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“Hydroxychloroquine in patients with COVID-19: A Systematic Review and meta- analysis.”

Awadhesh Kumar Singh, Akriti Singh, Ritu Singh, Anoop Misra

PII: S1871-4021(20)30136-3

DOI: https://doi.org/10.1016/j.dsx.2020.05.017

Reference: DSX 1672

To appear in: Diabetes & Metabolic Syndrome: Clinical Research & Reviews

Received Date: 6 May 2020

Revised Date: 7 May 2020

Accepted Date: 7 May 2020

Please cite this article as: Singh AK, Singh A, Singh R, Misra A, “Hydroxychloroquine in patients with COVID-19: A Systematic Review and meta-analysis.”, Diabetes & Metabolic Syndrome: Clinical Research & Reviews (2020), doi: https://doi.org/10.1016/j.dsx.2020.05.017.

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.

© 2020 Published by Elsevier Ltd on behalf of Diabetes India.

“Hydroxychloroquine in patients with COVID-19: A Sy stematic Review and

Meta-analysis.”

Types of article: Research article

Authors name in order: Awadhesh Kumar Singh, Akriti Singh, Ritu Singh,

Anoop Misra

Authors affiliation:

Awadhesh Kumar Singh: M.D; D.M, Senior Consultant, Diabetes &

Endocrinology, G.D Hospital & Diabetes Institute, Kolkata, West Bengal, India.

Akriti Singh: MBBS, Medical Resident, College of Medicine and JNM Hospital,

Kalyani, Nadia, West Bengal, India.

Ritu Singh: M.D, Senior Consultant, Gynaecology & Obstetrics, G.D Hospital &

Diabetes Institute, Kolkata, West Bengal, India.

Anoop Misra: M.D, Director, Fortis C-DOC Hospital for Diabetes and Allied

Sciences, New Delhi, India.

Highlights:

• The role of hydroxychloroquine in the treatment of COVID-19 is not fully

known.

• While initial studies with hydroxychloroquine showed some ray of hope,

recent studies that have emerged found either no benefit or a possible harm

in COVID-19.

• This meta-analysis showed no benefit on viral clearance, although a

significant increase in death was observed with hydroxychloroquine in

patients with COVID-19, compared to the control.

Abstract:

Backgrounds and Aims

The role of hydroxychloroquine (HCQ) in the treatment of COVID-19 is not fully

known. We studied the efficacy of HCQ compared to the control in COVID-19

subjects on a. viral clearance measured by reverse transcriptase polymerase chain

reaction (RT-PCR) and, b. death due to all cause.

Methods

PubMed, Scopus, Cochrane and MedRxiv database were searched using the

specific keywords up to April 30, 2020. Studies that met our objectives were

assessed for the risk of bias applying various tools as indicated. Three studies each

that reported the outcome of viral clearance by RT-PCR and death due to all cause,

were meta-analyzed by applying inverse variance-weighted averages of

logarithmic risk ratio (RR) using a random effects model. Heterogeneity and

publication bias were assessed using the I2 statistic and funnel plots, respectively.

Results

Meta-analysis of 3 studies (n=210) on viral clearance assessed by RT-PCR showed

no benefit (RR, 1.05; 95% CI, 0.79 to 1.38; p=0.74), although with a moderate

heterogeneity (I2=61.7%, p=0.07). While meta-analysis of 3 studies (n=474)

showed a significant increase in death with HCQ, compared to the control (RR,

2.17; 95% 1.32 to 3.57; p=0.002), without any heterogeneity (I2=0.0%, p=0.43).

Conclusions:

No benefit on viral clearance but a significant increase in mortality was observed

with HCQ compared to control in patients with COVID-19.

Keywords: Hydroxychloroquine, COVID-19, viral clearance, outcomes, death

1. Introduction:

Scientist and physicians are working at heightened pace to research the treatment

of coronavirus infection (COVID-19). Several potential candidate drugs have been

tried in COVID-19. From these list of candidate drugs, two anti-malarial drugs

came into limelight for following reasons. Initial studies found both chloroquine

(CQ) and its derivative hydroxychloroquine (HCQ) inhibits SARS-CoV-2

effectively in vitro [1-3]. This led clinicians to believe that both drugs may have

good potential in the treatment of COVID-19.

First report of human trial came from China. A commentary by Gao et al [4]

referring to 15 Chinese trials (whose complete results are still not available),

claimed benefit with CQ in inhibiting the exacerbation of pneumonia, improving

lung imaging findings, promoting a virus-negative conversion, and shortening the

disease in more than 100 patients. One study from these 15 Chinese trials,

conducted by Chen et al [5] later showed data of 62 patients and found that HCQ

significantly improved the clinical recovery (fever and cough) and pneumonia

assessed by chest CT scan, compared to the control. However, a close look into

this randomized control trial (RCT) found that the endpoints specified in the

published protocol differed from those reported. First, the trial was originally

supposed to report the results from two different dosage of HCQ on clinical and

radiological outcome, although only the report of higher dose HCQ was reported

finally. Second, the trial was stopped prematurely [6]. Another study from France,

a non-randomized trial of HCQ (n=36) by Gautret et al [7] also reported a

significant effect of HCQ and HCQ plus azithromycin (AZ) in lowering viral load

and viral clearance compared to control as measured by reverse-transcriptase

polymerase chain reaction (RT-PCR). However, this study was widely criticized

due to the poor trial design, unreliable conclusions, no clinical endpoints,

assessments made on day 6 despite a planned 10 days trial, different value of Cycle

threshold for RT-PCR, and derivation of results after excluding six patients from

the HCQ arm [8]. The publishing journal’s society also subsequently declared that

the trial by Gautret and Colleagues did “not meet the Society’s expected standard”

[9].

Nevertheless, based on these limited observational and anecdotal evidence, several

guidelines across the world allowed both these drugs in the treatment of COVID-

19 [10]. Interestingly, Indian Council of Medical research hurriedly issued a

guideline and additionally recommended the use of CQ and HCQ as a prophylactic

agent in the close contacts including the health care workers [11]. Surprisingly,

based on these emerging developments, US President while addressing the nation

on pandemic claimed CQ and HCQ as a “game changer” in the treatment of

COVID-19. The consequence of this announcement resulted in FDA issuing an

Emergency Use Authorization (EUA) to use both the drugs in the treatment of

COVID-19 on March 28, 2020. Historically, this new EUA represents the second

time when FDA has ever used any emergency authority to permit use of a

medication for an unapproved indication. Earlier, an investigational neuraminidase

inhibitor, peramivir was given similar EUA by FDA during the 2009-2010 for

severely ill patients with H1N1 influenza. Although later an RCT failed to show

any benefit of peramivir in severely ill hospitalized patients with influenza,

compared to the placebo. Nonetheless, peramivir is approved only for

uncomplicated influenza since 2014.

Since several newer studies of HCQ on COVID-19 have recently become

available, we aimed to study its effect on COVID-19 on two important objective

outcomes. These two important outcomes include – a. viral clearance by RT-PCR

negativity and, b. death due to all cause. In addition, we have also compiled the

results from all the studies that have studied the efficacy and safety of HCQ in

COVID-19, including non-controlled trials.

2. Methods:

This study was conducted in accordance with the Preferred Reporting Items for

Systematic Reviews and Meta-Analyses (PRISMA) [12]. However, this study has

not been registered in the International Prospective Register of Systematic Reviews

(PROSPERO).

2.1 Search strategy and Inclusion criteria:

Three authors (AKS, AS and RS) systematically searched the PubMed, Scopus,

Cochrane library and MedRxiv data base up to April 30, 2020. The key terms

searched were ‘‘Hydroxychloroquine’’ OR ‘‘HCQ’’ (All Fields) OR “viral

clearance” OR “death” OR “clinical recovery” AND COVID-19 OR SARS-CoV-

2. We retrieved all the studies conducted with hydroxychloroquine in patients with

COVID-19 that was compared to control and explicitly reported at least one

outcome of interest which include viral clearance by transcriptase polymerase

chain reaction (RT-PCR) and or death due to all cause.

We excluded case reports, preclinical studies, studies that did not report outcomes

with HCQ in COVID-19, and studies that did not compare the outcomes with HCQ

compare to placebo or control. The studies that met our predefined inclusion

criteria were screened by three authors (AKS, RS and AS), and the studies that

entirely fulfilled our inclusion criteria were retrieved with their supplementary

appendix for further review. Any ambiguity during study selection was resolved

by mutual discussion and consensus. One study whose full text was available in

Chinese (abstract in English) was translated to English by Google translator and

one study was retrieved through hand search. A detailed PRISMA flow-diagram

for the search strategy is included in figure 1.

2.2 Assessment of bias and Statistical Analysis:

Four reviewers (AKS, AS, RS and AM) independently assessed the studies for risk

of bias ascertained through Jadad checklist, ROBINS-I tool and Newcastle-Ottawa

scale for randomized, non-randomized and observational studies, wherever

appropriate [13-15] and any disagreements were resolved through mutual

discussion and consensus. Scoring of these studies on risk of bias tools have been

outlined in supplementary table 1. A detailed PRISMA checklist has been

appended in supplementary table 2.

Comprehensive meta-analysis (CMA) software Version 3, Biostat Inc. Englewood,

NJ, USA was used to calculate all the statistical analyses. Seven studies were

retrieved that reported any outcome with HCQ compared to the control in COVID-

19. Three studies each reported for viral clearance measure by RT-PCR and the

outcome of death due to any cause. We meta-analyzed the pooled data of primary

outcomes of 3 trials that reported the rate of PCR negativity, and 3 trials that

reported the difference in mortality between HCQ and control arm. Since one RCT

by Chen et al reported resorption of pneumonia on chest computed tomography

(CT) as a primary outcome but neither reported RT-PCR negativity, nor the

mortality outcome, thus we did not include this study in the meta-analysis,

however the outcome of this study shall be discussed.

Estimates from all the eligible studies have been combined by applying inverse

variance-weighted averages of logarithmic risk ratio (RR), using random-effects

analysis. Heterogeneity was measured using Higgins I² and Cochrane Q statistic

[16]. Heterogeneity was considered as low (I2 <25%) or moderate (25-50%) or high

(>50%). All the p reported here are two-sided and a p value of < 0.05 is considered

to be statistically significant. We also evaluated the potential publication bias by

applying funnel plots using the “trim and fill” adjustment, rank correlation test and

the Egger’s test.

3. Results:

The overview of results including the risk of bias from all the 7 studies that

compared HCQ to the control in COVID-19 have been summarized in table 1 [5, 7,

17-21]. The meta-data that was used in this metanalysis has been also represented

in table 2. Table 3 summarizes the safety and efficacy of all the 10 trials conducted

with HCQ in COVID-19, to date [5, 7, 17-24]. One RCT by Chen et al [5] that is

not included in this meta-analysis found “any improvement” in pneumonia were

significantly higher in HCQ arm, compared to the control (80.6 vs. 54.8%,

p=0.048). Moreover, significant improvement in chest CT (more than 50%

absorption of pneumonia) was increasingly observed in HCQ arm, compared to the

control (61.3 vs. 16.1%, p=not reported).

Nevertheless, the meta-analysis of 3 studies (n=210) that reported the rate of PCR

negativity (figure 2) found no benefit with HCQ, compared to the control (RR,

1.05; 95% CI, 0.79 to 1.38; p=0.74), although with a moderate heterogeneity

(I2=61.7%, p=0.07). After the adjustment of publication bias, the Trim and Fill

imputed the RR of 0.99 with 95% CI 0.69 to 1.42 (supplementary figure SF1).

However, the meta-analysis of 3 trials (n=474) that reported the mortality outcome,

showed a significant (2-fold) increase in death in HCQ arm (figure 3), compared to

the control (RR, 2.17; 95% 1.32 to 3.57; p=0.002), without any heterogeneity

(I2=0.0%, p=0.43) and publication bias (supplementary figure SF2).

4. Discussion:

To our knowledge, this would be the most updated meta-analysis to report the

effect of HCQ on viral clearance and mortality outcome, compared to the placebo

that included 6 studies. Additionally, we have also analyzed the results from all the

10 studies available that have studied the efficacy and safety of HCQ in patients

with COVID-19 (table 3).

A recent meta-analysis published by Sarma et al [25] have showed no difference in

viral clearance and composite of death or clinical worsening with HCQ, while a

significant improvement in radiological progression was observed, compared to the

control. However, the meta-analysis by Sarma et al seems to have overlooked the

raw data and mistakenly included the wrong denominators. For example – they

included number of patients for HCQ plus azithromycin (n=20) in their analysis,

rather than HCQ alone (n=14), for the denominator for viral clearance. Similarly,

the number of patients included for the composite of death or clinical worsening in

HCQ arm was also overlooked and mistakenly reported in denominator (n=20),

rather than the actual number (n=26). We believe that these differences could have

changed the outcomes.

We do acknowledge a number of limitations in our analysis that include lesser

number of patients overall, lack of individual patient data, combining the results of

RCT with other non-randomized studies and the inclusion of pre-print version of

some of the unpublished studies. Moreover, outcomes are not adjusted for multiple

confounding factors and no sensitivity analysis were made. Besides, this

metanalysis was not registered at PROSPERO.

While this meta-analysis found no benefit of HCQ in the treatment of COVID-19

on viral clearance and there was a 2-fold increase in death compared to the control

arm, this could have been skewed by the one larger study that have shown a

significant harm with HCQ, even when other smaller studies found no significant

difference. For example, the study by Magagnoli et al (n=368) [21] found that

there was no difference in the requirement of mechanical ventilator (MV) and

death in patients who were on MV. However, the risk of death from any cause was

higher in the HCQ group (adjusted hazard ratio 2.61, 1.10-6.17, p=0.03), compared

to the control. Since this study contributed more than 84% of weight in this pooled

meta-analysis of 3 studies, the signal of significant death appears to emerge.

Moreover, relatively elderly patients (mean age 68 year) and more sick (moderate

to severe COVID-19) patients were studied in Magagnoli et al study, compared to

all other studies. Therefore, the purported benefit of HCQ in early or mild

COVID-19 as observed in studies by Chen et al [5] and Gautret et al [7] cannot be

entirely ruled out, from the result of this meta-analysis. It is also possible that

HCQ may have some benefit in early and mild COVID-19 but possibly harmful in

moderate to severe COVID-19.

Nevertheless, none of these studies attributed the harm of HCQ directly linked to

the cardiac side effect. However, a recent double-blind RCT, Cloro-Covid-19

conducted by Borba et al [26] hinted of high lethality with the higher dosage of

chloroquine (CQ). Higher dose of CQ was associated with 39% death, compared to

15% death in lower dose arm. Fatality rate with high dose of CQ was as high as

60% in patients with underlying heart disease. QTc prolongation was significant in

19% of cases on high dose CQ compared to 11% in low dose CQ arm. Although no

signals of torsade de pointes were noted in this trial, it is believed that increase in

mortality in this trial could be attributed to the combination of CQ with

azithromycin (AZ) and oseltamivir or lopinavir/ritonavir, all of which can prolong

QTc interval [27]. Similarly, emerging studies from France and USA have

increasingly cautioned for QTc prolongation with both HCQ and HCQ plus AZ.

While Bessière et al [28] reported (n=40) a prolonged QTc in 93% of the patients

receiving either HCQ or HCQ plus AZ; Mercuro et al [29] reported QTc

prolongation (n=90) in 20% of patients treated with HCQ alone or HCQ plus AZ.

These findings underscores the safety of HCQ in the light of negligible benefit

observed in some of these studies.

Despite several limitations of this meta-analysis, we feel this finding would instill

some degree of skepticism and shall help in curbing the exuberant use of over

enthusiastically claimed “magical” drug. Hopefully, large randomized controlled

trial such as DISCOVERY (EudraCT 2020-000936-23) and RECOVERY (UK),

that is currently studying the effect of HCQ in COVID-19 and comparing it with

other anti-viral drugs will finally decide its fate. Meanwhile, we believe that any

prudent clinician would follow a pragmatic approach and shall apply these drugs

only after assessing the potential risk versus uncertain benefit.

5. Conclusions:

While no benefit on viral clearance demonstrated by HCQ compared to the control

in patients with COVID-19, a significant 2-fold increase in mortality with the HCQ

warrants its use if at all, with an extreme caution, until the results from larger

randomized controlled trials are available.

Funding: Not funded

Conflict of interest: Nothing to declare

Ethical permission: Not required as this analysis do not involve patients directly.

References:

1. Wang M, Cao R, Zhang L, Yang X, Liu J, Xu M, et al. Remdesivir and

chloroquine effectively inhibit the recently emerged novel coronavirus (2019-

nCoV) in vitro. Cell Res 2020;30:269-271.

2. Yao X, Ye F, Zhang M, Cui C, Huang B, Niu P, et al. In Vitro Antiviral Activity

and Projection of Optimized Dosing Design of Hydroxychloroquine for the

Treatment of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

Clin Infect Dis 2020;5801998:pii:ciaa237.

3. Liu J, Cao R, Xu M, Wang X, Zhang H, Hu H, et al. Hydroxychloroquine, a less

toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in

vitro. Cell Discov 2020;6:16.

4. Gao J, Tian Z, Yang X. Breakthrough: chloroquine phosphate has shown

apparent efficacy in treatment of COVID-19 associated pneumonia in clinical

studies. Biosci Trends 2020;14:72-3. 10.5582/bst.2020.01047 32074550

5. Chen Z, Hu J, Zhang Z, et al. Efficacy of hydroxychloroquine in patients with

COVID-19: results of a randomized clinical trial. Version 2. medRxiv

2020.03.22.20040758. [Preprint.] 10.1101/2020.03.22.20040758

6. Yan D, Zhang Z. Therapeutic effect of hydroxychloroquine on novel

coronavirus pneumonia (COVID-19). Chinese Clinical Trials Registry.

http://www.chictr.org.cn/showproj.aspx?proj=48880

7. Gautret P, Lagier JC, Parola P, etal . Hydroxychloroquine and azithromycin as a

treatment of COVID-19: results of an open-label non-randomized clinical trial. Int

J Antimicrob Agents 2020:105949. 10.1016/j.ijantimicag.2020.105949 32205204

8. Lenzer J. Covid-19: US gives emergency approval to hydroxychloroquine

despite lack of evidence. BMJ 2020;369:m1335. 10.1136/bmj.m1335 32238355

9. International Society of Antimicrobial Chemotherapy. Statement on IJAA paper.

Accessed April 23, 2020. https://www.isac.world/news-and-publications/official-

isac-statement

10. Singh AK, Singh A, Saikh A, Singh R, Misra A. Chloroquine and

hydroxychloroquine in the treatment of COVID-19 with or without diabetes: A

systematic search and a narrative review with a special reference to India and other

developing countries. Diabetes Metab Syndr. 2020 May-June; 14(3): 241–246.

11. Indian Council for Medical Research. Recommendation for empiric use of

hydroxychloroquine for prophylaxis of SARS-CoV-2 infection.

https://icmr.nic.in/sites/default/files/upload_documents/HCQ_Recommendation_2

2March_final_MM_V2.pdf. Accessed 3 April 2020.

12. Shamseer L, Moher D, Clarke M, Ghersi D, Liberati A, Petticrew M, et al.

Preferred reporting items for systematic review and meta-analysis protocols

(PRISMA-P) 2015: elaboration and explanation. BMJ 2015; 350: g7647

13. Jadad AR, Moore RA, Carroll D, et al. Assessing the quality of reports of

randomized clinical trials: is blinding necessary? Control Clin Trials 1996;17:1–

12.

14. Sterne JAC, Hernan MA, Reeves BC, Savoic J, Berkman ND, Viswanathan M.

et al. ROBINS-I: a tool for assessing risk of bias in non-randomised studies of

interventions. BMJ 2016;355:i4919

15. Stang A. Critical evaluation of the Newcastle-Ottawa scale for the assessment

of the quality of nonrandomized studies in meta-analyses. Eur J Epidemiol 2010;

25(9): 603-5.

16. Higgins JP, Thompson SG, Deeks JJ, Altman DG. Measuring inconsistency in

meta-analyses. BMJ 2003; 327: 557–60.

17. Jun C, Danping L, Li L, et al. A pilot study of hydroxychloroquine in treatment

of patients with common coronavirus disease-19 (COVID-19). J Zhejiang Univ

(Med Sci) 2020. doi: 10.3785/j.issn.1008-9292.2020.03.03

18. Tang W, Cao Z, Han M, Wang Z, Chen J, Sun W, et al. Hydroxychloroquine in

patients with COVID-19: an open-label, randomized, controlled trial. doi:

https://doi.org/10.1101/2020.04.10.20060558

19. Barbosa J, Kaitis D, Ryan F, Kim L, Xihui L. Clinical Outcomes of

Hydroxychloroquine in Hospitalized Patients with COVID-19: A Quasi-

Randomized Comparative Study. Bibliovid 2020. https://bibliovid.org/clinical-

outcomes-of-hydroxychloroquine-in-hospitalized-patients-with-covid-19-a-302

20. Mahevas M, Tran V-T, Roumier M, et al. No evidence of clinical efficacy of

hydroxychloroquine in patients hospitalized for COVID-19 infection with oxygen

requirement: results of a study using routinely collected data to emulate a target

trial. medRxiv 2020:2020.04.10.20060699.

21. Magagnoli J, Narendran S, Pereira F, Cummings T, Hardin HW, Sutton SS, et al.

Outcomes of hydroxychloroquine usage in United States veterans hospitalized with

Covid-19. doi: https://doi.org/10.1101/2020.04.16.20065920

22. Molina JM, Delaugeree C, Goff JL, et al. No evidence of rapid antiviral

clearance or clinical benefit with the combination of hydroxychloroquine and

azithromycin in patients with severe COVID-19 infection. Med Mal Infect (2020),

https://doi.org/10.1016/j.medmal.2020.03.006

23. Gautret P, Lagier JC, Parola P, et al. Clinical and microbiological effect of a

combination of hydroxychloroquine and azithromycin in 80 COVID-19 patients

with at least a six-day follow up: an observational study. IHU Méditerranée

Infection. 2020;27(1).

24. Million M, Lagier JC, Gautret O, et al. Early treatment of 1061 COVID-19

patients with hydroxychloroquine and azithromycin, Marseille, France. Bibliovid.

2020.

25. Sarma P, Kaur H, Kumar H, et al. Virological and clinical cure in COVID�19

patients treated with hydroxychloroquine: A systematic review and meta�analysis.

Journal of Medical Virology. Published on April 16, 2020.

https://doi.org/10.1002/jmv.25898

26. Borba MGS, Val FFA, Sampaio VS, et al; CloroCovid-19 Team. Effect of high

vs low doses of chloroquine diphosphate as adjunctive therapy for patients

hospitalized with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

infection: a randomized clinical trial. JAMA Open. 2020;3(4):e208857.

doi:10.1001/jamanetworkopen.2020.8857

27. Fihn SD, Perencevich E, Bradley SM. Caution Needed on the Use of

Chloroquine and Hydroxychloroquine for Coronavirus Disease 2019. JAMA

Network Open. 2020;3(4.23):e209035. doi:10.1001/jamanetworkopen.2020.9035

28. Bessière F, Roccia H, Delinière A, et al. Assessment of QT Intervals in a case

series of patients with coronavirus disease 2019 (COVID-19) infection treated with

hydroxychloroquine alone or in combination with azithromycin in an intensive

care unit. JAMA Cardiol. Published online May 1, 2020.

doi:10.1001/jamacardio.2020.1787

29. Mercuro NJ, Yen CF, Shim DJ, et al. Risk of QT interval prolongation

associated with use of hydroxychloroquine with or without concomitant

azithromycin among hospitalized patients testing positive for coronavirus 2019

(COVID-19) infection. JAMA Cardiol. Published online May 1, 2020. doi:10.

1001/jamacardio.2020.1834

30.

Table 1: Studies of HCQ compared to placebo in patients with COVID-19

Study Types of studies

Country Age (mean, years)

N Case Control Severity of COVID-19

HCQ dose/day X Days

Primary outcome

Secondary outcome

Improvement in Primary outcome

Improvement in Secondary outcome

Chen5 et al.* (ChiCTR 2000029559)

RCT China 44.7 62 31 31 Mild/ moderate

400 mg/d X 5D

Time to clinical recovery and improvement of pneumonia in chest CT

NR Yes NR

Jun17 et al.* (NCT04261517)

RCT China NR 30 15 15 Mild/ moderate

400 mg/d X 5D

Viral load by RT-PCR + vs. – at day 7

NR No NR

Tang18 et al.* (ChiCTR 2000029868)

RCT China 46 150 75 75 Mild/ Moderate (84%)

1200 mg/d X 3D, followed by 800 mg/d X 2 wks. (mild /moderate cases) or 3 wks. (severe cases)

Viral load by RT-PCR + vs. – at day 28

Clinical symptoms, normalization of laboratory parameters and chest radiology

No No. However, reduction in CRP and symptoms noted in HCQ arm in post- hoc analysis

Gautret7 et al.**

nRCT France 45.1 36 20# 16 Mild/ moderate

600 mg/d X 10D

Viral load by RT-PCR + vs. – at day 6

Improvement in symptoms, mortality

Yes NR

Barbosa19 et al.**

qRCT USA 62.7 63 32 31 Mild/ moderate

800 mg/d X 1-2D followed by 200-400 mg OD X 3-4D

Need to escalate respiratory support and rate of intubation at day 5

Change in lymphocyte count, NLR, and mortality

No, rather harm in HCQ arm

No, direction towards harm

Mahevas20 et al.***

Retro France 60 181 84 97 Pneumonia requiring O2 Rx

600 mg/d X 7D

ICU transfer or death from any cause at day 7

All-cause mortality at day 7, Occurrence of ARDS within 7 day

No No

Magagnoli21 Retro USA 68 368 210## 158 Mild/ NR Need for MV Death in No benefit. No

et al.*** moderate and death from any cause

patients on MV Risk of death due to any cause was higher in HCQ arm

Molina22 et al

POS France 58.7 11 11 0 Fever and O2 Rx (severe)

600 mg/d X 10D + AZ 500mg on day 1 and 250 mg 2-5 days

Viral load by RT-PCR + vs. – at day 5-6

NR No NR

Gautret23 et al

POS France 52.1 80 80 0 Mild (92%) /moderate

600 mg/d X 10D + AZ 500 mg on day 1 and 250 mg/d X 4D

Need for O2 therapy or ICU admission

Viral load, length of hospital stays

Yes Yes

Million 24 et al

POS France 43.6 1061 1061 0 Mild (95%) /moderate

600 mg/d X 10D + AZ 500 mg on day 1 then 250 mg/d X 4D

death, negative RT-PCR

NR Yes NR

* Quality assessed as 5/8 on Jadad checklist, ** Moderate quality on ROBINS I tool, *** Quality assessed as 7/8 on Newcastle-Ottawa Scale, #6 patients received HCQ plus AZ, ##113 received HCQ plus AZ, HCQ- hydroxychloroquine, AZ- azithromycin, RCT – randomized controlled trial, nRCT- Non-randomized controlled trial, qRCT- quasi- randomized controlled trial, RT-PCR- reverse transcriptase polymerase chain reaction, ICU- intensive care unit, ARDS- acute respiratory distress syndrome, MV- mechanical ventilators, NR- not reported, CT- computed tomography, D- days, d- daily, O2- oxygen, Rx- treatment, POS- prospective observational studies

Table 2: Meta-data for analysis and results

Study N Types of outcome assessed

Outcome assessed for, N

Events in HCQ arm, n

Total case on HCQ arm, N

Events in control arm, n

Total control arm, N

Relative risk, 95% CI, p value

Chen et al 62 Absorption pf pneumonia

62 25 31 17 31 1.47, 1.02-2.11, p=0.037

Jun et al 30 RT-PCR negativity 30 13 15 14 15 0.93, 0.73-1.18, p=0.55 Gautret et al 36 RT-PCR negativity 30# 8 14 2 16 4.57, 1.16-18.05, p=0.03 Tang et al 150 RT-PCR negativity 150 59 70 65 80 1.04, 0.90-1.20, p=0.622 Barbosa et al 63 Death 38* 2 17 1 21 2.47, 0.24-24.98, p=0.44 Mahevas et al 181 Death 181 3 84 4 97 0.87, 0.20-3.76, p=0.85 Magagnoli et al 368 Death 255## 27 97 18 158 2.44, 1.42-4.19, p=0.001 #6 patients on HCQ plus AZ not analyzed, ## 131 patients on HCQ plus AZ not analyzed, * 38 patients matched control analyzed, HCQ- hydroxychloroquine, AZ- azithromycin, CI- confidence interval

Table 3: Descriptive results, adverse events and limitation of all the trials done with Hydroxychloroquine as on April 30, 2020.

Study Details of primary and secondary outcome Result of primary and secondary outcome Adverse events noted

Limitations of the study

Chen5 et al i. Clinical recovery is defined as the return of body temperature (36.6 °C on the surface, ≤ 37.2 °C under the armpit and mouth or ≤ 37.8 °C in the rectum and tympanic Membrane) and cough relief, (slight or no cough) checked 3 times daily that maintained for more than 72 h. ii. Pulmonary recovery is defined at three levels as exacerbated, unchanged, and improved (moderately improved when less than 50 % of pneumonia were absorbed, and significantly improved, when more than 50 % pneumonia were absorbed in chest CT)

i. Recovery time from fever significantly shortened in the HCQ arm compared to control (2.2 vs. 3.2 days, p=0.0008). Cough remission time was significantly reduced in the HCQ arm compared to control (2.0 vs. 3.1 days, p=0.0016) ii. Improvement in pneumonia were significantly higher in HCQ arm compared to control (80.6 vs 54.8%, p=0.048). Significant improvement in chest CT were increasingly observed in HCQ arm compared to control (61.3 vs. 16.1%, p=nr).

i. Mild adverse reactions noted in 2 patients from HCQ arm. one developed a rash, and one had headache. ii. Four of 62 patients progressed to severe COVID-19, all from control arm and none from HCQ arm.

Protocol violation from original plan. Not reported the results from lower dose HCQ and premature stoppage of the trial. Detail use of other antivirals in control group is not available.

Jun17 et al Primary endpoint was negative RT-PCR of naso-pharyngeal for COVID-19 on days 7 after randomization

i. RT-PCR negativity at day 7 in throat swabs in HCQ arm versus control were similar (86.7 vs. 93.3% respectively, p>0.05). ii. Median duration from hospitalization to PCR negative were similar in HCQ arm and placebo (4 vs. 2 days respectively, p>0.05)]. The median time for fever normalization was similar (1 days) in both arms. iii. Radiological progression in CT chest was noted less in HCQ group compared to control (33.3 vs. 46.7% respectively, p=nr). .

Transient diarrhea and abnormal liver function were seen in 26.7% cases in HCQ arm compared to 20% in controls (p>0.05)

Manuscript available in Chinese language.

Tang18 et al i. The primary endpoint was PCR negativity for COVID-19 at day 28. ii. Secondary endpoints includes the improvement of clinical symptoms such as

i. No difference in PCR negative conversion rate between HCQ and control arm at day 28 (85.4 vs. 81.3%, p=0.341). The negative conversion time in HCQ arm and control were same (median 8 vs. 7 days; HR 0.846; 0.580-

Significantly higher adverse events noted in 30% of HCQ arm compared to 8.8% of control (p=0.001).

Selecting the virus negative conversion as the primary end-point might

fever (axillary temperature of ≤36.60C), normalization of SpO2 (>94% on room air), disappearance of respiratory symptoms (nasal congestion, cough, sore throat, sputum production and shortness of breath), normalization of CRP, ESR, IL-6, TNF-α level and lymphocyte count within 28-days. In addition, PCR negativity at day 4, 7, 10, 14 or 21.

1.234; p=0.341). ii. No difference in symptoms between two arms within 28-days. No difference in PCR negativity between two arms at day 4, 7, 10, 14 or 21. iii. A significantly greater reduction of CRP observed in HCQ arm compared to control (6.986 vs. 2.723 mg/l, p=0.045). A trend in more rapid recovery of lymphopenia also observed in HCQ arm compared to control. iv. Post-hoc analysis (confounding effects of anti-viral agents removed), found a significant improvement in symptoms in HCQ arm compared to control (HR 8.83, 1.09-71.3).

The most common adverse event was diarrhea in HCQ arm compared to control (10 vs. 0%, p=0.004). Blurred vision seen in 1 patient on HCQ.

not be the most appropriate outcome. Issues to ensure the fidelity to the protocol by investigators.

Gautret7 et al

i. Primary endpoint was negative RT-PCR for COVID-19 at day-6. ii. Secondary outcomes include virological clearance overtime, improvement in symptoms (temperature, respiratory rate, length of stay at hospital), mortality, and occurrence of side effects.

i. Negative RT-PCR for COVID-19 was significantly higher in HCQ arm (70 vs. 12.5% p= 0.001) compared to control at day 6. Combination arm of HCQ plus AZ had significantly higher PCR negativity compared to HCQ alone and control (100 vs. 57.1 vs. 12.5%, p<0.001) at day 6. ii. No other details available for secondary outcome

One patient died in HCQ arm on day 3 despite negative RT- PCR. One patient stopped HCQ due to GI side effect

Poor trial design, assessments made on day 6 despite a planned 10 days trial, different value of Cycle threshold for RT- PCR, and derivation of results after excluding six patients from the HCQ arm

Barbosa19 et al

i. Primary outcome - mortality, effect on escalation of respiratory support, ii. Secondary outcome - hematology benefits (absolute lymphocyte count and NLR)

i. Significantly higher respiratory support needed at day 5 in HCQ arm compared to control (p=0.013). HCQ treatment were independent predictors of escalation of respiratory support OR 7.18, (1.50-34.51, p=0.014). In a matched subgroup analysis (n=38) also shows escalated respiratory support in HCQ arm compared to control (p=0.041).

No torsade de pointes noted

i. Baseline requirement of O2 Rx or intubation were significantly higher in HCQ arm compared to control (p=0.012). ii. Major errors in

ii. Increased trend towards worsening of NLR in HCQ arm compared to control (p=0.051).

in Table 2. HCQ arm showing 31 patients and control arm 32 patients which is just reverse to table 1.

Mahevas20 et al

i. Primary outcome – composite of transfer to the ICU and or death from any cause within 7 days. ii. Secondary outcomes- all-cause mortality at day 7 and the occurrence of ARDS within 7 days.

i. Transfer to the ICU or died within 7 days were similar in HCQ arm compared to control (20.2 vs 22.1%; RR 0.91, 0.47–1.80). ii. Percentage of all-cause death at day 7 were similar in HCQ arm compared to control (2.8 vs. 4.6%; RR, 0.61, 0.13-2.89). iii. Percentage of patients who developed ARDS within 7 days were similar in HCQ arm and control (27.4 vs. 24.1%; RR 1.14, 0.65-2.00).

ECG changes were noted in 9.5% of cases in HCQ arm that caused HCQ discontinuation. ECG changes includes prolonged QTc, First- degree AV block and LBBB.

No random assignment, potential unmeasured confounders bias and no propensity match for some important prognostic variables.

Magagnoli21 et al

i. Primary outcomes were death from any cause and the need for mechanical ventilation ii. Secondary outcome was death on those on mechanical ventilator

i. Rates of death in the HCQ, HCQ+AZ, and control arm were 27.8%, 22.1%, 11.4%, respectively. Compared to control, the risk of death from any cause was higher in the HCQ group (adjusted HR 2.61, 1.10-6.17, p=0.03) but not in the HCQ+AZ group (adjusted HR 1.14, 0.56-2.32, P=0.72). ii. Rates of need of ventilation in HCQ, HCQ+AZ, and control arm were 13.3%, 6.9%, 14.1%, respectively. The risk of ventilation was similar in HCQ (adjusted HR 1.43, 0.53- 3.79, p=0.48), and HCQ+AZ arm (adjusted HR 0.43, 0.16-1.12, p=0.09), compared to control. iii. Secondary outcome of death in patients who required mechanical ventilation was similar in HCQ (adjusted HR 4.08, 0.77-21.70, p=0.10), and HCQ+AZ arm (adjusted HR 1.20, 0.25-5.77, p=0.82), compared to the control.

Nothing reported Non-randomized, retrospective, selection bias, residual confounding, only men, median age >65 years and majority were of Black ethnicity.

Molina22

et al Primary outcome was RT-PCR negativity at day 5-6

RT-PCR was positive in 80% of cases (95% CI 49–94) at days 5-6 after treatment.

One patient had prolonged QTc on HCQ+AZ and drug was stopped

Significant comorbidities present and majority of patient had severe COVID-19.

Gautret23

et al i. Primary outcome was need for O2 therapy or transfer to the ICU after at least three days of treatment. ii. Secondary outcome was PCR negativity and length of stay in the ID ward

i. Majority of patients (81.3%) had favorable outcome and were discharged. Only 15% required oxygen therapy. ii. RT-PCR was negative in 83% of cases at day 7, and 93% of cases at day 8. iii. Mean time for discharge was 4.1 days with a mean length of stay of 4.6 days.

Minor adverse events reported with HCQ including nausea, vomiting and blurred vision.

Results of six patients from previous trials by Gautret et al were included in this study also.

Million 24 et al

Endpoints were death, negative RT-PCR i. Good clinical outcome and negative RT- PCR were obtained in 91.7% within 10 days. Prolonged viral carriage was observed in 4.4% cases who had high viral load at diagnosis (p < 0.01), however viral culture was negative at day 10. All except one had negative PCR at day 15. ii. Poor outcome was observed in 4.3% and more associated with older age (OR 1.11), severe cases (OR 10.05) and use of selective beta-blockers and ARBs (p<0.05).

No cardiac toxicity was observed, although no details of assessment of cardiac toxicity is available.

Biased associated with all observational studies. Moreover, same groups of authors may have biased belief based on positive results from previous trials.

RT-PCR – reverse-transcriptase-polymerase-chain-reaction, ARDS- acute respiratory syndrome, HCQ- hydroxychloroquine, AZ- azithromycin, CI- confidence interval, ICU- intensive care unit, MV- mechanical ventilator, HR- hazard ratio, RR- relative risk, OR- odds ratio, nr- not reported, CT- computed tomography, ESR- erythrocyte sedimentation rate, CRP- c-reactive protein, IL- interleukin, TNF- tumor necrosis factor, O2- oxygen therapy, ID- infectious disease, ECG- electrocardiogram, AV- atrioventricular, LBBB- left bundle branch block

Conflict of Interest

We hereby declare that we have no conflict of interest related to this article

Awadhesh Kumar Singh

Akriti Singh

Ritu Singh

Anoop Misra