy, and ochronosis. , , , some authors suggest that hcqinduced hyperpigmentation is a marker of increased risk of retinopathy. , , conclusion hcq-induced hyperpigmentation of the skin is increasingly recognized as a side effect of hcq therapy. typically, hcqinduced hyperpigmentation presents as bluish, blue-grey macules/patches most commonly on the shins. to our knowledge, the condition has not been described in the pediatric age group. hcq has been used widely in the treatment of a variety of rheumatologic and dermatologic conditions and, recently, by some physicians in the prophylaxis and treatment of covid- . physicians who use hcq in children should be aware of this less-known side effect of hcq so that an accurate diagnosis can be made, and unnecessary investigations and inappropriate treatment can be avoided. confirms that this article has no conflicts of interest otherwise. this manuscript was sent out for independent peer review by the editor-in-chief. all other authors declare that they have no conflicts of interest relevant to this manuscript. the international committee of medical journal editors (icmje) potential conflicts of interests form for the authors is available for download at: https://www.drugsincontext.com/wp-content/uploads/ / /dic. - - -coi.pdf hydroxychloroquine-induced hyperpigmentation hydroxychloroquine-induced hyperpigmentation in systemic diseases: prevalence, clinical features and risk factors: a cross-sectional study of cases woman in grey: hydroxychloroquine-induced hyperpigmentation hydroxychloroquine-induced hyperpigmentation: a case report chloroquine and hydroxychloroquine toxicity hydroxychloroquine induced cutaneous pigmentation: a unique pattern hydroxychloroquine sulphate therapy of erosive oral lichen planus breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies diagnosis, treatment, and prevention of novel coronavirus infection in children: experts' consensus statement coronavirus disease (covid- ): latest developments in 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myopathy a case of exogenous ochronosis associated with hydroxychloroquine none. key: cord- -hidb q u authors: karatza, eleni; ismailos, george; marangos, markos; karalis, vangelis title: optimization of hydroxychloroquine dosing scheme based on covid- patients’ characteristics: a review of the literature and simulations date: - - journal: xenobiotica; the fate of foreign compounds in biological systems doi: . / . . sha: doc_id: cord_uid: hidb q u . during the recent covid- outbreak hydroxychloroquine (hcq) has been proposed as a safe and effective therapeutic option. however, a wide variety of dosing schemes has been applied in the clinical practice and tested in clinical studies. . an extended literature survey was performed investigating the pharmacokinetics, the efficacy and safety of hcq in covid- treatment. population pharmacokinetic models were retrieved from the literature and after evaluation and assessment one was selected in order to perform simulations. . the most commonly applied dosing schemes were explored for patients with different weights and different levels of hcq clearance impairment. model-based simulations of hcq concentrations revealed that high initial doses followed by low and sparse doses may offer significant benefits to patients by decreasing the viral load without reaching levels considered to produce adverse effects. for instance, the dosing scheme proposed for a kg adult with moderate covid- symptoms would be mg upon diagnosis, mg after h, mg after h, mg after h, followed by mg bid for d, followed by mg od for d. . based on the results from simulations performed and the currently published knowledge regarding hcq in covid- treatment, this study provides evidence that a high loading dose followed by sparse doses could offer significant benefits to the patients. during the recently emerged pandemic of coronavirus disease due to sars-cov- , hydroxychloroquine (hcq) has been proposed as a drug of choice (alpern & gertner, ) . covid- severity has been categorized based on symptoms by the who in mild, moderate and severe, while a life-threatening state of critical disease has been noted, as well (who, ) . most interestingly, it has been shown that mortality, as well as disease severity and immune response are closely related to the viral load of the patient (casadevall et al., ; liu et al., a; pujadas et al., ; wang et al., ; zheng et al., ) . up to this point, hcq has been used for the treatment of malaria or autoimmune diseases such as rheumatoid arthritis, discoid and systemic lupus erythematosus (plaquenil label, ; plaquenil smpc, ) . however, hcq seems to offer a dual beneficial action for covid- patients, as it exerts both immunomodulatory and antiviral effects (dro_ zd_ zal et al., ) . in fact, some recent studies have revealed the drug's in vitro antiviral activity paho/who, ; yao et al., ) . a wide variety of observational and randomized controlled trials (rct) worldwide are currently evaluating the efficacy of hcq for the treatment of covid- , implementing many different dosing schemes and patients with different levels of disease severity according to the who, ( ) categorization (alpern & gertner, ; cortegiani et al., ; paho/who, ) . several studies have yielded positive results, indicating that hcq may accelerate the alleviation of symptoms and diminish the length of hospital stay (arshad et al ; cavalcanti et al., ; chen et al., a; gautret et al. a,b; tang et al ; yu et al., ) , while others yielded negative results, indicating that hcq does not show any clinical effects in improving symptoms or was associated with increased length of hospital stay or disease progression (barbosa et al., ; chen et al., b; horby et al., ; molina et al ) . it has to be noted, that in many cases these studies report adverse effects promoted by hcq, such as prolongation of the qtinterval or diarrhea (gautret et al b; horby et al. ; tang et al., ) . in view of the high heterogeneity of the so far study outcomes, the who recommends not to use hcq outside the context of clinical trials (who, ) . in any case, due to the lack of a better option, hcq is currently included in the therapeutic protocols designed by numerous hospitals and health systems worldwide, for the treatment of covid- patients . in addition, the fda that initially approved the use of hcq for covid- (alpern & gertner, ) has recently revoked the emergency off-label use of this drug (fda, ) . it should be noted that currently there is not substantial evidence to prove the safety and efficacy of hcq in the treatment of covid- , and thus results from well-designed randomized trials are required for this drug's repurposing (elavarasi et al., ; singh-uttam et al., ) . indicatively, some dosing schemes that have been applied or evaluated in clinical studies are presented in table . considering all the above and based on the reported antiviral activity of the compound (dro_ zd_ zal et al., ; liu et al., b; yao et al., ) , there is currently a need to rationalize the administered dosing schemes in order to maximize the efficacy and safety of hcq in patients with covid- . eventually, additional time is needed in order to retrieve concrete results from new in vitro and in vivo studies specifically designed for hcq against covid- . however, in view of its long-standing clinical use a lot of data and experience has been accumulated for this drug. in this vein, modeling and simulation techniques have been proved very useful as they can combine all the available data from in vitro, preclinical and clinical studies (lav e et al., ; lowe et al., ) . thus, by implementing all the relevant knowledge that has been gathered so far and using modeling and simulation techniques it is possible to attain a better approximation to the optimum dosing scheme. the aim of the present study was firstly, to review all the relevant to hcq literature, focusing on covid- treatment and secondly, through simulations, to investigate the efficacy and safety of the dosing schemes of hcq for covid- treatment currently applied and to propose optimized dosing schemes. after oral administration hcq is rapidly and almost completely absorbed with bioavailability ranging from . to . . mean peak plasma concentrations (cmax) after a single dose of mg was approximately ng/ml, and mean time to peak plasma concentration (tmax) was . h. hcq was found to present linear pharmacokinetics in a therapeutic dose range (furst, ; plaquenil smpc, ) . hcq binds avidly to tissues, leading to large volumes of distribution, significant accumulation of the drug and low clearance. in fact, the increased amount of time needed for this accumulation to occur, accounts for the delayed appearance of its clinical effects but also of its adverse effects. approximately - % of the administered dose is bound to proteins, both albumin and alpha glycoprotein. hcq is metabolized by the liver with its main metabolite (desethylhydroxychloroquine) presenting some immunomodulatory activity (furst, ; munster et al., ) . the drug is primarily eliminated via the urine, where about % of the administered dose is recovered in h. following a single mg oral dose the half-life of hcq was found to be h ( . d) (plaquenil label, ; plaquenil smpc, ) . blood levels were found to vary significantly among individuals, with a variation in mean maximum/minimum concentration reaching %. this variability has been attributed barbosa et al., to the absorption and distribution rather than clearance characteristics of the drug (al-rawi et al., ; miller et al., ) . numerous factors have been shown to affect blood levels of hcq, such as genetic variants of the cyp enzyme family, age, gender, body weight, gastric emptying, and co-administration with immunosuppressants, such as corticosteroids (al-rawi et al., ; lee et al., lee et al., , . even though it has been reported that renal clearance of unchanged drug was approximately to % and did not correlate with creatinine clearance (miller et al., ) , renal insufficiency has been reported to increase the risk of toxicity (abdulaziz et al., ) . a total of eight population pharmacokinetic studies of hcq were identified in the literature (balevic et al., ; carmichael et al., ; haas et al., ; lim et al ; morita et al., ; rangwala et al., ; rosenfeld et al., ; vogl et al., ) . population parameter estimates and characteristics of the studies are presented in table . in the majority of studies assessed, samples were retrieved from patients suffering from severe diseases and receiving co-medications especially immunosuppressants that have been reported to alter hcq pharmacokinetics (lee et al., ) . in addition, given that hcq binds avidly to tissues, a fact that results in high volumes of distribution and slow clearance (furst, ) , a model with two compartments, a central one for plasma and tissues with an instantaneous distribution and a peripheral one standing for tissues where the drug distribution is slower seemed to be more appropriate for the description of hcq disposition. in this vein, the model of lim et al. ( ) was developed with samples retrieved from healthy volunteers and patients with vivax malaria that did not receive any other medications and resulted in a two compartmental model. also, the study of lim et al. ( ) that included the highest number of samples, i.e., a total of concentration measurements, from all the studies identified resulting in a two compartmental model, a fact that increases the reliability of the parameter estimates. in view of the above, the model developed by lim et al., ( ) was selected in order to perform the simulations. parameters estimated in this study, were similar to those obtained with other population pharmacokinetic models developed (rangwala et al., ; rosenfeld et al., ) , as well as to those retrieved after non-compartmental pharmacokinetic analysis (furst, ; munster et al., ) . body weight has been reported to affect significantly hcq in vivo concentrations (plaquenil smpc, ; lee et al., ) and several models have identified it as a statistically significant covariate affecting clearance allometrically (balevic et al., ; morita et al., ; vogl et al., ) . thus, weight has been included in the model used for simulations with an allometric exponent of . , as estimated in these studies. the maximum tolerated dose in adults with rheumatoid arthritis has been reported mg per day (munster et al., ) , while in patients with newly diagnosed glioblastoma multiforme the maximum tolerated dose reported was mg (rosenfeld et al., ) . in order to prevent retinopathy that hcq induces after long term use, a concentration range of - ng/ml has been proposed to be safe and effective in patients with systemic lupus erythematosus under chronic therapeutic treatment (durcan et al., ) . regarding its acute use, the most significant adverse effects to consider are cardiac, gastrointestinal, extrapyramidal and neuropsychiatric effects (plaquenil smpc, ; juurlink, ) . the cardiotoxicity of chloroquines has been shown to be dose-dependent, with mean increases in qtc of . ms after a chloroquine dose of mg and ms after a chloroquine dose of mg. it should be noted that many drugs co-administered with hcq for the treatment of covid- as azithromycin, ceftriaxone and fluoroquinoles have also been proved to promote prolongation of the qtc interval, increasing the risk of cardiotoxicity (briasoulis et al., ; juurlink, ; teng et al., ) . gastrointestinal (gi) toxicity of hcq has been studied in relation to blood levels in patients with rheumatoid arthritis followed for weeks. it was revealed that blood levels of ng/ ml promote gi adverse effects in % of patients, while ng/ ml in % of patients. this is a rather significant observation in terms of hcq treatment, as generally gi symptoms are the first and most frequent to occur constituting a warning, even for cardiotoxicity (munster et al., ; tang et al ) . risk factors related to hcq toxicity include old age, renal and liver disease, genetic variants, concomitant drug use, high body mass index and, obviously, high dose and long duration of treatment (marmor et al., ) . symptoms of hcq overdose manifest rapidly within minutes after administration. they include headache, visual disturbances, cardiovascular collapse, convulsions, and hypokalemia, cardiac rhythm and conduction disorders, including qt prolongation, torsade de pointes, ventricular tachycardia and ventricular fibrillation, followed by sudden potentially fatal respiratory and cardiac arrest (plaquenil label, ; de olano et al., ) . in view of the above, hcq levels of ng/ml, even though rather conservatory, were used as an upper limit, considering also the cardiotoxic effects of the drug, possible co-administered medications and impaired clearance due to co-morbidities (abdulaziz et al., ) . it should be noted that this concentration is far below the cytotoxic concentration of the drug (cc ¼ . lm or ng/ml) in green monkey kidney veroe cells . regarding cytotoxicity, yang et al., tested cytotoxicity of hcq in cell lines retrieved from heart, liver, kidney, retina, intestine and lung. this study identified that the lowest cytotoxic level of hcq was . lm or . ng/ ml, i.e., a concentration far below the safety threshold selected in the present study . in the study of liu et al. ( b) the % maximal effective concentration (ec ) of hcq against sars-cov- was estimated using the same cell line for different multiplicities of infections (mois) namely ( . , . , . , and . ) after treatment with the drug for h. the ec s found were . , . , . , . lm or . , . , . and . ng/ml, respectively . most interestingly, in another study performed by yao et al. ( ) , also in green monkey kidney veroe cells, a timedependency of the ec was demonstrated, with ec values corresponding to . lm or . ng/ml and . lm or . ng/ml after and h, respectively. this phenomenon was attributed to the fact that hcq is accumulated within the cells, and its actions present a delay to be manifested (yao et al., ) . besides its antiviral activity, hcq's therapeutic effects against covid- reside also on its immunomodulatory effects which are manifested with blood concentrations above ng/ml (durcan et al., ) . simulations were performed using the r function 'simulx' included in the 'mlxr . package (lavielle, ) . the model parameters used are summarized in table . interindividual variability was also taken into account and thus a population of patients was simulated. the dosing schemes explored were selected from published literature studies and simulations were performed assuming patients with weights of , and kg. then, the dosing scheme providing the most favorable profile was seeked. hcq is metabolized by the liver, while it is primarily excreted by the kidney. both renal insufficiency and impaired hepatic clearance, can reduce hcq's total clearance. it has been reported that renal clearance constitutes the % of total hcq's clearance (white et al., ) . covid- patients have been proved to present increased risk of renal impairment (cheng et al., ; naicker et al., ; ronco and reis ) , while extracorporeal membrane oxygenation (ecmo) has been showed to impair the drug's clearance (tukacs, ) . therefore, during simulations an intermediate and a high level of renal impairment reducing the total clearance of hcq by and by %, i.e., a population with an apparent clearance of . and . l/h, were also considered. simulations were performed in order to explore the expected blood levels of hcq upon administration of various dosing table . population parameters used for simulations (lim et al., schemes currently used in clinical practice, as well as, in order to propose an optimal dosing scheme in relation to patients' body weight, clearance impairment and covid- severity. the lower and upper bounds, selected herein, are in line with other published studies (arnold & buckner, ; perinel et al., ; yao et al., ) , while concentrations achieved with the simulations are in accordance with the dose-concentrations results presented in previously pharmacokinetic analyses (morita et al., ; rangwala et al., ; vogl et al., ) indicating the good predictability of our simulations. as it may be noted in figures and , low frequent doses of hcq result after a period of d in toxic levels even in patients with normal body weight, while high initial bolus doses do not increase blood levels significantly. this phenomenon is due to hcq pharmacokinetics that imply its accumulation into tissues leading to delayed clearance (furst, ) . in terms of efficacy, three factors should be considered for the determination of the appropriate hcq blood levels: (a) higher multiplicity of infection in vitro, i.e., higher viral load, results in higher ec , (b) increasing the duration of exposure to hcq results in lower ec that decreases from . ng/ml to . ng/ml in h, and (c) the immunomodulatory effect of the drug is promoted at concentrations over ng/ml. therefore, blood levels should ideally reach ng/ml during the first days upon diagnosis, especially in severe cases with high viral load, and be kept above ng/ ml for the following days (durcan et al., ; liu et al., b; yao et al., ) . on the other hand, levels exceeding ng/ml are considered more probable to promote gastrointestinal adverse effects, which seem to constitute a warning for cardiotoxicity as they occur first and are more common ( munster et al., ; gautret et al. a,b; plaquenil smpc, ) . in addition, this concentration is far below the concentration found to be cardiotoxic in vitro, i.e., . ng/ml. more interestingly, cardiotoxicity of chloroquines is known to be dosedependent, with mg of chloroquine, a far more toxic compound than hcq , promoting a prolongation of . ms, when prolongations less than ms are considered to be of low concern (us fda e , ). in general, significant prolongations of the qt interval with hcq have been noted with either high doses or with long termuse and frequent doses (gautret et al. a,b; jankelson et al., ; juurlink, ; saleh et al., ) . as a result, in the present study the dosing schemes proposed, were designed in order to avoid unnecessarily high and frequent doses. based on simulations among the currently published dosing schemes, the most adequate dose for a patient of or kg, even with % impaired clearance, would be mg once daily on day , followed by mg twice daily for d (scheme_ ) or mg upon diagnosis, mg h later, followed by mg bid for d (scheme_ ). for a patient of kg, mg bid for doses followed h later by mg od for - d (scheme_ ) seems to be the most adequate scheme. in this vein, for a patient of kg with %, a dose of mg per day for d (scheme_ ) seems to constitute a safe option, even though this dosing scheme is not expected to be effective enough to significantly decrease the viral load, during the first days of the treatment. through model-based simulations, optimal dosing schemes were developed (table and supplementary material) and the corresponding hcq blood concentrations versus time were retrieved (figures and ) . dosing schemes were designed in order to achieve a fast onset of "high" concentrations during the initial phase of the disease, since an initial higher viral load is anticipated, especially for patients with severe covid- , and then keep hcq blood levels below ng/ml and over ng/ml, at all times. this can be noted in figures and where the simulated hcq concentrations versus time profiles for volunteers are presented as % prediction intervals. in table the dosing scheme designed for a patient of kg with normal clearance is presented, while tables with the dosing schemes designed for patients of kg, kg, kg, kg, kg with % impaired clearance and kg with % impaired clearance are included in the supplementary material. it should be noted that some of the dosing schemes applied currently in the clinical setting start with a loading dose of mg. despite that, the rest of the treatment in most cases differs significantly, as in most cases doses that lead to drug accumulation and thus increase the possibility of adverse effects are widely noted in the literature. in addition, only one dose scheme is used in all cases and all patients, irrespectively of their body weight, hcq clearance or their condition. in this study, the need to reach higher concentrations in patients whose immune system is not effectively reducing the viral load, leading to more intense symptoms is addressed. in fact, it has been shown that viral load relates significantly to disease severity and immune response. high viral loads were linked to intense immune response, even in peripheral tissues, complicating the patient's condition and increasing the risk of mortality (casadevall et al., ; liu et al., a; pujadas et al., ; wang et al., ; zheng et al., ) . hcq in an appropriate dosage may be of benefit thanks to its dual action, i.e., immunomodulatory and antiviral. in addition, the possibility of impaired clearance is explored and its effect on the drug's levels estimated. based on the pharmacokinetics of hcq, the fact that the drug binds avidly to tissues and that patient's weight can significantly affect its clearance was taken under consideration. thus, depending on the patient and his/her condition, the dosing schemes proposed in this study actually present some significant differences compared to those currently applied (e.g., supplementary table s and s ), improving the possibility of a safer use of hcq, depending on patient's characteristics. a possible disadvantage of the dosing schemes proposed is their complexity, as they involve different doses and varying frequency of administration, especially during the first days upon diagnosis. however, based on the results from (gautret et al., a,b) . scheme_ : mg at diagnosis, mg h later, followed by mg bid for d used by the massachusetts general hospital (massachusetts general hospital, ). scheme_ : mg per day for d used by the italian society of infectious and tropical diseases lombardy section . scheme_ : mg orally per day for - d used by the central clinical task force, korea. . scheme_ : mg bid  doses then h later start mg od for - d used by the mount sinai health system, canada . scheme_ : mg bid for day , followed by mg bid for d. . scheme_ : mg once daily on day , followed by mg twice daily for d (perinel et al., ) red line: ng/ml, known to promote adverse effects to % of patients blue line: ng/ml, levels to achieve during the first doses. green line: ng/ml, minimum levels eliciting immunomodulatory effect. france (gautret et al., a,b) . scheme_ : mg at diagnosis, mg h later, followed by mg bid for d used by the massachusetts general hospital (massachusetts general hospital, ) . scheme_ : mg per day for d used by the italian society of infectious and tropical diseases lombardy section. . scheme_ : mg orally per day for - d used by the central clinical task force, korea. . scheme_ : mg bid  doses then h later start mg od for - d used by the mount sinai health system, canada. . scheme_ : mg bid for day , followed by mg bid for d . scheme_ : mg once daily on day , followed by mg twice daily for d (perinel et al., ) red line: ng/ ml, known to promote adverse effects to % of patients. blue line: ng/ml, levels to achieve during the first doses. green line: ng/ml, minimum levels eliciting immunomodulatory effect. simulations performed and the currently published data regarding hydroxychloroquine in covid- treatment, these schemes could offer significant benefits to the patients, while after the first d, in most cases they require only a "low" dose once or twice daily. a maximum of d of treatment is proposed in view of the course of covid- (harapan et al., ) . in fact, as it has been shown for the % of mild cases, viral clearance is achieved within d post-onset (liu et al., a) . however, clinical evaluation of patients has to be performed and decisions should be made on a case-by-case basis. therefore, continuation of a daily dose may be required for a longer period, keeping hcq concentrations steady. despite the high doses proposed upon diagnosis, the mtd of hcq is not exceeded. it should be noted that continuous ecg monitoring should be conducted, especially during the first-high dose phase, in patients with moderate and severe covid- . hcq concentrations in the lungs have been reported to be significantly higher than the corresponding ones in plasma (yao et al., ) . therefore, reaching hcq blood concentrations of ng/ml assures that the ec concentrations are achieved within the main organ affected by the disease (harapan et al., ) . in addition, there is some evidence that the virus attacks red blood cells, rendering them incapable of transporting oxygen (liu & li, ) and t cells, decreasing their number significantly (qin et al., ) . thus, a high loading dose providing an initial phase of high blood levels, followed by sparse small doses is anticipated to be both more efficacious and safer in comparison to a frequent low dose scheme. this can also be supported by both the pharmacokinetics of hcq and by its time-dependent ec , which resulted in -fold lower values after an incubation of h, as compared to an incubation period of h (yao et al., ) . consequently, in a dosage regimen with a high initial dose followed by small maintenance doses, the viral load is expected to decrease during the initial phase, while the virus becomes more susceptible to the drug's concentration, allowing, therefore, for a gradual decrease of the dose to be administered. regarding the immunomodulatory effect of the drug, it has been found to be exerted with relatively low hcq bloodlevels (range - ng/ml) (durcan et al., ) . it's worth mentioning that the aim of the present study was not to propose a cure for covid- . instead, given that, hcq is used both in the clinical setting and in clinical trials as an option for the management of covid- with a 'onedose fits all' approach, this study aimed to provide guidance on the dose to be selected depending on patient's characteristics. other studies, where modeling and simulation approaches have been used in order to identify an optimized dosing scheme for hcq in covid- treatment suggest, as well, that a higher dose upon diagnosis will significantly benefit the patients (arnold & buckner, ; fan et al., ; garcia-cremades et al ; perinel et al., ; white et al., ; yao et al., ) . however, in these studies the currently applied dosing schemes were not investigated, while they focused primarily on efficacy or on safety aspects. in addition, patients' characteristics were not taken into consideration and a specific dose was proposed for all cases. only in the study of white et al., ( ) renal impairment and body weight were taken under consideration. the remarkable effect on hcq's blood concentrations was made evident and the investigators addressed the importance of dose adjustment per weight (white et al., ) . in the present study, an integrated approach was used taking into consideration all the main aspects of treatment with hcq, while the proposed dosing schemes were designed by taking into consideration patient's weight, disease severity, and his/her hcq clearance. it is worth mentioning, that the significant advantage of a high loading dose has been demonstrated in an open-label, randomized, controlled trial including patients with covid- (tang et al., ) , while the safety of a sparse dosing strategy in order to avoid accumulation has also been indicated in a recently published study for chloroquine (karalis et al., ) , that exhibits similar chemical structure, mechanism of action and adverse effects with hcq . after an extensive literature survey and simulations performed, several dosing schemes of hcq have been proposed for the treatment of covid- in relation to patient's weight and disease severity that could indicate patient's viral load. a high initial dose followed by lower sparse doses seems to be the most appropriate approach to apply in this case, as it is postulated to be more effective and safer compared to small frequent doses. indeed, these dosing schemes were designed aiming to lower the viral load both in blood and in the lungs, without allowing for hcq accumulation that could lead to adverse effects. despite their complexity in terms of clinical practice we believe that they may offer significant advantages to clinicians coping with covid- . no potential conflict of interest was reported by the author(s). data sharing is not applicable to this article as no new data were created or analyzed in this study. hydroxychloroquine: balancing the need to maintain therapeutic levels with ocular safety: an update steady-state pharmacokinetics of hydroxychloroquine in patients with cutaneous lupus erythematosus off-label therapies for covid- -are we all in this together? hydroxychloroquine for treatment of sars-cov- infection? improving our confidence in a model-based approach to dose selection treatment with hydroxychloroquine, azithromycin, and combination in patients hospitalized with covid- pharmacokinetics of hydroxychloroquine in pregnancies with rheumatic diseases clinical outcomes of hydroxychloroquine in hospitalized patients with covid- : a quasirandomized comparative study qt prolongation and torsade de pointes induced by fluoroquinolones: infrequent side effects from commonly used medications population pharmacokinetics of hydroxychloroquine in patients with rheumatoid arthritis sars-cov- viral load and antibody responses: the case for convalescent plasma therapy hydroxychloroquine with or without azithromycin in mild-to-moderate covid- efficacy of hydroxychloroquine in patients with covid- : results of a randomized clinical trial url: medrxiv preprint posted april , . 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corrected qt interval in patients with sars-cov- infection drug repurposing approach to fight covid- chloroquine and hydroxychloroquine in the treatment of covid- with or without diabetes: a systematic search and a narrative review with a special reference to india and other developing countries hydroxychloroquine in patients with mainly mild to moderate coronavirus disease : open label, randomised controlled trial torsades de pointes and qt prolongation associations with antibiotics: a pharmacovigilance study of the fda adverse event reporting system pharmacokinetics and extracorporeal membrane oxygenation in adults: a literature review e clinical evaluation of qt/ qtc interval prolongation and proarrhythmic potential for non-antiarrhythmic drugs -questions and answers (r ) guidance for industry kinetics of viral load and antibody response in relation to covid- severity covid- prevention and treatment: a critical analysis of chloroquine and hydroxychloroquine clinical pharmacology cytotoxicity evaluation of chloroquine and hydroxychloroquine in multiple cell lines and tissues by dynamic imaging system and pbpk model. (pre-publication. not peer-reviewed) in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus hydroxychloroquine application is associated with a decreased mortality in critically ill patients with covid- . medrxiv viral load dynamics and disease severity in patients infected with sars-cov- in zhejiang province, china key: cord- -l sahv t authors: takla, michael; jeevaratnam, kamalan title: chloroquine, hydroxychloroquine, and covid- : systematic review and narrative synthesis of efficacy and safety date: - - journal: saudi pharm j doi: . /j.jsps. . . sha: doc_id: cord_uid: l sahv t the covid- pandemic has required clinicians to urgently identify new treatment options or the re-purposing of existing drugs. of particular interest are chloroquine (cq) and hydroxychloroquine (hcq). the aims of this systematic review are to systematically identify and collate studies describing the use of cq and hcq in human clinical trials and to provide a detailed synthesis of evidence of its efficacy and safety. of clinical trials, % showed no significant difference in the probability of viral transmission or clearance in prophylaxis or therapy, respectively, compared to the control group. among observational studies employing an endpoint specific to efficacy, % concurred with the finding of no significant difference in the attainment of outcomes. three-fifths of clinical trials and half of observational studies examining an indicator unique to drug safety discovered a higher probability of adverse events in those treated patients suspected of, and diagnosed with, covid- . of the total papers focusing on cardiac side-effects, % found a greater incidence of qtc prolongation and/or arrhythmias, % found no evidence of a significant difference, and % mixed results. the strongest available evidence points towards the inefficacy of cq and hcq in prophylaxis or in the treatment of hospitalised covid- patients. in line with the picot format (riva et al., ) of framing subjects for clinical research, this study centres on answering the question: 'in patients suspected of, and diagnosed with, covid- , how efficacious, relative to standard symptomatic care, and safe are cq and hcq in patients at risk or suspected of, and diagnosed with, covid- ?' the subsequent elaboration of the systematic review and narrative synthesis adhered to the preferred reporting items for systematic reviews and meta-analyses (prisma) guidelines (l. et al., ) for evidence-based assessment of published research. in light of the current global health emergency, and the requisite rapid turnover of publications to meet the consequently urgent need to obtain and analyse the results that they present, several authors have resorted to the use of preprint servers to disseminate their findings. despite the evident shortfalls inherent in referring to data, whose quality has not been peer-reviewed, the present paucity of published original research on the efficacy and safety of cq and hcq in covid- patients demands exceptional measures. as such, this systematic review will take into account non-peer-reviewed work, provided that they have been submitted to a preprint server, where they are available in open-access form. nevertheless, given its focus on data quality, this review will make unambiguous every instance in which data from such sources are used. therefore, on august , medrxiv and biorxiv, along with pubmed and web of science, acted as the databases for the initial search of items relevant to the picotformatted question. the preliminary use of the search terms "covid", "chloroquine", and "hydroxychloroquine" yielded a large number of results that bore little relevance to the research topic. combining such terms into phrases -"covid" and "chloroquine", and "covid" and "hydroxychloroquine" in the title or abstract -and requiring the term "outcome" or "outcomes" anywhere in a free full text appearing within the last year considerably focused the responses. the subsequent application of identical phase stenography to each database ensured internal consistency. as this review aims to establish the weight of evidence for the use of a therapy in patients, data able to answer such a question must derive from primary research. moreover, owing to the international extent of the present health crisis, any imposition of an original language requirement would exclude useful and otherwise rare resources. as such, following the collation of items in mendeley and the removal of duplicates, application of these criteria excluded unique items for which there was either no english version or no original data. screening of the resulting papers against the criteria established by the picotformatted question -namely, the requirement that data be collected from covid- patients treated with cq and/or hcq -included only controlled trials and observational studies. of all case series considered, only those directly assessing cq or hcq safety addressed each facet of the question and were thus included. having applied the exclusion and inclusion criteria to all search results and removing duplicates at all stages where necessary, two investigators independently reviewed the final repertoire of studies. rather than merely verifying the relevance and scope of the material in the final library, holistic analysis of each item of research in line with the framework set out in the checklist of review criteria provided by the task force of academic medicine and gea-rime committee (bordage et al., ) ensured stringent appraisal of study quality. indeed, the identification of -among other facets of robust research -appropriate study design, statistical analysis, and quality control (table ) permitted only papers with sufficient scientific merit to pass onto the data extraction stage. among the research items constituting the final library for analysis, there exists a wide variation in study design, results, and, crucially, the extent to which each distinct aspect of the picot-formatted question is answered. as such, a specialised data extraction table collates and summarises the most important information in every paper (table ). in particular, emphasis on the different sample sizes and structures, symptomatic stages, doses of drug used, primary and/or secondary outcomes and overall design limitations, facilitates both clarity and caution when making comparisons between the sets of results presented. the results of each search, and the number failing and passing exclusion and inclusion criteria, respectively, have been summarised in a flowchart ( figure ). proposals; systematic reviews and/or meta-analyses; and three models of covid- spread, pathological dynamics, and/or therapy. likewise, of those unique items found on the preprint servers, were excluded by the same criteria, distributed categorically as follows: systematic reviews or meta-analyses; five study proposals; three commentaries; two models; and one literature review. as part of the search attrition methodology, parallel application of the inclusion criteria to each set of remaining unique results left and sixteen items from the first two, and preprint, databases, respectively. of these studies, three were duplicates, culminating in a library of papers from all four databases. the use of criteria created in accordance with the aforementioned checklist of review criteria elaborated by the gera-rime committee, meeting of which was judged to be indicative of scientific rigour, excluded two (mehra et al., ), (molina et al., ) of the remaining items (table ) . however, it is noteworthy that, of the studies approved by an ethics committee, explicitly waivered informed consent -another basic tenet of data collection from patient trials -citing the retrospective nature of their research. confusingly, three studies claiming ethical board approval failed to explicitly mention consent (sbidian et al., ) , (arshad et al., ) , , while one investigation (boulware et al., ) only sought consent after randomised allocation had already taken place. as a result of the relatively broad scope of the research question, the authors of the papers passing quality appraisal employed a variety of study types, therapeutic doses, and primary and/or secondary outcomes (table ) . it is thus essential to distinguish results by study design in order to prevent invalid inferences drawn from comparison of data sets. although the research question requires the use of cq and/or hcq in patients suspected of, and diagnosed with, covid- , there exists a range of possible approaches to the collection of data obtained from such patients. the gold standard of primary clinical research into the efficacy and safety of drugs administered to humans is the randomised controlled trial (rct), evidence from which may be further buttressed by masking of subjects, experimenters, or both, as well as the use of a placebo in the control group. however, only % of items passing quality appraisal were clinical trials, of which, though all were randomised, only one (boulware et al., ) employed either masking or an exclusively placebo control. while unacceptable under normal circumstances, the absence of both masking and a placebo is admissible in light of the ethical violation that would otherwise result from the use of either in the context of patient consent being unlikely. indeed, the paper reporting use of masking and a placebo was likely able to do so given the asymptomatic presentation of the uninfected patients constituting the sample from which the control group derived. the remaining % of papers were observational studies, of which the vast majority retrospectively searched hospital databases to collect clinical data obtained by following up on cohorts of patients from the time they received cq and/or hcq, or the standard of care only, until a defined end-point. despite % using a case-control structure, a minority ( %) constituted case series focusing on the cardiac safety of drugs administered to hospitalised covid- patients for a given duration of time. the deliberate absence of a specified dose in the research question accounts for the diversity of administration regimens among the papers. indeed, the doses used largely reflect the studies being conducted on different dates, which, in turn, influences the relative sway of either federal healthcare guidelines or the results of prior clinical research on regimen selection. every clinical trial tested a distinct dosing scheme: a loading dose of mg hcq per day for three days, followed by a maintenance dose of mg per day for two or three weeks if symptoms are mild/moderate or severe, respectively (w. tang et al., ); mg hcq once and mg six to eight hours later for one day, then mg per day for four days (boulware et al., ) ; mg hcq twice in six hours, then mg at hrs on day one, followed by mg twice a day for nine days, or until discharge (horby et al., ) ; mg hcq once on day one, then mg per day for six days (mitja et al., ) ; and mg twice a day on day one, followed by mg twice a day for four days (kamran et al., ). likewise, each clinical trial treated its control group differently, from: a placebo folate tablet administered in a regimen identical to that of the treatment group (boulware et al., ) , to only a standard of care, itself varying with clinical centre and national guidelines. by contrast, the retrospective and often multi-centre nature of many observational studies has resulted in % using variable or undeclared doses, some collecting data from some patients taking azithromycin in combination with the hcq. the remaining % of items relied on highly divergent dosing regimens (table ) only on day one, then mg per day for nine days (arshad et al., ) ; or twice per day on day one, followed by mg per day for four days (geleris et al., ) , with or without azithromycin. only three sets of authors also analysed data for patients taking cq variably (hsia et al., ) , or, more commonly, at a full dose of mg twice a day, either: for one day, followed by half-dose for four days, with or without mg azithromycin per day for five days (saleh et al., ) ; or at a half-dose for the duration of treatment (huang et al., a) . similarly, the % of observational studies with a control treated the group differently, giving standard of care without, or with declaration of additional antivirals, antibiotics, or both. given the vast array of possible measures of cq and hcq efficacy and safety in covid- patients, the different authors outlined distinct primary and/or secondary outcomes. the primary outcome in % of clinical trials was c t > for pcr amplification of sars-cov- rna, indicating either prophylaxis or viral clearance. likewise, % of observational studies directly measured a specific indicator of efficacy other than mortality rate, using similar outcomes to the clinical trials, as well as the duration of hospital stay, need for mechanical ventilation, and probability of transfer to an intensive care unit (icu). as regards direct measurement of safety, all clinical trials and % of observational studies explicitly recorded adverse events as an indicator of cq and/or hcq safety in covid- patients, with % of the total actively focusing on cardiac pathology. notably, % of studies used mortality rate as a key end-point. in isolation, however, risk of death could be indicative of either safety or efficacy. as such, this review reports the findings on mortality rate separately from those pertaining to outcomes that are specific measures of one of efficacy or safety. of the clinical trials providing data on a specific indicator of cq and/or hcq efficacy in patients suspected of, and diagnosed with, covid- , all showed no significant difference in the probability of viral transmission or clearance in the prophylactic (boulware et al., ) , (mitja et al., ) (horby et al., ) , and dosage across the range of papers. in contrast, only % of observational studies employing an endpoint specific to efficacy recorded no significant difference in the attainment of outcomes, such as duration of hospital stay, need for mechanical ventilation, and probability of transfer to an intensive care unit (icu), between covid- patients given a range of cq and/or hcq doses, and the control groups. indeed, of the remaining papers, % found evidence of a higher probability of discharge rate (sbidian et al., ) , viral clearance and shorter symptom duration (huang et al., a) in a therapeutic context, and a lower incidence of sars-cov- infection in a prophylactic context (bhattacharya et al., although % of clinical trials found evidence of higher mild adverse drug-related events in the treatment group, none of those specifically focusing on cardiac-side effects discovered any significant difference relative to the control. likewise, % of observational studies examining an indicator unique to drug safety discovered a higher probability of adverse events in those treated patients suspected of, and diagnosed with, covid- . of those retrospective studies measuring cardiac side-effects, % failed to find a significantly different incidence between the treatment and control groups, while another % indicated a significantly greater probability, with qtc prolongation the most common finding, in addition to its potentially lethal consequences of vt and cardiac arrest. in contrast, one such paper presented mixed findings, with an elevated risk of cardiac events despite no apparent rise in the risk of qtc prolongation. of the total studies using mortality rate as a key end-point, % reported no significant change in the risk of death, while % showed a depression, in treated relative to control patients. interestingly, one investigation ( %) yielded a mixed result of raised mortality risk in patients given hcq only, but no significant difference in those coadministered azithromycin (magagnoli et al., ) . the absence of a pharmacological treatment tailored to covid- has rendered urgent the search to find alternative therapies by repositioning drugs with the theoretical potential to alleviate symptoms. however, hypothetical plausibility is insufficient grounds for translation into clinical practice. indeed, any therapeutic repurposing must only proceed in light of strong evidence for the pre-clinical basis, and clinical efficacy and safety, of the drug in question. this review finds that, while such evidence certainly exists for the former, it does not for the latter, calling into question any clinical use of cq and/or hcq in covid- patients prior to the collection and analysis of high-quality rct data. pre-clinical studies performed in vitro provide strong evidence for the theoretical utility of cq and hcq in inhibiting all stages of viral entry, maturation, and spread. in vitro, cq blocks infection both at, and after, entry of sars-cov- into vero e cells, with an ec of . µm . indeed, although therapeutic doses of cq do not seem to alter s protein glycosylation (vincent et al., ) , whose pattern is distinct from that of sars-cov- (kumar et al., ), they may inhibit biosynthesis of sialic acid (kwiek et al., ) , n-glycosylation of ace , as well as downregulating the expression of picalm (wolfram et al., ) in the clathrin-dependent endocytosis machinery. furthermore, immunofluorescence analysis of the amount of nucleoprotein in distinct vesicular compartments of the host cell has demonstrated that treatment of infected cells with cq and hcq stalls transfer of viruses from early to late endosomes . in fact, by increasing the ph of the early endosome, cq has the potential to reduce acid-dependent proteolytic cleavage of the s protein by cathepsin and tmprss , thereby inhibiting viral uncoating, genomic replication and particle maturation (wang et al., ) . despite its similar effect on viral distribution, as well as its comparable cytotoxicity (liu and li, ), to cq, hcq appeared to amplify and enlarge the late endosomes, implying a slightly distinct mechanism of action. furthermore, there exists conflicting evidence for the relative in vitro efficacy of the two drugs (yao et al., ) . it is nonetheless clear that the initial basis for investigating the translatability of cq and/or hcq to the treatment of hospitalised covid- patients was predicated on highquality evidence for its pre-clinical antiviral efficacy. that cq and hcq can reduce viral entry, trafficking, and budding in vitro constitutes evidence of translational potential relies on the underlying assumption that symptom severity importantly, however, in covid- patients, symptoms subsequent to the causal infection result firstly from the initial cytokine wave of the innate immune response , then a state of immunodeficiency and lymphopenia (hadjadj et al., ) , and, finally, a potentially lethal cytokine storm (f. . the causal distinction between these symptomatic phases highlights not only the difficulty in repurposing a single drug for use at all time-points, but also the need to approach with caution the comparison of trial data collected from patients given drugs at different times post-infection. the sample of one of the first clinical trials performed on patients testing positive upon pcr amplification of sars-cov- rna comprised asymptomatic patients ( %), as well as those with upper ( %) and lower ( %) respiratory tract infections, thereby capturing the range of symptom severity. after days of treatment, patients given hcq alone had a higher probability of viral clearance compared to those given the standard of care only ( % vs. %), rising to % in patients also given azithromycin (gautret et al., ) . that the authors additionally discovered a greater drug effect on patients with upper and lower respiratory tract infections than on asymptomatic individuals raises the possibility that the potential therapeutic benefit of hcq in covid- patients lies in its capacity for immunomodulation. on a theoretical level, the anti-inflammatory effects of hcq render such an effect possible. indeed, through alkalinisation of early endosomes, cq and hcq could impair: pamp-induced activation of tlr and tlr (saitoh and miyake, ) , and, by extension, mmp- expression (lim et al., ) ; antigen presentation by major histocompatibility complexes (mhcs) (roche and furuta, ) , (guerriero, ) ; prostaglandin and thromboxane production (nosál' and jančinová, ) ; and t and b cell activation (goldman et al., ) , differentiation, and proliferation (yang et al., ) . importantly, both sars-cov- , (conti et al., ) and related coronaviruses, such as sars-cov- (wang et al., ) and mers-nevertheless, many observational studies support the absence of significant evidence cq and/or hcq efficacy compared to that of standard of care alone. furthermore, the homogeneity of, and correction for, baseline characteristics in the case and control cohorts further buttresses the reliability of the evidence presented by studies with sample sizes ranging from , (geleris et al., ) to , (s. . one such study, however -with a sample size of , -used a treatment cohort with a significantly lower age but greater symptomatic disease compared to the control (ip et al., ) . similarly, the vast majority of papers contending that cq and/or hcq delayed sars-cov- clearance in covid- patients were severely underpowered and exhibited significant differences in sample structure and/or comorbidities, nullifying their influence on the conclusion of this review (mahévas et al., ). as such, it is reasonable to conclude that, at present, the highest quality evidence does not support the efficacy of either cq or hcq in the prophylaxis or treatment of patients at high risk of, or diagnosed with, covid- , relative to the standard in-hospital management of symptoms. nevertheless, the completion of more rcts will be necessary to further substantiate this claim. robust evidence for the safety of an otherwise efficacious drug is a prerequisite for its widespread application in any clinical setting. in light of the unsubstantiated benefit of cq and/or hcq administration in patients with covid- , there exists an even more compelling imperative to ensure that any compassionate use did, and does, not contribute to excess mortality. despite their adequate safety records in an anti-inflammatory context, as aminoquinolines, cq, and its derivative, hcq, are proarrhythmic (khobragade et al., ) . arrythmias arise from an imbalance of the normal physiological variables influencing the activation and inactivation kinetics of the cardiac ion channels that permit the transmembrane currents forming the foundation of the cardiac action potential. in a healthy milieu intérieur, the waveform of this cardiac action potential is quadriphasic (noble, ) . electrical diastole (iv) precedes a rapid depolarisation ( ), after which a refractory period is followed by a slow hyperpolarisation (i), a ms plateau (ii), and then a period of repolarisation (iii) to resting membrane potential (e m ). by blocking -in order of increasing potency -the delayed (i kr ) and inwardly-rectifying (i k ) k + currents (sánchez-chapula et al., ) , and the latter preferentially at depolarised e m , cq and hcq significantly prolong the qt interval and slow ventricular conduction, thereby predisposing to early-after-depolarisation and, by extension, torsades de pointess. combined with their tonic block of voltage-gated na + and ltype ca + currents at low channel opening frequencies, qt interval prolongation thus significantly increases the risk of potentially fatal vts. indeed, despite the only randomised clinical trials examining prophylaxis having found no increases in adverse cardiac events upon hcq administration to patients at risk of covid- (mitja et al., ) (boulware et al., ) , there is some evidence that aminoquinoline treatment predisposes those diagnosed with confirmed infections to tachyarrhythmia. in fact, a significant association of high doses of cq with lethality in patients with severe symptoms forced the premature termination of a rct (borba et al., ) . despite this relationship with mortality risk disappearing upon correction for age, there remained a significantly higher proportion of patients in the high ( %) compared to the low ( %) dose group with qtcf > ms, including three percent of patients who experienced vt before death. however, given the abortion of the study, as well as the coadministration of qt-prolonging oseltamivir (hama and bennett, ) confounding the causal link to cardiac side-effects, these data, alone, are insufficient to conclude that cq is unsafe in covid- patients. notably, the only paper failing this review's quality appraisal suggested a significant excess mortality in patients treated with cq and/or hcq relative to controls. a highlypowered observational study of , patients with no significant differences in comorbidities between groups, it claimed that patients given cq (four percent) or hcq (six percent) alone had a significantly augmented risk of de novo in-hospital ventricular arrhythmias, compared to controls (three-tenths of one percent) given standard therapy, including remdesivir (mehra et al., ). however, within one day of investigative journalists' discovery of inconsistencies in the reporting of the data from international hospitals involved in the study, over signatories penned an open letter to the authors of the paper and the journal in which it was published, also criticising the statistical analysis and lack of ethics review. despite the study's consequent correction of the continental assignment of one hospital, which had no impact on its overall findings, questions remain over the lack of transparency of the company that managed the original databases. on june , three of the four co-authors retracted the paper (mehra et al., n.d.), thereby necessitating caution when interpreting its original findings. nonetheless, a more recent observational study of , patients suspected of, or diagnosed with, covid- , has elucidated a significantly higher proportion of cardiac-attributable mortality in the hcq treatment ( %) than the control ( %) group. moreover, despite being individually underpowered, several smaller retrospective database searches focusing specifically on the qtc duration have consistently and independently supported a significant prolongation in hospitalised covid- patients treated with a range of cq and/or hcq doses (saleh et al., ) , (mercuro et al., ) . regardless, drawing causal inferences from such observational studies is inadvisable given the lack of randomisation and absence of a placebo in the control groups, leaving the data susceptible to unmeasured confounders. by contrast, the most highly powered rct found no evidence of a significant excess of de novo cardiac arrhythmias in covid- patients of varying severity given hcq for days, or until discharge (horby et al., ) . likewise, many of the largest retrospective case- control studies passing quality appraisal demonstrated an absence of a statistically significant difference in the incidences of vts, fibrillation, or sudden cardiac deaths (scds) between hospitalised patients administered hcq and those provided with the standard of care alone (a. k. , (arshad et al., ) . in any case, from a theoretical standpoint, the cardiac safety risk of cq and hcq use is unlikely uniform among covid- patients. indeed, there is a significant positive correlation between baseline qtc and age (reardon and malik, ) , qtc prolongation and anti-dysrhythmic, antipsychotic or macrolide antibiotic co-administration (al-khatib et al., ) , and qt interval dispersion and mortality risk in type ii diabetes mellitus (giunti et al., ) . given the relative risk conferred by both older age ("the epidemiological characteristics of an outbreak of novel coronavirus diseases in china," ) and type ii diabetes mellitus (richardson et al., ) on symptom severity and consequent probability of hospitalisation of covid- patients, the data for in-hospital cq/hcq safety may not be extrapolable to many infected individuals in the population due to selection bias (henderson and page, ) . as such, the wide variation in average sample patient age, symptom severity and drug dosing regimen (table ) further complicates inferences of reliable agreement between the papers. therefore, as of aug , there is a lack of strong evidence that, relative to standard in-hospital management of symptoms, the use of cq and hcq to treat hospitalised covid- patients has been either distinctly safe or unsafe. the substantiation, of presently unclear strength, for the tendency of the aminoquinolines to prolong qtc in this therapeutic context may still provide cause for concern in patients whose comorbidities predispose them to vts. the ongoing inquiries into the validity of data sets indicating a lack of drug safety, however, should preclude definitive judgment on the matter until the completion of more high-quality rcts. crucial to the understanding of the conclusions drawn in this systematic review is an appreciation of its many limitations, which relate to both the search methodology and data analysis. insofar as peer-reviewed publications are concerned, this review searched two databases to yield only unique results, leaving the authors to also seek the findings of papers on two preprint servers. despite facilitating the collection of a more representative sample of current research on the subject in question, the absence of documented expert scrutiny ought to prevent their data from influencing clinical decisions. nevertheless, to compensate for the lack of peer review, rigorous application of the quality appraisal criteria established by the gea-rime committee and the task force of academic medicine ensured that only data from adequately designed studies were taken into account. importantly, however, that peer-reviewed journal material was no longer a prerequisite for inclusion may have slightly reduced the effects of positive publication bias (mlinarić et al., ) on the results of this systematic review. in light of the recent investigation into the integrity of the data collection and statistical analysis of the largest observational study to date (mehra et al., ) , it is also necessary to advocate caution in the interpretation of results, even if the paper in which they are presented has undergone peer review. indeed, worries concerning the decline in review duration and even scrutiny, as a result of the rush to publish, should elicit wariness in considering even the most fundamental of underlying assumptions; namely, that a data set is valid. however, the predominant shortcoming of the review is its inability to completely disentangle the large differences in study design when making comparisons between different data sets from the included papers. indeed, despite stressing the obvious invalidity of cutting across distinct sample sizes, baseline characteristics, drug doses, and individual limitations, a systematic review, by nature, does exactly that. the categorisation of the results and data analysis by randomisation, covid- symptom severity, and hcq/cq dosage constitutes an attempt to reduce this problem of comparative inferences as greatly as possible. on march , the who announced the launch of an international phase iii-iv rct, with four arms, measuring the efficacy and safety of: ( ) remdesivir; ( ) lopinavir and ritonavir; ( ) lopinavir, ritonavir, and ifn-(beta); and ( ) cq or hcq (kupferschmidt, ). in the meantime, amidst a dearth of high-quality evidence from completed randomised clinical trials, the u.s. food and drug administration (fda) issued an emergency use authorisation of cq and hcq in covid- patients (piller, ) . since then, data from the most robust of the completed case-control studies have failed to find any significant evidence of the efficacy of cq and/or hcq in the treatment of hospitalised covid- patients. meanwhile, on may , in the midst of a large number of research groups finding evidence of significant qtc prolongation upon administration of aminoquinoline drugs, a recent large retrospective observational study indicated a possible lack of drug (particularly cardiac) safety in this clinical context. on may , the who suspended the fourth arm of the solidarity trial, citing these safety concerns. in the uk, the medicines and healthcare products regulatory agency (mhra) followed suit, calling the copcov trial to a halt. less than one week later, however, an international coalition of scientists raised concerns regarding the integrity of the collection and analysis of the inaccessible data on which the suspension was based, and the responsible paper has since been retracted. the mhra has authorised the copcov trial to recommence. following the who's recontinuation of the solidarity trial arm, interim results provided little evidence of a reduction in mortality risk for hospitalised covid- patients administered cq and/or hcq, ultimately leading to its indefinite discontinuation on july . the urge to begin all international and national clinical trials arose from the necessary desire to rapidly compensate for the prior and present scarcity of randomised data on the efficacy and safety of cq and/or hcq in patients infected with sars-cov- . in the absence of evidence for the safety of cq and hcq in covid- patients, the fda's initial decision to authorise their use in hospitalised patients was of questionable scientific prudence. indeed, this review finds that, despite data from different in vivo studies conflicting and even contradicting each other, the strongest evidence does not support the efficacy of either cq or hcq in the prophylaxis or treatment of patients at high risk of, or diagnosed with, covid- , relative to the standard in-hospital management of symptoms. likewise, there is a lack of strong evidence that the use of cq and hcq to treat hospitalised covid- patients has had a distinct effect on safety outcomes. yet it is precisely because of this dearth of evidence that there still exists a pressing demand for rcts. in fact, the saga ensuing from the hastiness in accepting the findings of the now-retracted observational study is a case in point of the need to collect randomised clinical data in order to substantiate or contend any claims of drug efficacy and/or safety. long qt and hydroxychloroquine; a poorly recognised problem in rheumatology patients what clinicians should know about the qt interval chloroquine and covid- : a light at the end of the tunnel, or is it another train? electron treatment response to hydroxychloroquine and antibiotics for mild to moderate covid- : a retrospective cohort study from south korea treatment with hydroxychloroquine, azithromycin, 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reduce the likelihood of coronavirus- (cov- or sars-cov- ) infection and lung inflammation mediated by il- the spike glycoprotein of the new coronavirus -ncov contains a furin-like cleavage site absent in cov of the same clade who declares covid- a pandemic an interactive web-based dashboard to track covid- in real time structural and molecular modelling studies reveal a new mechanism of action of chloroquine and hydroxychloroquine against sars-cov- infection hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial observational study of hydroxychloroquine in hospitalized patients with covid- increased qt nterval dispersion predicts -year cardiovascular mortality in type diabetic subjects: the population-based casale monferrato study hydroxychloroquine inhibits calcium signals in t cells: a new mechanism to explain its immunomodulatory properties the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- macrophages: their untold story in t cell activation and function impaired type i interferon activity and exacerbated inflammatory responses in severe covid- patients the mechanisms of sudden-onset type adverse reactions to oseltamivir tissue distribution of ace protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis temporal dynamics in viral shedding and transmissibility of covid- appraising the evidence: what is selection bias? sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor effect of hydroxychloroquine in hospitalized patients with covid- : preliminary results from a multi-centre, randomized qt prolongation in a diverse, urban population of covid- patients treated with hydroxychloroquine, chloroquine, or azithromycin preliminary evidence from a multicenter prospective observational study of the safety and efficacy of chloroquine for the treatment of covid- treating covid- with reply to gautret et al. : a bayesian reanalysis of the effects of hydroxychloroquine and azithromycin on viral carriage in patients with covid- hydroxychloroquine and tocilizumab therapy in covid- patients -an observational study phylogenetic analysis and structural modeling of sars-cov- spike protein reveals an evolutionary distinct and proteolytically sensitive activation loop chloroquine and hydroxychloroquine for covid- : implications for cardiac safety clearing the fog: is hcq effective in reducing covid- progression: a randomized controlled trial. medrxiv . . electrocardiographic safety of daily hydroxychloroquine mg plus azithromycin mg as an ambulatory treatment for covid- patients in cameroon dealing with the positive publication bias: why you should really publish your negative results no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe covid- infection a modification of the hodgkin-huxley equations applicable to purkinje fibre action and pacemaker potentials cationic amphiphilic drugs and platelet phospholipase a (cpla ) a short therapeutic regimen based on hydroxychloroquine plus azithromycin for the treatment of covid- in patients with non-severe disease. a strategy associated with a reduction in hospital admissions and complications inhibition of chikungunya virus infection in cultured human muscle cells by furin inhibitors: impairment of the maturation of the e surface glycoprotein hydroxychloroquine sulfate in prevention of thromboembolic phenomena in surgical patients former fda leaders decry emergency authorization of malaria drugs for coronavirus pulmonary embolism in covid- patients: awareness of an increased prevalence the blood-retinal barrier in chloroquine retinopathy hydroxychloroquine protects the annexin a anticoagulant shield from disruption by antiphospholipid antibodies qt interval change with age in an overtly healthy older population presenting characteristics, comorbidities, and outcomes among patients hospitalized with covid- in the new york city area what is your research question? an introduction to the picot format for clinicians the ins and outs of mhc class ii-mediated antigen processing and presentation regulatory molecules required for nucleotide-sensing toll-like receptors the effect of hydroxychloroquine and azithromycin on the corrected qt interval in patients with sars-cov- infection blockade of currents by the antimalarial drug chloroquine in feline ventricular myocytes hydroxychloroquine with or without azithromycin and in-hospital mortality or discharge in patients hospitalized for covid- infection: a cohort study of , in-patients in france chloroquine and hydroxychloroquine in the treatment of covid- with or without diabetes: a systematic search and a narrative review with a special reference to india and other developing countries outcomes of hydroxychloroquine treatment among hospitalized covid- patients in the united states-real-world evidence from a federated electronic medical record network chloroquine is a potent inhibitor of sars coronavirus infection and spread sars coronavirus entry into host cells through a novel clathrin-and caveolae-independent endocytic pathway remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro antibody-dependent sars coronavirus infection is mediated by antibodies against spike proteins a chloroquine-induced macrophage-preconditioning strategy for improved nanodelivery cryo-em structure of the -ncov spike in the prefusion conformation characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china ocular toxicity of hydroxychloroquine hydroxychloroquine inhibits the differentiation of th cells in systemic lupus erythematosus exuberant elevation of ip- , mcp- and il- ra during sars-cov- infection is associated with disease severity and fatal outcome in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) low dose of hydroxychloroquine reduces fatality of critically ill patients with covid- low dose of hydroxychloroquine reduces fatality of critically ill patients with covid- d-dimer levels on admission to predict in-hospital mortality in patients with covid- clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study no funding received for this study mt wrote the first draft and edited all subsequent drafts of the manuscript. kj designed the study and edited subsequent drafts of manuscript. horbymention of ethical approval, but explicit waiver of informed consent no serious side-effects observed.absence of a placebo. standard of care unusually consisted of zinc, and vitamins c and d.overwhelmingly male sample prevents generalisation to both sexes.a subset of day pcr negatives retested positive on day , suggesting either false negatives on day due to variable kit sensitivity, or false positives on day as non-replicable viral nucleic acid is detected by pcr after days. no significant difference in mortality by days between hcq only and control.significantly higher mortality risk for patients given hcq + azithromycin compared to control.significantly higher discharge rate in the group given hcq only. lack of randomisation.limited sample size for the hcq + azithromycin subgroup necessitates caution in interpreting higher mortality risk compared to control.dosage may have varied from guidelines due to physician discretion.significantly higher proportion of younger, male and smoking patients in the treatment cohorts than the control, though no significant differences in biological parameters. no significant difference in mortality.significantly higher proportion of mortality attributable to cardiac causes in patients treated with hcq ( %) compared to the control group ( %).similar incidence of arrhythmias and cardiomyopathy in treated ( % and %) and control ( % and %) patients.lack of randomisation.significantly lower age, but later presentation in clinical course and greater symptomatic disease in the treatment group.possible misclassification due to manual abstraction of her data.possible sampling bias due to use of a convenience sample for data collection. considerable imbalance in the sample size of the treatment and control groups.interferon application reached % in the control, but % in the treatment group.among patients who died, significantly longer hospital stay for the hcq ( days) compared to the control ( days) group.significant reduction in plasma il- in hcq, but not control patients. among patients with il- > pg ml - , hcq treatment, but not control treatment reversed the trend after days, and significantly reduced fatality. significantly higher probability of viral clearance by days and in the treatment group.significantly shorter duration of fever symptoms in the treatment group.no serious adverse events observed in the treatment group. however, higher incidence of gi disturbances.significantly lower incidence of adverse events in patients in the treatment group on halfdose, compared to those given the full dose.lack of randomisation.only patient in the treatment, compared to patients in the control, group experienced aggravated (i.e. moderate to severe) symptoms.impossibility of dissociating the effects of cq from those of other antiviral drugs used prior to its administration. however, patients given cq within days of symptom onset still exhibit faster viral clearance. higher probability of prescribing hcq ± azithromycin to patients with more severe metabolic, haematological, and ventilatory symptoms of covid- .significant differences in demography, vital signs, prescription drug use, comorbidities, and disease severity. however, all adjusted by propensity score. lack of randomisation.small sample size, with large imbalance between treatment ( ) and control ( ) groups limiting the number of factors included in the propensity score model.a few patients in the treatment group ( . % of total) also received treatment with lopinavir/ritonavir, though duration of use included in multivariate cox analysis.baseline systolic abp and haematocrit varied significantly between treatment and control group, even after matching. % of hcq patients experienced averse ecg modifications requiring cessation of treatment after a median of days. among them, % had a qtc prolongation > ms (including > ms in one patient). one of these patients presented with a st degree av block after days despite a lack of concomitant proarrhythmic medication. small sample size.lower probability of co-administration of azithromycin in the hcq ( %) compared to the control ( %) group. higher probability of co-administration of amoxicillin and clavulanic acid in the hcq ( %) compared to the control ( %) group.hcq patients had lower prevalence of comorbidities, except hepatic cirrhosis.the covariates exceeding the standardised difference threshold were excluded from the final propensity score model. imbalance in the number of hcq patients between centres not taken into account by the propensity score model. in the treatment group. as gi disturbances ( %), skin rash ( %), and headache ( %).no serious adverse effects observed.atypically low prevalence of comorbidities in atypically young demographic.no taking into account of differences in behaviourassociated risk. significantly higher probability of qtc prolongation in any treatment group as compared to baseline.significantly higher mortality risk with qtc prolongation > ms.no significant differences in the prevalence of specific arrhythmic events, such as atrial fibrillation, tachycardia or torsade de pointes.lack of randomisation.small sample size.inclusion only of patients requiring hospitalisation increases risk of selection bias.higher probability of ecg-monitored patients being in a specialised care unit with more testing availability. % and % of patients were coadministered methadone and ondansetron, respectively, both of which prolong qtc, creating a confounder effect. % of patients ceased hcq before day due to arrhythmic and gi adverse events, as well as a case of hypoglycaemia.lack of randomisation.small sample size.possible underestimation of qtc due to short followup period. possible role of covid- -associated myocarditis and/or stress cardiomyopathy in observed adverse events.baseline qtc was shorter in hcq + azithromycin compared to hcq only patients.most patients had at least cardiac comorbidity, and were taking or more drugs prolonging qtc. significantly longer time to viral clearance than patients given lopinavirritonavir and mg day - , respectively.no significant difference in delay until improvement of disease symptoms. no significant difference in the incidence of adverse events.significantly lower incidence of lymphopenia and hyperbilirubinaemia than in patients given lopinavirritonavir.lack of randomisation.small sample size.significantly greater probability of confirmed pneumonia on ct scans in patients given lopinavirritonavir than those given hcq. no observed side-effects of hcq.no significant changes in plasma counts of leukocytes, lymphocytes, or concentrations of crp and ferritin in either hcq or control groups.lack of randomisation.small sample size.no direct measurement of qtc prolongation.hcq group had both significantly higher comorbidities and d-dimer levels.abbreviations: rct = rct; os = observational study; scd = sudden cardiac death; mcot = mobile cardiac outpatient telemetry ( ) acquisition of submitted, but not yet peerreviewed, items from a preprint server; and ( ) application of exclusion and inclusion criteria.the removal of duplicates at each step, where necessary, left unique items to pass onto the quality appraisal stage.