Hydroxychloroquine with or without macrolide and standard of care versus standard of care alone for COVID-19 cases: a systematic review and meta-analysis

Background: Coronavirus disease (COVID-19) pandemic has been a global health threat. The speci�c treatment of this disease has not yet been approved. In this review, we aimed at assessing the role of hydroxychloroquine with/without macrolide in terms of e�cacy and adverse effects against the standard of care. Methods: Pubmed, Medline, Google Scholar, Cochrane Library, and Clinicaltrials.gov were searched for the quantitative and qualitative synthesis of 13 studies using PRISMA guidelines for a proper review. Assessment of heterogeneity was done using the I-squared (I 2 ) test and �xed/random effect analysis was done to determine the odds/risk ratio among the selected studies. Results: Meta-analysis of our study demonstrated no signi�cant differences in improvement for the virological cure (RR 0.95, 0.67-1.34), whereas a signi�cant relationship was there in radiological progression (pneumonia resolution) (RR 1.40, 1.03-1.91) between the two arms. There are 1.52 times the odds of intubation during treatment (CI 0.61-3.77), 1.08 times the risk of mortality (CI 0.65-1.79), and about 2.21 times increased risk of development of adverse effect (OR 2.21, 0.95-5.17). Though overall it is of no statistical signi�cance, clinical relevance to thinking while using the treatment for COVID-19 is advised. Among randomized controlled trials, the treatment group has 3.5 times (OR 3.48, 1.64-7.42) higher risk of developing adverse effects. There is 2.5 times the likelihood of severe arrhythmias and QT prolongation (OR 2.49, 1.67-3.70) on the treatment arm compared to control. Conclusion: Hydroxychloroquine with/without macrolide has shown no bene�cial effect in viral clearance, survival rates while shows signi�cant relation with the radiological improvement compared to standard of care but may increase the risk of intubation, overall side effects, and cardiac complications like arrhythmias and QT prolongation. Thus utilizing this treatment needs to be judged


Background
COVID-19 pandemic has spread to the whole world and has affected millions of people.During COVID-19 cases surging up, de nitive treatment for cure or prevention is not yet con rmed with a handful of evidence [1].Due to the inadequate and uncertainty of the available therapeutic option for the cure of COVID-19, the focus is also targeted towards preventive measures for breaking the chain of transmission and even towards vaccine development simultaneously with other drug trials [2].For quick drug development and treatment modalities; repurposing of many prior available medications is under trial.In this regard, an anti-malarial agent like hydroxychloroquine (HCQ) is one of the proposed and most interesting treatment options for COVID-19 [1,[3][4][5][6].HCQ acts as an anti-in ammatory molecule during cytokine storm in COVID-19 [7].
Meanwhile, concern arises regarding the clinical e cacy and safety of HCQ with or without macrolide as a treatment option for COVID-19.Hydroxychloroquine use can give rise to mild non-speci c adverse effects like nausea, vomiting, headache to severe arrhythmias affecting multiple systems [5,8].QT intervals prolongation is the commonest cardiac consequence of HCQ with drug-like azithromycin (AZT), for which the patient needs to be monitored carefully before other dire consequences like ventricular arrhythmias [9][10][11].Metabolic derangements like hypoglycemia may also occur as other adverse consequences [12].The increment in the dose of HCQ as compared to its regular use, which may be needed to reach SARS-CoV-2 inhibitory concentration, predisposes more to adverse effects [13,14].Thus, we decided to evaluate the e cacy and safety concerns of HCQ with or without macrolide in COVID-19 cases.The objective is to assess differences in virological clearance, radiological improvement, overall adverse effects, severe arrhythmias, and death rate between the treatment group (HCQ with or without macrolide and standard of care) and control group (standard of care).

Methods
We used PRISMA for the systematic review of the available literature [15].

Types of studies
We included studies conducted to determine the safety and e cacy of HCQ with/without azithromycin in addition to standard of care (SOC) for COVID-19 diagnosed cases based on guidelines with a comparison control arm were included in the present meta-analysis.

Types of participants
COVID-19 diagnosed cases based on guidelines who were enrolled either in HCQ with/without AZT in addition to standard of care or standard of care alone.

Types of interventions
HCQ with/without AZT in addition to standard of care is taken as the treatment arm and standard of care alone as a control arm.

Types of outcome measures
Clinical improvement of HCQ with/without AZT in the treatment of COVID-19; mortality rate between treatment and control group; adverse effects occurred during treatment; intubation and mechanical ventilation requirements were outcomes of interest.

Outcomes
Clinical improvement measured as virological cure and radiological progression (pneumonia resolution), overall death between treatment and control arm, overall adverse effects occurred during treatment, and de-novo severe ECG changes in the form of QT prolongation or ventricular arrhythmias leading to the necessity to stop treatment or requiring management of the adverse cardiac event, intubation and mechanical ventilation requirement between treatment and control arm were compared.

Search methods for identi cation of studies
Two reviewers (DBS and ER) have independently searched and evaluated the quality of the studies using COVIDENCE and extracted data for quantitative and qualitative synthesis, which is reviewed by another reviewer for the resolution of any possible con ict (SK).Assessment of bias and cross-checking of selected studies was done by another reviewer (PB).

Electronic searches
The electronic search strategy is provided in supplementary le 1.

Data collection and analysis
We searched electronic databases like Pubmed, Medline, Google Scholar, Cochrane Library, clinicaltirals.gov,and WHO clinical trial registry.We also checked the references section from published review articles on-screen and identify additional studies for our analysis.Extracted data for quantitative synthesis was analyzed using RevMan 5.3.Heterogeneity was assessed using the I 2 test, and the xed/random-effects model was used when appropriate for the pooling of studies.

Selection of studies
Due to the inadequate number of published randomized control trials and small-sized published Randomized Clinical Trials (RCTs), we also included prospective/retrospective observational studies with a comparison between HCQ with/without AZT in addition to standard of care and standard of care alone in the present meta-analysis.We excluded 8 studies lacking a control group receiving standard of care in addition to the treatment arm.We excluded 3 studies in which the outcome was compared between HCQ with/without AZT to HCQ alone or AZT alone, as our study is focused on comparing HCQ with/without AZT against the standard of care alone.We excluded the recently retracted paper of Mehra published in Lancet due to data discrepancy [16].We also excluded commentaries, viewpoints, reviews, in-vitro studies, editorials, letters to editors, protocols, and studies done in the pediatric population.

Data extraction and management
Selected studies were evaluated for the quality of the studies and among them having the outcome of interest were included for quantitative synthesis.

Assessment of risk of bias in included studies
The Cochrane ROB tool was used to analyze the risk of bias in our included RCTs shown in gure 1.We used the NHLBI (National Heart, Lung, and Blood Institute) quality assessment tool to assess the risk of bias in our retrospective studies and cohort studies [17].Risk-of-bias plots have been created through the RevMan 5.3.

Assessment of heterogeneity
Heterogeneity assessment was done using the I 2 test.Interpretation of I 2 test was done based on the Cochrane Handbook for Systematic Reviews of Interventions as follows: "0% to 40%: might not be important; 30% to 60%: may represent moderate heterogeneity; 50% to 90%: may represent substantial heterogeneity; 75% to 100%: considerable heterogeneity.The importance of the observed value of I 2 depends on (i) the magnitude and direction of the effect and (ii) the strength of evidence for heterogeneity (e.g.P-value from the chi-squared test, or a con dence interval for I 2 )."

Assessment of reporting biases
Predetermined outcome reporting documentation assessed to assess reporting bias.

Data synthesis
Statistical analysis was performed using RevMan 5.3 software.Risk Ratio (RR)/ Odds Ratio (OR) was used for outcome estimation whenever appropriate with 95% Con dence Interval (CI).The xed/randomeffects model was used according to heterogeneities.

Subgroup analysis and investigation of heterogeneity
In the case of heterogeneity, the inverse variance, random-effect model, and exclusion of outlier studies based on weight tried.Forest plots were presented to visualize the degree of variation between studies.

Sensitivity analysis
In our study, a sensitivity analysis was performed by examining the effect of studies on the results based on their weight when considered signi cant, outlier studies were excluded, and the analysis was re-run.

Qualitative synthesis
A total of 1385 studies were identi ed after electronic database searching.After 399 duplicates were removed, we screened the title and abstracts of 986 studies.808 studies were excluded and 178 articles were seen for full-text eligibility.A total of 164 studies were excluded with de nite reasons mentioned in the PRISMA ow diagram in gure 2. We included a total of 13 studies in our study.The summary of the 13 studies is discussed in table 2.00  A loading dose of 1200 mg HCQ followed by a maintenance dose of 400 mg OD

Quantitative synthesis of treatment outcome
In the present meta-analysis, we have compared randomized and non-randomized studies to extract outcome on virological clearance, radiological progression, overall death, development of adverse effects (severe, minor), cardiac complications (QT-prolongation, de-novo arrhythmias), the requirement of intubation, and mechanical ventilation.Among the included studies in the meta-analysis, we found there is mild-substantial heterogeneity; which may be due to clinical and variability in study design and the risk of bias among studies which could not be omitted fully may be due to acute surge to COVID-19 cases having diversity in presenting and getting treatment due to pandemic.HCQ with or without AZT regimen and SOC versus SOC only; effectiveness Among the treatment group HCQ with or without AZT in addition to SOC versus SOC, we have compared the duration of virological clearance (negative RT-PCR) and radiological progression.

Virological clearance
The meta-analysis of RR for HCQ with or without AZT in addition to SOC effectiveness compared with SOC alone using a random effect model among randomized and non-randomized studies showed that there were no signi cant differences between the two arms (RR 0.95, 95% CI 0.67 to 1.34; participants = 250; studies = 4; I 2 = 76%).Moreover, there is no signi cant risk difference (RD) between the two groups for the virological cure of HCQ with or without AZT in COVID-19 patients (RD 0.04, 95% CI -0.27 to 0.34) (Figure 3a).

Sensitivity analysis for HCQ with or without AZT in addition to SOC effectiveness compared with SOC alone
To evaluate the impact of inverse RRs as well as studies' weight on the meta-analysis results, we conducted sensitivity analyses as according to the substantial relative weight of Tang et al. on the metaanalysis, by excluding that studies, as follows no signi cant change, was observed (RR 1.15, 95% CI 0.53 to 2.48).(Supplementary le 2/ gures 1, 2).

Radiological progression (pneumonia resolution)
Among the two studies reported radiological improvement, overall RR for HCQ with or without AZT in addition to SOC compared with SOC alone using a xed-effect model showed that there was signi cant improvements among treatment arm (RR 1.40, 95% CI 1.03 to 1.91; participants = 92; studies = 2; I 2 = 0%).Additionally, there is signi cant RD between the two groups for radiological progression for pneumonia resolution among HCQ with or without AZT in COVID-19 patients (RD 0.22, 95% CI 0.03 to 0.41) (Figure 3b).

HCQ with or without AZT regimen and SOC versus SOC only; mortality
The meta-analysis of death outcome in comparative randomized and non-randomized studies showed no signi cant differences for mortality rate between HCQ with or without AZT regimen and standard treatment group compared with SOC alone (RR 1.08, 95% CI 0.65 to 1.79; participants = 4012; studies = 9; I 2 = 83%; RD -0.01, 95% CI -0.08 to 0.07) (Figure 3c).

Sensitivity analysis for HCQ with or without AZT in addition to SOC on mortality compared with SOC alone
To evaluate the impact of inverse RRs as well as studies' weight on the meta-analysis results, we conducted sensitivity analyses as according to the substantial low relative weight excluding four studies <50 participants in treatment groups; Barbosa 2020 [26], Gautret 2020 [29], Lee 2020 [25], and Yu 2020 [24]; with total 702 participants from all 4 studies on the meta-analysis.By excluding those studies, no signi cant changes were observed(RR 1.28, 95% CI 0.75 to 2.17) (Supplementary le 2/ gures 3, 4).While swapping events between the treatment group with the control group also did not show signi cance in preventing from dying [(RR (Non-event) 0.93, 95% CI 0.86 to 1.01] (Supplementary le 2/ gure 5).

HCQ with or without AZT regimen and SOC versus SOC only; mortality among RCTs
The meta-analysis of death outcome in randomized studies using xed effect showed no signi cant differences for mortality rate between HCQ with or without AZT regimen and standard treatment group compared with SOC alone (RR 2.23, 95% CI 0.35 to 14.37; participants = 80; studies = 2; I 2 = 0%; RD 0.05, 95% CI -0.05 to 0.16) (Figure 3d).

HCQ with or without AZT regimen and SOC versus SOC only; intubation and mechanical ventilation
The meta-analysis on intubation rate and mechanical ventilation among randomized and nonrandomized studies showed no signi cant differences between HCQ with or without AZT regimen and SOC versus SOC only about the odds of intubation during treatment (OR 1.52, 95% CI 0.61 to 3.77; participants = 3506; studies = 6; I 2 = 89%) (Figure 3e).
Excluding the three studies, Barbosa 2020 [26], Lee 2020 [25], and Yu 2020 [24] with substantial low relative weight (<50 participants in the treatment group) showed no signi cant odds of intubation during/after the beginning of treatment among HCQ with or without AZT regimen and SOC on the metaanalysis sensitivity (OR 1.72, 95% CI 0.51 to 5.83) (Supplementary le 2/ gure 7).

HCQ with or without AZT regimen and SOC versus SOC only: overall adverse effects
The meta-analysis of among randomized and non-randomized studies showed that the odds of having overall adverse effects among those under HCQ with or without AZT regimen addition to SOC regimen was approximately 2.2 times higher than SOC only taking individuals without HCQ regimen though it was not statistically signi cant (OR 2.21, 95% CI 0.95 to 5.17) (Figure 3f).Patients under HCQ with or without AZT regimen and SOC regimen were having 2 times higher odds of having severe adverse effects, though it is also not statistically signi cant (OR 2.08, 95% CI 0.40 to 10.74) (Figure 3f).
We conducted sensitivity analyses as according to the substantial relative weight of Rosenberg 2020 [18] (>500 participants in events) on the meta-analysis, by excluding that study also no statistically signi cant ndings observed on odds of having Overall adverse effects among HCQ with or without AZT regimen and SOC on the meta-analysis (Overall: OR 2.22, 95% CI 0.61 to 8.04) (Supplementary le 2/ gure 8).

HCQ with or without AZT regimen and SOC versus SOC only: overall adverse effects among RCTs
The meta-analysis of among randomized controlled trials showed that the odds of having overall adverse effects among those under HCQ with or without AZT regimen addition to SOC regimen was approximately 3.5 times higher than SOC only taking individuals without HCQ regimen (OR 3.48, 95% CI 1.64 to 7.42; participants = 284; studies = 4; I 2 = 0%; RD 0.14, 95% CI 0.06 to 0.22) (Figure 3g).

Arrhythmias and cant QT-prolongation
The meta-analysis of non-randomized studies showed that the odds of having Arrhythmias and signi cant QT-prolongation among those under HCQ with or without AZT addition to SOC regimen were approximately 2.5 times higher than SOC only taking individuals without HCQ regimen (OR 2.49, 95% CI 1.67 to 3.70; participants = 1400) (Figure 3h).While for sensitivity analysis done using random effect and inverse variance, it showed no signi cant odds of developing arrhythmias and QT prolongation (Supplementary le 2/ gure 9).

Clinical trials
are 207 trials registered focusing on the safety and e cacy of hydroxychloroquine for COVID-19 treatment along with different parameters around the globe as of 30 May 2020 [31] (Supplementary table 2).Among these,

Discussion
COVID-19 pandemic has become an alarming issue as it continues to spread all over the world and affect people globally.About the impact this has made over global health, studies dedicated to nding out an effective treatment for this issue remain inadequate.Therefore, to contribute to establishing an evidence-based treatment for such an issue, this meta-analysis was done including 13 studies from all over the globe.The drug hydroxychloroquine (HCQ) has been used as an antimalarial and anti-rheumatic drug for a long time and its side effects have been studied well over time.Although its repurposed use in the treatment of the newly discovered viral pandemic, COVID-19 necessitates us to look further into the clinical e cacy of this drug in curing patients.Although few meta-analyses have been done, we found that they were limited to small sample sizes and had some errors in data entry as well.These factors aided in the rationale for conducting this study.
The primary outcome for the meta-analysis was the mortality rate between the treatment and control group, 9 studies were included which showed no statistically signi cant difference between the two groups (RR 1.08, 95% CI 0.65 to 1.79).This contrasts with Singh's meta-analysis which showed an increased risk of death (RR, 2.17; 95% 1.32 to 3.57) in patients taking HCQ [32].Our analysis contained 9 studies compared to Singh's study and a larger sample size may have led to differences in results.Our study, however, was similar to Chacko's meta-analysis where no difference in mortality was seen (OR: 1.41, 95% CI: 0.76-2.62;p = 0.28) [33].
Evidence of adverse effects was seen in the treatment group compared to the control group with 7 studies (1756 cases) included.Overall adverse effects were present 2.2 times more in the treatment group, although statistical signi cance was not established (OR 2.21, 95% CI 0.95 to 5.17), though clinical relevance needs to be judged while using HCQ with or without AZT in treatment.However, the overall adverse effect among the treatment arm is approximately 3.5 times (OR 3.48, CI 1.64-7.42)higher than the control arm while taking 4 RCTs only, but the power is low because of small-sized studies.The risk of severe side effects was 2.08 times more in the treatment group without statistical signi cance (OR 2.08, 95% CI 0.4 to 10.74).Chacko and Ren both found a statistically signi cant increased side effect risk with the use of hydroxychloroquine [33,34].Even though the results were not proven statistically, the presence of side effects must be kept in mind while the use of HCQ is considered.Cardiac adverse effects have been an important concern with the use of HCQ and a signi cant result establishes the need for caution while using the drug.The statistically signi cant result showing 2.49 times higher odds of having arrhythmias and signi cant QT prolongation in cases treated with HCQ with/without AZT with SOC (OR 2.49, 95% CI 1.67-3.70).It is in concordance with multiple studies that have shown an increased risk of QT among patients taking hydroxychloroquine [9,10].Before Mehra et al's paper retraction, when we analysed data including from that paper showed 10 times higher odds of having arrhythmias and signi cant QT prolongation which has also been attached with the other portions of the analysis in the supplementary le [Supplementary le 3][16].
The secondary outcomes for the meta-analysis were clinical improvement where no signi cant difference (RR or RD) was observed based on the virological cure (negative RT-PCRs), while it showed improvement in radiological progression where 4 studies (250 cases) and 2 studies (92 cases) were included respectively.No improvement in virological clearance was similarly seen in Sarma, Singh, and Chacko's meta-analysis [32,33,35].Our nding of radiological improvement among patients taking HCQ with/without AZT is similar to Chacko and Sarma's analysis of improvement in CT ndings following intake of HCQ [33,35].Forest plot g (C:-18; HCQ : -27; HCQ + AZT : Figures

Table 1 :
-NHLBI quality assessment tool for observational cohort and cross-sectional studies Good if they ful lled 60-100% of the tool items, Fair if 50-59% or Poor if 0-49%.

Table 2 :
Summary of included studies [31]ials have recently been completed.A total of 103 trials are recruiting participants, 74 trials have not yet started recruiting, 9 trials are active but not recruiting participants, and 10 trials are enrolling by invitation.Out of these, 16 trials are observational and the rest are RCTs.Altogether, 6 trials are either suspended or terminated or withdrawn due to different reasons.According to the location provided, around 42 countries are regulating trials on such subjects, where the USA is at the highest position conducting most of the 52 trials followed by France which is managing 24 trials.The largest trial is RCT conducted in Thailand with 40,000 cases, while the smallest trial is an observational type conducted in Belgium with 12 participants[31].