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. 2024 Sep 12;21(9):e1004428. doi: 10.1371/journal.pmed.1004428

Evaluation of hydroxychloroquine or chloroquine for the prevention of COVID-19 (COPCOV): A double-blind, randomised, placebo-controlled trial

William H K Schilling 1,2,*, Mavuto Mukaka 1,2, James J Callery 1,2, Martin J Llewelyn 3,4, Cintia V Cruz 1,2, Mehul Dhorda 1,2, Thatsanun Ngernseng 1, Naomi Waithira 1,2, Maneerat Ekkapongpisit 1, James A Watson 2,5, Arjun Chandna 2,6, Erni J Nelwan 7,8, Raph L Hamers 2,9, Anthony Etyang 2,10, Mohammad Asim Beg 11, Samba Sow 12, William Yavo 13, Aurel Constant Allabi 14, Buddha Basnyat 2,15, Sanjib Kumar Sharma 16, Modupe Amofa-Sekyi 17, Paul Yonga 18, Amanda Adler 19, Prayoon Yuentrakul 1, Tanya Cope 1, Janjira Thaipadungpanit 1,20, Panuvit Rienpradub 1, Mallika Imwong 1,21, Mohammad Yazid Abdad 1,2, Stuart D Blacksell 1,2, Joel Tarning 1,2, Frejus Faustin Goudjo 22, Ange D Dossou 23, Abibatou Konaté-Touré 13, Serge-Brice Assi 24, Kra Ouffoué 25, Nasronudin Nasronudin 26,27, Brian Eka Rachman 26,27, Pradana Zaky Romadhon 26,27, Didi Darmahadi Dewanto 28, Made Oka Heryana 28, Theresia Novi 28, Ayodhia Pitaloka Pasaribu 29, Mutiara Mutiara 30, Miranda Putri Rahayu Nasution 30, Khairunnisa Khairunnisa 30, Fauzan Azima Dalimunthe 29, Eka Airlangga 31, Akmal Fahrezzy 31, Yanri Subronto 32, Nur Rahmi Ananda 33, Mutia Rahardjani 9, Atika Rimainar 9, Ruth Khadembu Lucinde 10, Molline Timbwa 10, Otieno Edwin Onyango 10, Clara Agutu 10, Samuel Akech 2,10, Mainga Hamaluba 2,10, Jairus Kipyego 18, Obadiah Ngachi 18, Fadima Cheick Haidara 12, Oumar Y Traoré 12, François Diarra 12, Basudha Khanal 16, Piyush Dahal 16, Suchita Shrestha 15, Samita Rijal 15, Youssouf Kabore 34, Eric Adehossi 35, Ousmane Guindo 34, Farah Naz Qamar 36, Abdul Momin Kazi 36, Charles J Woodrow 37,38, Steven Laird 39, Maina Cheeba 17, Helen Ayles 17,40, Phaik Yeong Cheah 1,2, Walter R J Taylor 1,2, Elizabeth M Batty 1,2, Kesinee Chotivanich 1,20, Sasithon Pukrittayakamee 1,20, Weerapong Phumratanaprapin 20, Lorenz von Seidlein 1,2, Arjen Dondorp 1,2, Nicholas P J Day 1,2, Nicholas J White 1,2; on behalf of the COPCOV Collaborative Group
Editor: Jens-Ulrik Jensen41
PMCID: PMC11392261  PMID: 39264960

Abstract

Background

Hydroxychloroquine (HCQ) has proved ineffective in treating patients hospitalised with Coronavirus Disease 2019 (COVID-19), but uncertainty remains over its safety and efficacy in chemoprevention. Previous chemoprevention randomised controlled trials (RCTs) did not individually show benefit of HCQ against COVID-19 and, although meta-analysis did suggest clinical benefit, guidelines recommend against its use.

Methods and findings

Healthy adult participants from the healthcare setting, and later from the community, were enrolled in 26 centres in 11 countries to a double-blind, placebo-controlled, randomised trial of COVID-19 chemoprevention. HCQ was evaluated in Europe and Africa, and chloroquine (CQ) was evaluated in Asia, (both base equivalent of 155 mg once daily). The primary endpoint was symptomatic COVID-19, confirmed by PCR or seroconversion during the 3-month follow-up period. The secondary and tertiary endpoints were: asymptomatic laboratory-confirmed Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection; severity of COVID-19 symptoms; all-cause PCR-confirmed symptomatic acute respiratory illness (including SARS-CoV-2 infection); participant reported number of workdays lost; genetic and baseline biochemical markers associated with symptomatic COVID-19, respiratory illness and disease severity (not reported here); and health economic analyses of HCQ and CQ prophylaxis on costs and quality of life measures (not reported here).

The primary and safety analyses were conducted in the intention-to-treat (ITT) population. Recruitment of 40,000 (20,000 HCQ arm, 20,000 CQ arm) participants was planned but was not possible because of protracted delays resulting from controversies over efficacy and adverse events with HCQ use, vaccine rollout in some countries, and other factors. Between 29 April 2020 and 10 March 2022, 4,652 participants (46% females) were enrolled (HCQ/CQ n = 2,320; placebo n = 2,332). The median (IQR) age was 29 (23 to 39) years. SARS-CoV-2 infections (symptomatic and asymptomatic) occurred in 1,071 (23%) participants. For the primary endpoint the incidence of symptomatic COVID-19 was 240/2,320 in the HCQ/CQ versus 284/2,332 in the placebo arms (risk ratio (RR) 0.85 [95% confidence interval, 0.72 to 1.00; p = 0.05]).

For the secondary and tertiary outcomes asymptomatic SARS-CoV-2 infections occurred in 11.5% of HCQ/CQ recipients and 12.0% of placebo recipients: RR: 0.96 (95% CI, 0.82 to 1.12; p = 0.6). There were no differences in the severity of symptoms between the groups and no severe illnesses. HCQ/CQ chemoprevention was associated with fewer PCR-confirmed all-cause respiratory infections (predominantly SARS-CoV-2): RR 0.61 (95% CI, 0.42 to 0.88; p = 0.009) and fewer days lost to work because of illness: 104 days per 1,000 participants over 90 days (95% CI, 12 to 199 days; p < 0.001). The prespecified meta-analysis of all published pre-exposure RCTs indicates that HCQ/CQ prophylaxis provided a moderate protective benefit against symptomatic COVID-19: RR 0.80 (95% CI, 0.71 to 0.91). Both drugs were well tolerated with no drug-related serious adverse events (SAEs). Study limitations include the smaller than planned study size, the relatively low number of PCR-confirmed infections, and the lower comparative accuracy of serology endpoints (in particular, the adapted dried blood spot method) compared to the PCR endpoint. The COPCOV trial was registered with ClinicalTrials.gov; number NCT04303507.

Interpretation

In this large placebo-controlled, double-blind randomised trial, HCQ and CQ were safe and well tolerated in COVID-19 chemoprevention, and there was evidence of moderate protective benefit in a meta-analysis including this trial and similar RCTs.

Trial registration

ClinicalTrials.gov NCT04303507; ISRCTN Registry ISRCTN10207947.

In a randomised controlled trial, William Schilling and co-workers investigate potential prevention of COVID-19 by chloroquine or hydroxychloroquine.

Author summary

Why was this study done?

  • At the beginning of the Coronavirus Disease 2019 (COVID-19) pandemic, there was an urgent need to find ways to prevent COVID-19.

  • Laboratory studies showed that the related 4-aminoquinolines, chloroquine (CQ), and hydroxychloroquine (HCQ), which had been used widely for over 50 years, had antiviral activity against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

  • HCQ proved ineffective in the treatment of hospitalised patients, and individual RCTs testing COVID-19 prophylaxis did not show benefit of HCQ. However, a meta-analysis of trial data did suggest some efficacy in preventing COVID-19.

  • Current evidence-based guidelines using data from the same studies recommend strongly against the use of HCQ for prophylaxis.

  • This study aimed to provide a definitive answer whether or not pre-exposure use of these drugs could prevent COVID-19.

What did the researchers do and find?

  • The COPCOV study was a double-blind placebo-controlled evaluation of CQ and HCQ COVID-19 chemoprevention. It was the largest pre-exposure prophylaxis study in COVID-19.

  • We found that CQ and HCQ were well tolerated and safe in prophylaxis. There was some evidence for protection against symptomatic COVID-19, and a reduction in workdays lost to illness.

  • Our updated meta-analysis of all chemoprevention studies in COVID-19 confirms that chemoprophylaxis with CQ or HCQ is well tolerated, safe, and provides a moderate beneficial effect in preventing COVID-19.

What do these findings mean?

  • Although CQ or HCQ are unlikely to be used in COVID-19 prevention at this stage, they could have been deployed with benefit earlier, and they might have value in future pandemics.

  • Randomised controlled trial (RCT) evidence is essential in evaluating therapeutics in the context of a pandemic.

  • Trials should be facilitated and protected so that evidence is generated rapidly and evidence-based policies can be implemented without delay to allow timely interventions.

Introduction

In the 4 years since the start of the Coronavirus Disease 2019 (COVID-19) pandemic, the majority of the world’s population has been infected. It is estimated conservatively that over 6.9 million people have died from COVID-19 [1]. At the beginning of 2020, there were no vaccines and no specific treatments, and there was substantial global concern about the projected consequences of the developing pandemic. Many existing medicines were proposed as potential therapeutics (“repurposing”). Prominent among these were the 4-aminoquinolines, chloroquine (CQ), and hydroxychloroquine (HCQ), as they had been used widely for decades in the prevention and treatment of malaria and rheumatological conditions, and they had in vitro activity against both SARS-CoV-1 and SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) [2,3]. After initial claims of benefit, their use rapidly became politicised and controversial. This unhelpful milieu was worsened in May 2020 by a prominent false claim of lethal cardiovascular toxicity [4]. The clinical trials evaluating the preventive and curative efficacy of the 4-aminoquinolines were caught, and in many cases damaged, by the controversies and regulatory decisions. Soon afterwards, large randomised controlled trials (RCTs) in patients hospitalised with COVID-19 showed definitively that HCQ treatment did not reduce mortality [5,6]. Although it has become clear that antivirals are most effective early in COVID-19, when viral burdens are highest, whereas anti-inflammatory drugs are beneficial in late disease (hospitalised patients), the negative results from the large RCTs in severe COVID-19 were extrapolated to indicate a lack of efficacy for HCQ in all stages of COVID-19 infection [7,8]. Nevertheless, HCQ was recommended widely as COVID-19 chemoprevention [9]. For the chemoprevention clinical trials attempting to provide definitive evidence the widely publicised controversies and negative regulatory responses adversely affected recruitment and study conduct. Despite this, some investigators did successfully complete their RCTs [10–23]. Taken together, these studies point towards moderate preventive efficacy even though individually most were underpowered to demonstrate benefit [24], but the evidence is far from conclusive. In contrast, most authorities recommend against HCQ [7,25]. As a result, there still remains substantial uncertainty regarding the true efficacy of HCQ in COVID-19 prophylaxis. This study’s aim was to characterise the efficacy, tolerability, and safety of HCQ/CQ pre-exposure prophylaxis in the prevention of COVID-19.

Methods

Study design

COPCOV was a multinational double-blind, randomised, placebo-controlled trial of COVID-19 chemoprevention (Fig 1). Potential investigators in 76 countries across the world were contacted to seek their interest and ability to conduct the study and follow the protocol (Fig A1 in S1 Appendix). Hydroxychloroquine was evaluated in Africa and the United Kingdom (UK), and chloroquine was evaluated in Asia. As the drugs have comparable in vitro activities, mode of action, and pharmacokinetic properties, they were considered equivalent [26,27].

Fig 1. COPCOV study participant flowchart.

Fig 1

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Participants

Initially, in the rapidly spreading pandemic, the focus was on protecting healthcare workers but, as the study progressed, the inclusion criteria were widened (S1 Appendix). The study recruited unvaccinated healthy, nonpregnant, adults aged between 17 and 70 years who were at risk of COVID-19, agreed to the study procedures, could be followed reliably for up to 5 months, and had ready access to an internet-enabled smartphone. Participants with any underlying disease or contraindication to taking 4-aminoquinolines were excluded.

Randomisation and masking

The trial statistician (MM) generated 2 separate permuted-block randomisation sequences in blocks of 10 stratified in sets of 400 drug kits (each kit comprised 10 blister packs of 10 tablets containing either drug or identical placebo) packed into boxes of 50 (hydroxychloroquine and placebo: Accord Healthcare, London, UK) and 200 (chloroquine and placebo: Utopian Pharmaceutical Co., Bangkok, Thailand) kits by Piramal Healthcare (UK) and Utopian, respectively, according to company regulations. The assignments were concealed from investigators, research staff, and participants. Kits were in sequence of randomisation and allocation was by opening in sequential order. The Statistical Analysis Plan (SAP) was completed and signed before the database lock and subsequent unblinding (S1 Statistical Analysis Plan).

After education about the study and provision of voluntary written informed consent, participants were randomised to receive either CQ or HCQ (depending on study site) or identical matched placebos (1:1 randomisation). The tablets were film-coated to conceal the taste and prevent unblinding. A loading dose of 10 mg base/kg (four 155 mg tablets for a 60 kg subject) was followed by 155 mg daily (equivalent to 250 mg chloroquine phosphate or 200 mg hydroxychloroquine sulphate) for 3 months.

Procedures

At the initial visit, participants were examined and baseline screening blood samples were taken. Participants were instructed how to use the mobile ‘phone application (ePRO, Axiom Real-Time Metrics, ON, Canada) and were asked to record their temperature twice daily (an oral electronic thermometer was provided) and symptoms at least once daily. Thereafter, each participant was reviewed in person each month. Serology serum samples were taken on days 0 and 90, or at the last visit if the participant left the study earlier. Contingency dried blood spot samples (DBS) were taken for drug measurement on days 0, 30, 60, and 90 (the analysis of drug concentrations will be reported separately). If symptoms consistent with COVID-19 occurred, the participant was asked to alert the study team by ‘phone, so that nose and throat swabs could be taken. These were stored at −80°C. The methods for SARS-CoV-2 PCR diagnosis and serological quantification of SARS-CoV-2 spike protein IgG antibodies are described in the S1 Appendix. Vaccines were deployed increasingly during the trial. Vaccinated participants were reviewed and an “end of study” serum sample was taken before, or within 3 days, of their vaccination, and were censored from the trial on receiving the first dose of vaccine.

Procedures for case identification, management, and subsequent isolation followed local and national guidelines. Continuation of the blinded study medication in confirmed COVID-19 cases was at the discretion of the attending health care professional. Overall trial monitoring was conducted by the Mahidol Oxford Tropical Medicine Research Unit Clinical Trials Support Group.

Outcomes

The primary outcome was symptomatic laboratory-confirmed COVID-19 defined as symptoms consistent with COVID-19 and laboratory evidence of SARS-CoV-2 infection (S1 Appendix). Laboratory evidence was defined by a prespecified hierarchy: first by a nose and/or throat swab PCR positive for SARS-CoV-2; second, if the PCR was negative, failed, or not done, by seroconversion (S-protein IgG antibody) based on baseline and end of study paired sera [28]; and third, only if paired sera were unavailable or uninterpretable, by seroconversion from the contingency DBS in an adapted assay. The prespecified primary endpoint determination algorithm code is provided in the GitHub repository.

A Serology Endpoint Assessment Committee (SEAC) comprising 2 external experts with extensive SARS-CoV-2 serology experience was convened to adjudicate on equivocal serology endpoints (SAP: S1 Appendix). Their judgements before trial unblinding were included in the database and regarded as final.

Secondary outcomes

There were 3 prespecified secondary outcomes: asymptomatic laboratory-confirmed SARS-CoV-2 infection; severity of COVID-19 symptoms; and all-cause PCR-confirmed symptomatic acute respiratory illness (including SARS-CoV-2 infection).

Tertiary outcomes

There were 3 generic prespecified trial tertiary outcomes: the participant reported number of workdays lost; genetic and baseline biochemical markers associated with symptomatic COVID-19, respiratory illness, and disease severity (not reported here); and health economic analyses of HCQ and CQ prophylaxis on costs and quality of life measures (not reported here).

In addition, we prespecified that a meta-analysis of previously published randomised hydroxychloroquine COVID-19 pre-exposure chemoprevention studies and the current study should be conducted (S1 Appendix and Methods A2 in S1 Appendix).

Statistical analysis

At the beginning of the pandemic, we did not know what would be the subsequent incidence of COVID-19, and estimated conservatively a 90-day incidence of 3%. We therefore planned to enrol 20,000 participants in Asia (chloroquine-based randomisation) and 20,000 in Europe/Africa (hydroxychloroquine-based randomisation). This sample size allowed for approximately 20% loss to follow-up, withdrawals, protocol deviations and non-adherence, and provided 80% power to detect a 23% reduction in symptomatic COVID-19 incidence for each drug individually with 95% confidence. Unfortunately, there were protracted delays and difficulties with recruitment as the study was starting in 2020. These were related to adverse publicity and withdrawal of regulatory approvals resulting from falsified claims of frequent serious cardiotoxicity [4,29]. The Data Safety and Monitoring Board (DSMB) then acknowledged that the original sample size would no longer be achievable, but recommended study continuation, with pooling of the HCQ and CQ results. The primary outcome was subsequently changed to include seroconversion. This resulted in a 4-fold higher event rate than initially forecasted (approximately 12%). Assuming a continued 12% event rate in the control arm a total sample size of 4,600 had >80% power to detect the previously targeted treatment effect (i.e., 23% reduction).

The primary outcome included all participants and was analysed using intention-to-treat (ITT) with a two-sided p-value <0.05 considered significant. A secondary per protocol (PP) analysis excluded participants as described in the CONSORT diagram (Fig 1) and in the SAP (S1 Appendix). Secondary and tertiary endpoints were analysed using the ITT population. Fisher’s exact test was used to compare treatment effects between groups. Risk ratios were obtained from a log-binomial model. Kaplan–Meier survival curves were estimated for the time to PCR-confirmed symptomatic COVID-19 and all-cause respiratory infection and were tested using the log-rank test. The number of workdays lost was analysed under a zero-inflated Poisson regression model. For convergence reasons, we fitted this model using a Bayesian framework in the brms R package (which uses stan to estimate posterior distributions under weakly informative priors). Data analysis was performed using Stata 17.0, StataCorp, College Station, Texas, United States of America and R version 4.2.2. All code and data required to reproduce the primary analysis are provided in https://github.com/jwatowatson/COPCOV. The study is reported according to the CONSORT guidelines for reporting of a randomised trial (S1 CONSORT Checklist).

Trial support and role of the funding source

The COPCOV trial was approved by all local ethics committees and the Oxford Tropical Research Ethics Committee (OxTREC: 25–20) and was sponsored by the University of Oxford. The DSMB met before and during the trial and reviewed all serious adverse events (SAEs). The study sponsor, funder, and the drug manufacturers had no input into the study design, conduct, oversight, analysis, or reporting. The authors vouch for the completeness and accuracy of the data. After critical review all authors agreed to submit the manuscript for publication.

Results

The COPCOV study was conducted in 26 sites in 11 countries (Benin, Côte d’Ivoire, Indonesia, Kenya, Mali, Nepal, Niger, Pakistan, Thailand, UK, and Zambia) (Table A1 and Fig A2 In S1 Appendix) and ran between 29 April 2020 and 10 March 2022 (Fig A3 in S1 Appendix). A total of 4,652 adult participants (Table 1) were randomised to hydroxychloroquine or chloroquine (HCQ: 1,299, CQ: 1,021: total: 2,320) or corresponding matched placebos (N = 2,332) (Fig 1). The median age of the participants was 29 years (interquartile range, 23 to 39). The population was generally healthy; 4.8% (225/4,652) reported having a chronic disease. There were no significant differences in tolerability, safety, or efficacy between chloroquine and hydroxychloroquine.

Table 1. Demographic details of the COPCOV study participants.

Chloroquine/hydroxychloroquine Placebo Total
N = 2,320 N = 2,332 N = 4,652
Age (years), median (IQR) 29 (23–39) 29 (24–39) 29 (23–39)
Sex, n (%)
Male 1,252 (54.0) 1,267 (54.3) 2,519 (54.1)
Female 1,067 (46.0) 1,064 (45.6) 2,131 (45.8)
Not specified 1 (0) 1 (0) 2 (0)
Temperature (°C), mean (SD) 36.4 (0.4) 36.4 (0.5) 36.4 (0.5)
Weight (kg), mean (SD) 64.8 (14.2) 65.3 (14.7) 65.1 (14.4)
Height (cm), mean (SD) 164 (9) 164 (10) 164 (9)
BMI (kg/m2), median (IQR) 23.1 (20.3–26.9) 23.3 (20.5–26.9) 23.2 (20.4–26.9)
Smoking, n (%)
Yes 374 (16.12) 395 (16.94) 769 (16.53)
Never smoked 1,789 (77.11) 1,772 (75.99) 3,561 (76.55)
Former smoker 157 (6.77) 165 (7.08) 322 (6.92)
COVID-19 in household, n/N (%) 263 (11.3) 273 (11.7) 536 (11.5)
Existing comorbidities
Chronic pulmonary disease (not asthma), n/N (%) 5 (0.2) 0 (0) 5 (0.1)
Asthma (physician diagnosed), n/N (%) 22 (0.9) 18 (0.8) 40 (0.9)
Chronic kidney disease, n/N (%) 0 (0) 0 (0) 0 (0)
Liver disease, n/N (%) 1 (0.04) 1 (0.04) 2 (0.04)
AIDS/HIV, n/N (%) 37 (1.6) 34 (1.5) 71 (1.5)
Diabetes, n/N (%) 21 (0.9) 15 (0.6) 36 (0.8)
Hypertension, n/N (%) 43 (1.9) 41 (1.8) 84 (1.8)
Cancer, n/N (%) 1 (0.04) 1 (0.04) 2 (0.04)
Condition requiring immunosuppressive drugs, n/N (%) 1 (0.04) 0 (0) 1 (0.02)
Ischaemic heart disease, n/N (%) 2 (0.09) 0 (0) 2 (0.04)
High cholesterol, n/N (%) 5 (0.2) 7 (0.3) 12 (0.3)
Any chronic condition, n (%) 118 (5.1) 107 (4.6) 225 (4.8)
Baseline symptoms
Fever, n (%) 0 (0) 0 (0) 0 (0)
Cough, n (%) 5 (0.2) 3 (0.1) 8 (0.2)
Sore throat, n (%) 0 (0) 1 (0.04) 1 (0.02)
Runny nose (Rhinorrhoea), n (%) 0 (0) 1 (0.04) 1 (0.02)
Wheezing, n (%) 1 (0.04) 0 (0) 1 (0.02)
Anosmia, n (%) 0 (0) 0 (0) 0 (0)
Chest pain, n (%) 0 (0) 1 (0.04) 1 (0.02)
Muscle pain (myalgia), n (%) 2 (0.09) 5 (0.2) 7 (0.2)
Joint pain (Arthralgia), n (%) 4 (0.2) 4 (0.2) 8 (0.2)
Shortness of breath on exertion, n (%) 0 (0) 3 (0.13) 3 (0.06)
Shortness of breath at rest, n (%) 0 (0) 0 (0) 0 (0)
Fatigue/malaise, n (%) 0 (0) 3 (0.13) 3 (0.06)
Itching, n (%) 0 (0) 1 (0.04) 1 (0.02)
Headache, n (%) 1 (0.04) 5 (0.2) 6 (0.1)
Dizziness, n (%) 2 (0.09) 3 (0.1) 5 (0.1)
Visual disturbance, n (%) 0 (0) 1 (0.04) 1 (0.02)
Abdominal pain, n (%) 0 (0) 2 (0.09) 2 (0.04)
Poor appetite, n (%) 1 (0.04) 1 (0.04) 2 (0.04)
Nausea, n (%) 0 (0) 2 (0.09) 2 (0.04)
Vomiting, n (%) 0 (0) 1 (0.04) 1 (0.02)
Diarrhoea, n (%) 1 (0.04) 0 (0) 1 (0.02)
Skin rash, n (%) 0 (0) 2 (0.09) 2 (0.04)
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Primary outcome

Symptomatic, laboratory-confirmed COVID-19 during the 3-month follow-up period occurred in 524 participants (11.3%); 240/2,320 (10.3% [95% CI, 9.2 to 11.7]) received HCQ/CQ and 284/2,332 (12.3% [95% CI, 10.9 to 13.6]) received placebo (risk ratio (RR) 0.85 [95% CI, 0.72 to 1.00; p = 0.05]) (Table 2). Symptomatic COVID-19 was PCR-confirmed in 24/2,320 HCQ/CQ recipients (1.0% [95%CI, 0.7 to 1.5]) and 56/2,332 (2.4% [95% CI, 1.8 to 3.1]) placebo recipients (RR 0.43 [95% CI, 0.27 to 0.69; p < 0.001]) (Fig 3). Among those with paired sera, symptomatic COVID-19 was diagnosed by seroconversion in 211/1,462 (14.4% [95% CI, 12.7 to 16.3]) HCQ/CQ recipients and 245/1,498 (16.4% [95%CI, 14.5 to 18.3]) placebo recipients (RR 0.88 [95% CI, 0.74 to 1.05], p = 0.2) (Table 2). In the remaining cases analysed by DBS (i.e., without paired sera), symptomatic COVID-19 was diagnosed in 25/280 (8.9% [95% CI, 5.9 to 12.9]) HCQ/CQ recipients and 26/297 (8.8 [95% CI, 5.8 to 12.6]) placebo recipients (RR 1.02 [95% CI, 0.60 to 1.72; p = 0.9]). Primary outcome data were missing for 1,114 participants (24%) for the serology component, mainly because of loss to follow-up (Fig 2). Asymptomatic infection was also common, occurring in 524 participants, thus the overall proportion of SARS-CoV-2 infection within the 3-month study was 23% (1,071 of 4,652).

Table 2. Prespecified endpoints of the COPCOV trial in the intention to treat population.

Outcome Chloroquine/hydroxychloroquine
(N = 2,320)		 Placebo
(N = 2,332)		 Risk ratio
(95% CI)		 Fisher’s exact P-value
Total participant days 181,263 184,688
Primary endpoint: Symptomatic laboratory-confirmed COVID-19. n (%); 95% CI 240/2,320
10.3 (9.1 to 11.7)		 284/2,332
12.2 (10.9 to 13.6)		 0.85
(0.72 to 1.00)		 0.051
PCR-confirmed diagnosis. n/N (%); 95% CI 24/2,320
1.0 (0.7 to 1.5)		 56/2,332
2.4 (1.8 to 3.1)		 0.43
(0.27 to 0.69)		 <0.001
Serology confirmed diagnosis (serum).
n (%); 95% CI		 211/1,462
14.4 (12.7 to 16.3)		 245/1,498
16.4 (14.5 to 18.3)		 0.88
(0.74 to 1.05)		 0.154
Serology confirmed diagnosis (DBS).
n (%); 95% CI		 25/280
8.9 (5.9–12.9)		 26/297
8.8 (5.8 to 12.6)		 1.02
(0.60 to 1.72)		 1.000
Secondary endpoints:
Asymptomatic SARS-CoV-2 infection.
n (%); 95% CI		 267/2,320
11.5 (10.2 to 12.9)		 280/2,332
12.0 (10.7 to 13.4)		 0.96
(0.82 to 1.12)		 0.617
All SARS-CoV-2 infection n (%); 95% CI 507/2,320
21.9 (20.2 to 23.6)		 564/2,332
24.2 (22.5 to 26.0)		 0.90
(0.81 to 1.00)		 0.060
All-cause respiratory illness*. n (%); 95% CI 44/2,320
1.9 (1.4 to 2.5)		 73/2,332
3.1 (2.5 to 3.9)		 0.61
(0.42 to 0.88)		 0.009
Severity score. Median (IQR) 20.0
(5–85)		 21.5
(5–89)		 NA 1.000
Tertiary endpoint:
Participant reported workdays lost		 700/181,263 932/184,688 NA 0.0002**
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*PCR-confirmed respiratory infection including COVID-19.

**Assessed by a zero-inflated Poisson regression model.

Missing outcomes in the primary endpoint ITT analysis were treated as not having had COVID-19 during the study period.

See Fig 2 for Venn diagram depicting breakdown of numbers analysed.

Fig 3.

Fig 3

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All-cause PCR-confirmed respiratory infections (left) and PCR-confirmed COVID-19 (right) over time in the HCQ/CQ recipients (green) and placebo recipients (pink). All-cause respiratory infection was a secondary endpoint. The majority (68%, 80/117) of infections were SARS-CoV-2. Log-rank test p-values are shown. Patients are right censored at the date of last visit. COVID-19, Coronavirus Disease 2019; CQ, chloroquine; HCQ, hydroxychloroquine; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2.

Fig 2. Venn diagram depicting the breakdown of numbers analysed.

Fig 2

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Those with no serology data were either those who dropped out of the study before day 30, and had no end of study samples, or had missing end of study samples, or those judged unreliable by the serology endpoint assessment committee. This group differed from the PP population, with some overlap.

Prespecified secondary outcomes

Asymptomatic COVID-19 occurred in 267/2,320 (11.5% [95% CI, 10.2 to 12.9]), HCQ/CQ recipients and 280/2,332 (12.0% [95% CI, 10.7 to 13.4]) placebo recipients (RR: 0.96 [95% CI, 0.82 to 1.12; p = 0.6]). In symptomatic patients, there were no significant differences in symptom severity scores (20.0 (5–85) versus 21.5 (5–89)). All-cause respiratory illness (mainly COVID-19) occurred in 44/2,320 (1.9% [95% CI, 1.4 to 2.5]) HCQ/CQ recipients and 73/2,332 (3.1% [95% CI, 2.5 to 3.9]) placebo recipients (RR: 0.61 [95% CI, 0.42 to 0.88; p = 0.009]) (Fig 3 and Table A8 in S1 Appendix).

Prespecified tertiary outcomes

Under a zero-inflated Poisson regression model, the mean number of workdays lost over 90 days of chemoprophylaxis was 337 (95% CI, 279 to 398) per 1,000 participants in HCQ/CQ recipients and 441 (95% CI, 370 to 515) per 1,000 in placebo recipients; a mean difference of 104 days (95% CI, 12 to 199). Similar results were observed in the ITT and per protocol analyses for all comparisons (Tables A2–A3 in S1 Appendix). Tests for genetic and biochemical markers for symptomatic COVID-19, respiratory illness or disease severity, were not conducted, as suitable specific tests were not available. A health economic analysis will be reported separately.

Safety and tolerability

The study medications were well tolerated. One HCQ recipient was hospitalised with confirmed COVID-19. There were 10 SAEs in 9 HCQ/CQ recipients and 8 SAEs in 8 placebo recipients but none were considered drug-related (Table 3 and Table A4 in S1 Appendix) and no one died. Overall, 218/2,320 (9.4%) HCQ/CQ recipients had at least 1 adverse event (AE) compared to 242/2,332 (10.4%) placebo recipients (p = 0.3). Fewer HCQ/CQ recipients had severe AEs (31/2,320; 1.3%) than placebo recipients (58/2,332; 2.5%; p = 0.005). Twelve HCQ/CQ recipients (0.52%) discontinued chemoprophylaxis either because of side-effects (N = 10) or inability to comply with study procedures (N = 2), versus 4 (0.17%) in the placebo group (p = 0.05).

Table 3. Safety and tolerability of the COPCOV study medications.

Adverse events Chloroquine/hydroxychloroquine Placebo Fisher’s exact P-value
Number of subjects N = 2,320 N = 2,332
Total participant days 181,263 184,688
Number of subjects with at least 1 AE, n (%)
Total adverse events, nE (%)		 218 (9.4)
578 (24.9)		 242 (10.4)
656 (28.1)		 0.260
Participants with at least 1 SAE, n (%):
Total events, nE (%):		 9 (0.4)
10 (0.4)		 8(0.3)
8 (0.3)
Deaths, n/N, (%) 0 (0) 0 (0)
Possible, probable, or definite drug related SAEs, n/N, (%) 0 (0) 0 (0)
Grading of adverse events, n E /N, (%) Moderate
(grade 2)
N = 2,320 		Severe
(grade 3)
N = 2,320 		Moderate
(grade 2)
N = 2,332 		Severe
(grade 3)
N = 2,332 		P-value for total severe AEs between groups
Symptoms
Number of adverse events** 547 (23.6) 31 (1.3) 598 (25.6) 58 (2.5) 0.005
Itching 7 (0.3) 0 (0) 11 (0.5) 1 (0)
Headache 85 (3.7) 5 (0.2) 85 (3.6) 11 (0.5)
Dizziness 30 (1.3) 1 (0) 20 (0.9) 0 (0)
Visual disturbance 3 (0.1) 0 (0) 7 (0.3) 0 (0)
Abdominal pain 22 (0.9) 5 (0.2) 19 (0.8) 6 (0.3)
Poor appetite 3 (0.1) 0 (0) 5 (0.2) 0 (0)
Nausea 19 (0.8) 0 (0) 19 (0.8) 2 (0.1)
Vomiting 9 (0.4) 1 (0) 8 (0.3) 1 (0)
Diarrhoea 22 (0.9) 0 (0) 17 (0.7) 2 (0.1)
Skin rash 6 (0.3) 0 (0) 3 (0.1) 1 (0)
Other 341 (14.7) 19 (0.8) 404 (17.3) 34 (1.5)
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*There were no participants with grade 4 AEs.

**SAEs are reported separately in the same table.

SAE, serious adverse event.

Prespecified meta-analysis

Including this study and the 11 previously published randomised hydroxychloroquine COVID-19 pre-exposure chemoprevention studies (Fig 3), the prespecified meta-analysis showed an overall protective efficacy against symptomatic COVID-19 (RR 0.80 [95% CI, 0.71 to 0.91; p = < 0.001]) [11,12,15–23]. Although the studies reported slightly different outcomes, used different doses and for different durations, there was no clear between-study heterogeneity or evidence of publication bias (Fig A4 in S1 Appendix).

Discussion

This large multinational randomised double-blind COVID-19 chemoprevention trial provides strong evidence of the safety of hydroxychloroquine and chloroquine given daily for 3 months (average 2.4 mg base/kg/day). The trial ended below its original recruitment objective but, because of the higher than anticipated incidence of COVID-19, it was able to provide evidence relevant to protective efficacy. For the trial’s primary endpoint (incidence of symptomatic laboratory-confirmed COVID-19), the apparent benefit observed for HCQ/CQ treatment is consistent with the aggregated results of previous smaller studies [11,12,15–23], all but one of which had a lower incidence of symptomatic COVID-19 in the HCQ treatment arm (Fig 4). Incorporating the COPCOV trial data in a prespecified meta-analysis of pre-exposure RCTs, in which there was little heterogeneity across the studies and no evidence of publication bias, suggests a moderate protective benefit (RR 0.80 [95% CI 0.71 to 0.91]). In other prespecified comparisons in the COPCOV study, HCQ/CQ chemoprevention was associated with fewer adverse events, fewer all-cause PCR-confirmed respiratory infections (mostly COVID-19), and a reduction in workdays lost, but it did not affect the incidence of asymptomatic infections or the severity of respiratory illness (although no patients required hospitalisation for hypoxia).

Fig 4. Prespecified meta-analysis of 4-aminoquinoline COVID-19 pre-exposure chemoprevention RCTs based on individual study primary endpoints according to the method of Garcia-Albeniz and colleagues [24].

Fig 4

Open in a new tab

Schilling 2024 refers to the current study. The size of the grey squares centred at the treatment effect estimates are proportional to the study weight. Risk ratios were determined for all studies based on the reported data, apart from Seet and colleagues, which was cluster randomised and a recalculated adjusted RR was used. See Methods A2 in S1 Appendix for further details. CQ, chloroquine; HCQ, hydroxychloroquine; RCT, randomised controlled trial; RR, risk ratio.

Despite this study’s size, and the combined evidence, there is still substantial uncertainty as to the true prophylactic benefit of these 4-aminoquinolines in COVID-19. The large difference between the groups in rates of PCR-confirmed diagnosis was not mirrored by a similar difference in seroconversion-based diagnosis. This could be a chance finding from PCR (as numbers were relatively small) or, more likely, results from imprecision of serodiagnosis diluting the power of the study. The DBS samples, used to determine seroconversion when paired sera were unavailable, may have provided unreliable results. The extraction of serum from DBS is variable and inefficient and, unlike the serum-based assays [28], the seroconversion thresholds have not been fully validated, although they have been used in other COVID-19 studies [30]. The absence of a significant protective effect against asymptomatic infection may indicate that protective benefit is proportional to the viral burden and thus disease severity, but it could also reflect the imprecision of the seroconversion-based endpoint.

The tolerability and safety of these well-established 4-aminoquinoline medicines is further reinforced in this double-blind comparison [31]. This was a point of controversy early in the pandemic. Initial reports described a range of toxicities that created a climate of concern and adverse opinion. This trial provides no support for these claims, and it supports the generally good short-term safety profile of these drugs [31]. It challenges the World Health Organization “living” guidelines on COVID-19 chemoprevention which emphasised toxicity in recommending against hydroxychloroquine. The same guideline also stated that prophylactic use “probably has a small or no effect on laboratory-confirmed COVID-19” with “moderate certainty,” and has no effect on mortality with “high certainty” [27]. Yet, the meta-analysis reported here suggests significant benefit. No deaths have been reported in any of the pre-exposure prophylaxis studies, and the drugs have been well tolerated with very low rates of treatment discontinuation (Figs A5–A6 in S1 Appendix) [32].

Few drugs have excited such controversy as hydroxychloroquine in COVID-19. Bureaucracy, politicisation, and polarised debate obstructed the undertaking of clinical trials needed to provide objective evidence early in the pandemic. Now, with increasing vaccine coverage, declining viral virulence and availability of effective medicines, there is little reason to recommend the 4-aminoquinolines for COVID-19 chemoprevention. But earlier in the pandemic mortality was much higher, there were no vaccines or drugs, and healthcare systems were under intense strain as their workforces were depleted by illness. If these results had been available then, a case could have been made to deploy these moderately effective, inexpensive, available, and safe 4-aminoquinolines more widely. To put the moderate prophylactic efficacy of HCQ/CQ in perspective, the WHO stated initially that COVID-19 vaccines with a protective efficacy of at least 30% would be considered [33]. In those early days, the rapid spread and the initial high mortality of COVID-19 in the absence of proven effective therapies or vaccines created global concern but, despite the seriousness of the pandemic, there were major obstacles to the conduct of urgently needed clinical research, particularly in low resource settings. In addition, the endorsement of unproven medicines and remedies (including hydroxychloroquine), and later warnings and revocations by regulatory authorities [29], the associated politicisation, and the intense media scrutiny excited controversy and polarised views. All of these factors compromised objective scientific evaluation.

There are significant limitations to this study. The final sample size was substantially smaller than intended which limits confidence in the preventive effect estimates. At the beginning of the pandemic we did not know what the incidence of COVID-19 would be, nor what magnitude of preventive benefit would be considered enough to warrant use of these medicines in prophylaxis. The trial was conducted in many resource-limited settings where collection of respiratory swab specimens and PCR diagnosis was difficult, so only a small proportion of cases could be confirmed by PCR. Seroconversion over 3 months is a less precise measure of symptomatic infection. In particular, the DBS method of assessment, although employed in other studies [30], was not validated and did not perform well in this study. A significant proportion of participants were lost to follow-up (Fig 1) although these were balanced across arms (Fig A7 in S1 Appendix) and the PP analysis corresponded well with that of the ITT population (Tables A2–A3 in S1 Appendix). In addition, some participant outcomes were not evaluable because the end-of-study results were either unavailable (early drop-out), or were considered to be unreliable by the independent Serology Endpoint Assessment Committee (Table 2 and Table A5 in S1 Appendix). Although the inclusion of the DBS assessment was prespecified in the composite endpoint, this likely reduced the precision of the endpoint ascertainment and thus the estimate of preventive efficacy (Table 2). The study primary endpoints evaluated in the meta-analysis were slightly different, but meta-analysis of the PCR-confirmed infections indicates consistent evidence of moderate protective benefit (Fig A6 in S1 Appendix).

In summary, this large double-blind, placebo-controlled, trial showed that hydroxychloroquine and chloroquine prophylaxis was safe and well tolerated, and combined with data from other similar trials provided some evidence that laboratory-confirmed symptomatic COVID-19 might be reduced. The totality of evidence from this and other RCTs suggests a moderate preventive benefit. This knowledge could have proved beneficial earlier in the COVID-19 pandemic when vaccines and treatments were lacking, and mortality and morbidity were high. RCTs should be supported during pandemics.

Supporting information

S1 CONSERVE Checklists. CONSERVE Checklists.

(DOCX)

pmed.1004428.s001.docx (19.7KB, docx)
S1 CONSORT Checklist. CONSORT 2010 checklist of information to include when reporting a randomised trial.

(DOCX)

pmed.1004428.s002.docx (62.1KB, docx)
S1 Protocol. Chloroquine/hydroxychloroquine prevention of coronavirus disease (COVID-19) in the healthcare setting; a randomised, placebo-controlled prophylaxis study (COPCOV).

(DOCX)

pmed.1004428.s003.docx (183.3KB, docx)
S1 RiskOfBias Assessment. Risk of Bias Assessment.

(XLSX)

pmed.1004428.s004.xlsx (12.5KB, xlsx)
S1 Statistical Analysis Plan. Statistical Analysis Plan.

(PDF)

pmed.1004428.s005.pdf (1.3MB, pdf)
S1 Text. Membership of COPCOV Collaborative Group.

(XLSX)

pmed.1004428.s006.xlsx (15KB, xlsx)
S1 Appendix. Table A1.

List of COPCOV study sites. Table A2. Baseline characteristics in the COPCOV trial (Per Protocol Analysis). Table A3. Outcomes of Chloroquine/Hydroxychloroquine and Placebo Pre-exposure Prophylaxis against COVID-19 in the COPCOV study (Per Protocol Analysis). Table A4. Summary of Serious Adverse Events in the COPCOV study. Table A5. Primary and secondary outcomes of Chloroquine/Hydroxychloroquine Therapy for Pre-exposure Prophylaxis against COVID-19 (missing outcomes treated as not having had COVID-19 during the study period) ITT–Results presented as “Risk differences.” Table A6. Outcomes of Chloroquine/Hydroxychloroquine and Placebo Pre-exposure Prophylaxis against COVID-19 in the COPCOV study (removing cases for which the SEAC judged that a study endpoint could not be determined). Table A7. Summary characteristics of previously published pre-exposure prophylaxis studies considered for meta-analysis. Table A8. Listing of causes of PCR-confirmed respiratory illness. Fig A1. Atlas showing those countries in which investigators were contacted to enquire whether they would be interested in, and able to join the COPCOV study. Fig A2. Atlas showing the location of the COPCOV trial sites which recruited participants, the 4-aminoquinoline tested, and the approximate numbers recruited. Fig A3. Graph showing cumulative enrollment over time (per week) by country. Fig A4. Funnel plot showing the 4-aminoquinoline COVID-19 pre-exposure chemoprevention RCTs included in the prespecified meta-analysis, and the relationship between point estimate risk ratios for the primary outcome and the corresponding standard errors. Fig A5. Meta-analysis of 4-aminoquinoline COVID-19 pre-exposure chemoprevention RCTs based on individual study primary endpoints using Risk Of Bias tool (RoB 2). Fig A6. Meta-analysis of the safety and tolerability outcomes in COVID-19 chemoprevention RCTs using the same methodology as reported in the WHO living guideline [24]. Fig A7. Meta-analysis of adverse events leading to treatment discontinuation reported in double-blind, placebo-controlled, 4-aminoquinoline COVID-19 pre-exposure chemoprevention RCTs. Fig A8. Graph showing cumulative loss to follow-up (LTFU) for Hydroxychloroquine/ Chloroquine arms and Placebo arm.

(DOCX)

pmed.1004428.s007.docx (5.6MB, docx)

Acknowledgments

We are very grateful to all the participants of this study and the many nurses, technicians, laboratory scientists, doctors, and support staff who helped with the study.

Abbreviations

COVID-19

Coronavirus Disease 2019

CQ

chloroquine

DBS

dried blood spot

DSMB

Data Safety and Monitoring Board

HCQ

hydroxychloroquine

ITT

intention-to-treat

PP

per protocol

RCT

randomised controlled trial

RR

risk ratio

SAE

serious adverse event

SAP

Statistical Analysis Plan

SARS-CoV-2

Severe Acute Respiratory Syndrome Coronavirus 2

SEAC

Serology Endpoint Assessment Committee

Data Availability

The data underlying the results presented in the study are available from https://github.com/jwatowatson/COPCOV.

Funding Statement

This research was funded by the Wellcome Trust [Grant number 221307/Z/20/Z] through the COVID-19 Therapeutics Accelerator.

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Decision Letter 0

Richard Turner

25 Aug 2023

Dear Dr Schilling,

Thank you for submitting your manuscript entitled "Hydroxychloroquine or chloroquine prevention of COVID-19 (COPCOV) a double-blind, randomised, placebo-controlled trial" for consideration by PLOS Medicine.

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Decision Letter 1

Richard Turner

8 Oct 2023

Dear Dr. Schilling,

Thank you very much for submitting your manuscript "Hydroxychloroquine or chloroquine prevention of COVID-19 (COPCOV) a double-blind, randomised, placebo-controlled trial" (PMEDICINE-D-23-02452R1) for consideration at PLOS Medicine.

Your paper was discussed among the editors and sent to independent reviewers, including a statistical reviewer. The reviews are appended at the bottom of this email and any accompanying reviewer attachments can be seen via the link below:

[LINK]

In light of these reviews, we will not be able to accept the manuscript for publication in the journal in its current form, but we would like to invite you to submit a revised version that addresses the reviewers' and editors' comments fully. You will recognize that we cannot make a decision about publication until we have seen the revised manuscript and your response, and we expect to seek re-review by one or more of the reviewers.

In revising the manuscript for further consideration, your revisions should address the specific points made by each reviewer and the editors. Please also check the guidelines for revised papers at http://journals.plos.org/plosmedicine/s/revising-your-manuscript for any that apply to your paper. In your rebuttal letter you should indicate your response to the reviewers' and editors' comments, the changes you have made in the manuscript, and include either an excerpt of the revised text or the location (eg: page and line number) where each change can be found. Please submit a clean version of the paper as the main article file; a version with changes marked should be uploaded as a marked up manuscript.

In addition, we request that you upload any figures associated with your paper as individual TIF or EPS files with 300dpi resolution at resubmission; please read our figure guidelines for more information on our requirements: http://journals.plos.org/plosmedicine/s/figures. While revising your submission, please upload your figure files to the PACE digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at PLOSMedicine@plos.org.

We hope to receive your revised manuscript by Oct 30 2023 11:59PM. Please email us (plosmedicine@plos.org) if you have any questions or concerns.

***Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.***

We ask every co-author listed on the manuscript to fill in a contributing author statement, making sure to declare all competing interests. If any of the co-authors have not filled in the statement, we will remind them to do so when the paper is revised. If all statements are not completed in a timely fashion this could hold up the re-review process. If new competing interests are declared later in the revision process, this may also hold up the submission. Should there be a problem getting one of your co-authors to fill in a statement we will be in contact. YOU MUST NOT ADD OR REMOVE AUTHORS UNLESS YOU HAVE ALERTED THE EDITOR HANDLING THE MANUSCRIPT TO THE CHANGE AND THEY SPECIFICALLY HAVE AGREED TO IT. You can see our competing interests policy here: http://journals.plos.org/plosmedicine/s/competing-interests.

Please use the following link to submit the revised manuscript:

https://www.editorialmanager.com/pmedicine/

Your article can be found in the "Submissions Needing Revision" folder.

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Please ensure that the paper adheres to the PLOS Data Availability Policy (see http://journals.plos.org/plosmedicine/s/data-availability), which requires that all data underlying the study's findings be provided in a repository or as Supporting Information. For data residing with a third party, authors are required to provide instructions with contact information for obtaining the data. PLOS journals do not allow statements supported by "data not shown" or "unpublished results." For such statements, authors must provide supporting data or cite public sources that include it.

Please let me know if you have any questions, and we look forward to receiving your revised manuscript.

Sincerely,

Richard Turner PhD

Consulting editor, PLOS Medicine

plosmedicine@plos.org

-----------------------------------------------------------

Requests from the editors:

In the text and supplementary material, several organizations are mentioned in the context of provision or packaging of study drugs (e.g., "Accord Healthcare" and "Piramal"). We feel that all such contributions should mentioned in the financial disclosures (submission form).

We ask you to adapt the competing interest statement (submission form) to name relevant members of PLOS Medicine's editorial board among the authors.

We suggest adapting the title as follows: "Evaluation of Hydroxychloroquine or chloroquine for prevention of COVID-19 (COPCOV): A double-blind, randomised, placebo-controlled trial

Please adapt the short title to contain brief details of the study.

Please ensure that the revised abstract follows CONSORT; if secondary endpoint outcomes are to be reported, all should be included to avoid an appearance of cherry-picking.

Please combine the "Methods" and "Findings" subsections of the abstract.

Please add a new final sentence to the combined subsection, which should begin "Study imitations include ..." or similar and should quote 2-3 of the study's main limitations.

Please quote aggregate demographic details for study participants in the abstract.

In place of the "Research in Context" section, please substitute a new "Author Summary" section in PLOS Medicine style. You may find it helpful to consult one or two recent research papers in the journal to get a sense of the preferred style.

Noting comments from the referees on the statement "Published studies suggest clinical benefit", please adapt the abstract and main text to reflect the implications of the published individual studies.

Please adapt the final sentence of the Introduction (main text; "We present the results ...") to state the study's aim.

Please restructure the Discussion section (main text) so that the first paragraph summarizes the study's main findings, with these being discussed in subsequent paragraphs.

Please remove all trademarks from the paper.

Please remove the information on funding from the title page and from the end of the main text. In the event of publication, this information will appear in the article metadata, via entries in the submission form.

In the reference list, please convert italics to plain text.

Please add accessed dates to online references such as reference 1.

Noting reference 2, please list 6 author names followed by "et al.", where appropriate.

Please use the journal name abbreviation "PLoS ONE".

Please adapt the title for Fig 1 to "Participant flowchart" or similar.

Please include a completed CONSORT checklist as a supplementary file, labelled "S1_CONSORT_Checklist" or similar and referred to as such in the Methods section (main text).

In the checklist, please refer to individual items by section (e.g., "Methods") and paragraph number, not by line or page numbers as these generally change in the event of publication.

Is there a formal study protocol that you can attach as "S2_Protocol"?

Please rename the attached statistical analysis plan "S3_Statistical Analysis Plan" or similar.

Comments from the reviewers:

*** Reviewer #1:

Statistical review

This paper reports a RCT comparing pooled Hydroxychloroquine + chloroquine vs placebo for prevention of covid-19 infection. Although the sample size enrolled was much lower than target, the trial is still very large and provides useful evidence about the positive effect of the drugs. The trial was reported well and I had only some minor comments:

1. Abstract "The primary endpoint was symptomatic COVID-19": perhaps add 'during the follow-up period.' to clarify that this wasn't time-to-event.

2. Abstract: The secondary outcomes reported in the abstract do not align perfectly with the secondary outcomes in the clinicaltrials.gov registration page (or later in the paper). If those outcomes were collected it would be good to report them in the abstract.

3. Abstract: "Clinical trials need to be supported and protected, particularly in pandemic emergencies." - Although I see the rationale for this statement after reading the paper's discussion, I didn't see how this followed from the rest of the abstract so would leave it out.

4. Page 9: were any covariates adjusted for in the log-binomial models?

5. Page 9: was there non-negligible loss-to-follow-up (the sample size calculation planned for 20%) and if so how was this handled in the ITT analysis?

6. Page 10 "In symptomatic patients there were no differences in symptom severity scores." - I would add no significant difference (and preferably would include the median score in each arm, similarly to other outcomes have summary by arm).

7. Page 10 "a mean difference of 104 days (95% CI, 12 to 199)." - is there a reason the authors did not provide a p-value for this outcome? Other secondary outcomes have them.

James Wason

*** Reviewer #2:

In this double-blind, placebo controlled, multi-national, randomised trial the authors test whether a three month chemoprophylaxis with hydroxychloroquine or chloroquine can reduce the incidence of clinical COVID-19. Several secondary outcomes were also reported

The trial was planned for 40,000 participants, of whom 4,652 were recruited, after which point the trial had to close due to lack of recruitment.

The hypothesis is clearly stated, and, despite the premature closure, I believe, within reasonable clinical certainty, can conclude on the hypothesis.

The results seem clear, and the wording in the interpretation, are well-balanced by the authors. They also manage to balance the limitations.

In all aspects, apart from the premature closure, this is a very high quality RCT.

I strongly agree with the authors on their point that especially during crises like the COVID-19 pandemic, stakeholders should stand up for the completion of RCT´s since, at the end of the day, this design is the only that can inform us solidly, when conducted rigorously.

I have few comments:

The authors state :

"This allowed for approximately 20% loss to follow up, withdrawals, protocol deviations and non-adherence, and provided 80% power to detect a 23% reduction in symptomatic COVID-19 incidence (from an estimated 3%) for each drug individually with 95% confidence. There were protracted delays and difficulties with recruitment as the study was starting in 2020"

This seems extremely ambitious, however, sometime the "perfect becomes enemy of the good", insofar as the chance of completing extremely ambitious projects decreases with the increasing target sample size. I speak of bitter personal experience…. As far as I can see, the sample size calculation wanted to detect a difference between approx. 3% to approx. 2.4%. In light of the pandemic threat and the situation when the trial was planned, I am a little puzzled: would a protective effect as small as this actually have been clinically relevant in stopping the pandemic? Could the authors elaborate on this in the discussion

In the discussion, first part: "Few drugs have excited such controversy as hydroxychloroquine in COVID-19. Bureaucracy, politicisation, unfounded opinions, false claims and outright fraud conspired against the conduct of the clinical trials needed to provide objective evidence early in the pandemic"

I agree, this was outrageous. However, I feel, the results of the trial should be respected in the first lines of the discussion. This is an opinion, I´m aware, however, maybe the authors should "kill this darling", at least regarding the prioritizing of messages in the discussion. Maybe such a statement (that may for some readers be controversial) should be placed less prominently. This is said by a trialist who had a very public controversy with my national medicines agency (for closing down a HC trial during the first wave) so I do feel much the same as in this statement, nevertheless, maybe only a minority of the readers think this is key ? Just a comment, I guess the authors have had some relevant discussions on this…

Further:

"This trial provides no support for these claims, and it supports the generally good short-term safety profile of these drugs.30 It challenges the World Health Organization "living" guidelines on COVID-19 chemoprevention which emphasised toxicity in COPCOV Lancet V2 Page 12 of 26 recommending against hydroxychloroquine. The same guideline also stated that prophylactic use "probably has a small or no effect on laboratory-confirmed COVID-19" with "moderate certainty" , and has no effect on mortality with "high certainty".26 Yet no deaths were reported in any of the pre-exposure prophylaxis studies, while the drugs have been well-tolerated with very low rates of treatment discontinuation (Figures S5, S6).31"

This is extremely important, which I strongly agree on, and actually it raised serious concern on the WHO handling of the pandemic on a very key point. I do not think the aftermath in WHO has handled this erroneous and very harmful reaction at a crucial point in the pandemic in a relevant way.

Later: "Now, in early 2023, with increasing coverage of effective vaccines," - possibly change to "Now, with increasing…" to strengthen generalizability of the statement

Another point. I think the discussion lacks a discussion of the interesting and very relevant finding of a reduction in the incidence of respiratory infections. Focus on this would make the results relevant in a future context, it indicates a general effect in respiratory infections.

Limitations are very well discussed.

Finally, I want the congratulate the authors with a very well-planned, well conducted RCT, despite of the unreasonable resistance and irrelevant political decisions that made life very hard for people who really wanted to inform the world with precise data, on important issues during the COVID-19 pandemic.

*** Reviewer #3:

The authors present the results of the long-awaited COPCOV study, which evaluated chloroquines as chemoprophylaxis for COVID-19. This may sound outdated now that we fight the pandemic with vaccines, but there were times when vaccines did not exist and the repurpose of existing drug was the best option to tackle the pandemic. Its results will not have a direct impact on the management of the current pandemic, but will help addressing challenges that caught the scientific community off-guard when the pandemic hit in 2020. The design and implementation of the study are sound

Major comments

- Figure 2 should contain the primary outcome.

- Please refer to the primary outcome as "symptomatic, laboratory-confirmed COVID-19" throughout the manuscript. That terminology is used in methods when the primary outcome is described, but not in the results section, which is confusing.

- I find the "primary outcome" section in results confusing:

o It starts reporting SARS-CoV-2 infection, which is not a primary outcome (it is actually an outcome that is not described in methods). Given that PCR tests were not done systematically, how is SARS-CoV-2 infection different from "symptomatic covid-19"? This needs to be clarified in methods.

o Then it reports symptomatic [laboratory-confirmed] covid-19 which is the primary outcome

o And then it reports comparisons that of non-primary outcomes: pcr-confirmed symptomatic covid-19, symptomatic covid-19 diagnosed by seroconversion, DBS symptomatic COVID-19.

o So, if you read this section fast (as most of us do), it is hard to identify the main result of the study. I think some rearrangement can make this section clearer.

* Describe time to vaccination (KM curve) by arm. How were these patients handled?

* Make sure that the KM estimations are truly an ITT analysis, i.e., that patients who discontinue the drug are not censored (which is what the SAP says in page 14, but that is not an ITT analysis)

* It is odd that the main analysis is not a time-to-event analysis (e.g. by using a Kaplan Meier estimator). This means that the authors assume there is not right-censoring, when in fact there is according to Figure S7. I don't think that the results will change substantially, but I think a time-to-event analysis should be provided too (maybe as supplemental).

* I think a KM curve for the main outcome with the following three arms should be added (maybe as supplemental): control, chloroquine, hydroxychloroquine.

* Effect estimates in an absolute scale (i.e., risk differences) should be provided.

Regarding the meta-analysis:

* Addressing the risk of bias of the component studies is standard practice in meta-analyses. See, for example Supp Table 1 of Garcia-Albeniz et al. I think it would be good to use the Cochrane risk of bias 2.0 tool to address the risk of bias of those studies not already evaluated for bias in the study by Garcia-Albeniz et al.

* Figure 3:

o The confidence interval by Seet et al does not match the original publication (0.44-0.97)

o The estimate by Polo et al should be 0.49 (0.00-2.29)

o I would not include the article by McKinnon et al in this meta-analysis because of the same reason that Garcia-Albeniz (reference 23) used: although the authors do not label the study as post-exposure prophylaxis, they report that "60% [of study participants] reported contact with a COVID-19 positive patient before study entry

o The estimate by Vijayaraghavan should be 0.85, 95% CI 0.35 to 2.07

o The upper limit by Llanos-Cuentas should be 7.11

o For consistency with Garcia-Albeniz et al, I think the authors should estimate the confidence interval of the meta-analyzed risk ratio using both a fixed (common) effect model and a random effects model.

o Figure 3 should have a note explaining the meaning of the grey squares around the point estimates

o I think it would be good to complement the meta-analysis with the risk difference estimates, similar to Supp Figure 1 of Garcia-Albeniz et al.

* Figure S5

o The estimate by Polo et al should be 0.49 (0.00-2.29)

Minor comments:

- If the sample size allows for an informative analysis, it would be nice to see a subgroup analysis of only healthcare workers

- Results from the drug measurement described in the sixth line of "Procedures" section are not reported

- I don't see the need to report p-values or concluding that "there were/were not statistically significant differences" because I consider this an outdated practice. https://www.nature.com/articles/d41586-019-00857-9

- There is a section "Interpretation of results" in the supplementary materials, that is inside the meta-analysis section and it reads like it does not belong there.

*** Reviewer #4:

This randomized controlled trial investigated the effect of using hydroxychloroquine (HCQ) as a preventive tool against symptomatic covid-19 (main endpoint). This study found that there is a borderline difference in the incidence of covid-19 RR=0.85 (0.72-1.00), p=0.05 and there was no effect against asymptomatic COVID-19 (RR: 0.96 (95% CI, 0.82 to 1.12), p=0.6) nor for all SARS-COV-2 infections in ITT analyses. In per protocol analysis, the primary outcome showed no effect.

The strengths of this study include the larger number of participants included (4600) compared to other clinical trials on hydroxychloroquine as a preventive medication. The trial took place in multiple countries.

My main concern is that the preventive effect is only observed among confirmed SAS-CoV-2 PCR (with a small number of cases confirmed by PCR) but there is no effect when the diagnosis is based on seroconversion or based on all SARS-CoV-2 cases or only asymptomatic cases. These drugs may have a small or no effect on COVID-19. 29% (13-14% loss to follow-up) of the initial randomized population have been excluded from per protocol analyses. Additionally, timing of this study may may no longer be as important now that vaccines are available and the variants of SARS-CoV-2 are different.

The description and the methods used in the meta-analysis raise some questions.

Main analysis:

1) "The study took place in multiple countries, including the United Kingdom, Asia, and Africa." Is there a geographical effect? Standard of cares, exposure to the SRAS-CoV-2 (according to preventive policies), population are variable. It could be interesting to have analyses per continent or broad geographic areas

2) "The recruitment for the study took place between April 2020 and March 2022. Various variants, such as Alpha and Delta of the SARS-CoV-2, emerged during this period. Are these differences being considered? Regarding the vaccines, their effectiveness varied depending on the variants."

3) "The analyses combine two different molecules (CQ and HCQ). Is there a difference in their effects?"

4) "How was adherence to the intervention assessed? Plasma concentrations of HCQ or CQ were not measured."

5) "The power calculation was originally presented for a total of 40,000 participants. However, only 4,652 people were ultimately randomized. What is the expected detectable effect size?

6) The authors have longitudinal data with almost 2 years of follow-up. Why is the primary analysis based on a Fisher exact test? I wonder if the results would be different with a survival model and Kaplan-Meier analysis (Kaplan-Meier was performed for respiratory infections but not for COVID-19). The main result for the primary outcome is borderline significant (RR=0.85 [0.72-1.00], p=0.05), it is difficult to interpret this result. Results for all SARS-CoV-2 infection suggest no effect of CQ and CHQ.

7) Table 2 mentions missing data for the primary outcome. What was the number of missing data? This should be specified.

8) In the discussion, statement is not correct "This protective effect is consistent with previous studies10,11,14-22" I looked at the first references mentioned: ref 10, Syed et al. 2021 writes "It is concluded that the PrEP HCQ does not significantly prevent COVID-19 among high-risk HCPs." Abella et al. 2021 wrote "there was no clinical benefit of hydroxychloroquine administered daily for 8 weeks as pre-exposure prophylaxis in hospital-based HCWs exposed to patients with COVID-19." Same for Rajasingham et al. 2021: Pre-exposure prophylaxis with hydroxychloroquine once or twice weekly did not significantly reduce laboratory-confirmed COVID-19 or COVID-19-compatible illness among healthcare workers.

9) In the Abstract, « Published studies suggest clinical benefit" is not right. Several randomized trials have shown no effect of HCQ on covid19 as treatment or preventive tool… These results could be compared with negative results from previous meta-analysis studying HCQ as preventive tool (Hong et al. 2023). Previous meta-analyses of RCTs on HCQ as treatment also reported no effect on COVID-19.

Hong H, Friedland A, Hu M, Anstrom KJ, Halabi S, McKinnon JE, Amaravadi R, Rojas-Serrano J, Abella BS, Portillo-Vázquez AM, Woods CW, Hernandez AF, Boulware DR, Naggie S, Rajasingham R. Safety and efficacy of hydroxychloroquine as prophylactic against COVID-19 in healthcare workers: a meta-analysis of randomised clinical trials. BMJ Open. 2023 Jun 16;13(6):e065305. doi: 10.1136/bmjopen-2022-065305. PMID: 37328184; PMCID: PMC10276967.

10) I wonder whether the protocol was pre-registered on an open-platform.

Meta-analysis: The meta-analysis suggests a protective effect. It is important to underline that this study has a weight of 61%.

it is unclear what RR (or Odds Ratio or Hazard Ratios ; adjusted or not) was used. A table describing the individual included studies could be useful to understand what was the intervention, the dose, the placebo, the duration, and the endpoint, study population (general population vs health workers).

1) Heterogeneity could be added in the main text

2) In the supplementary material, it is not clear what meta-analysis model has been used: fixed or random effect model?

3) This meta-analysis pooled different interventions: I looked at the studies with the most important weight in the meta-analysis. The meta-analysis pooled different endpoints across the studies (PCR positive or probable covid-19, symptomatic covid only, infections…). I have not checked all the included studies but some of them included a different placebo (Vitamin C)

4) In Rajasingham 2020, the Hazard ratio is .74 (95% CI, 0.45-1.20) for COVID-19 compatible with symptoms and the forest plot in this manuscript reported slightly different values 0.74 (95% CI, 0.50-1.10). Placebo was Vitamin C.

5) In Seet 2022 trial, the placebo was Vitamin C. The value used in the forest plot is 0.70 [0.47 ; 1.04]. Was it recalculated for symptomatic COVID-19? In the original study of Seet, Odds Ratio are provided and not Relative Risk. And the aOR is different: 0.70 [0.44 ; 0.97] for SARS-CoV2-infection.

6) I also wonder whether there is a dose-effect of HCQ/CQ (using meta-regression)

I suggest to add line numbering in the .pdf

To conclude, I wonder to what extent a prevention policy for SARS-CoV-2 using hydroxychloroquine can be implemented and useful now that vaccines are available (as it is mentioned in the discussion). The recommendation would be to advise the general population to take hydroxychloroquine regularly every day. Is this sustainable and feasible? Unlike vaccines, which have shown a low preventive effect, where we only need one injection and boosters.

***

Any attachments provided with reviews can be seen via the following link:

[LINK]

Decision Letter 2

Richard Turner

26 Nov 2023

Dear Dr. Schilling,

Thank you very much for submitting your revised manuscript "Evaluation of hydroxychloroquine or chloroquine for prevention of COVID-19 (COPCOV): a double-blind, randomised, placebo-controlled trial" (PMEDICINE-D-23-02452R2) for consideration at PLOS Medicine.

Your paper was discussed among the editors and re-seen by three of our independent reviewers, including the statistical reviewer. The reviews are appended at the bottom of this email and any accompanying reviewer attachments can be seen via the link below:

[LINK]

We will not be able to accept the manuscript for publication in the journal in its current form, but would like to invite you to submit a further revised version that addresses the editors' comments fully. Again, we will be unable to make a decision about publication until we have seen the revised manuscript and your response, and we may seek re-review by one or more of the reviewers.

In revising the manuscript for further consideration, your revisions should address the specific points made by each reviewer and the editors. Please also check the guidelines for revised papers at http://journals.plos.org/plosmedicine/s/revising-your-manuscript for any that apply to your paper. In your rebuttal letter you should indicate your response to the reviewers' and editors' comments, the changes you have made in the manuscript, and include either an excerpt of the revised text or the location (eg: page and line number) where each change can be found. Please submit a clean version of the paper as the main article file; a version with changes marked should be uploaded as a marked up manuscript.

In addition, we request that you upload any figures associated with your paper as individual TIF or EPS files with 300dpi resolution at resubmission; please read our figure guidelines for more information on our requirements: http://journals.plos.org/plosmedicine/s/figures. While revising your submission, please upload your figure files to the PACE digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at PLOSMedicine@plos.org.

We hope to receive your revised manuscript by Dec 11 2023 11:59PM. Please email us (plosmedicine@plos.org) if you have any questions or concerns.

***Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.***

We ask every co-author listed on the manuscript to fill in a contributing author statement, making sure to declare all competing interests. If any of the co-authors have not filled in the statement, we will remind them to do so when the paper is revised. If all statements are not completed in a timely fashion this could hold up the re-review process. If new competing interests are declared later in the revision process, this may also hold up the submission. Should there be a problem getting one of your co-authors to fill in a statement we will be in contact. YOU MUST NOT ADD OR REMOVE AUTHORS UNLESS YOU HAVE ALERTED THE EDITOR HANDLING THE MANUSCRIPT TO THE CHANGE AND THEY SPECIFICALLY HAVE AGREED TO IT. You can see our competing interests policy here: http://journals.plos.org/plosmedicine/s/competing-interests.

Please use the following link to submit the revised manuscript:

https://www.editorialmanager.com/pmedicine/

Your article can be found in the "Submissions Needing Revision" folder.

Please ensure that the paper adheres to the PLOS Data Availability Policy (see http://journals.plos.org/plosmedicine/s/data-availability), which requires that all data underlying the study's findings be provided in a repository or as Supporting Information. For data residing with a third party, authors are required to provide instructions with contact information for obtaining the data. PLOS journals do not allow statements supported by "data not shown" or "unpublished results." For such statements, authors must provide supporting data or cite public sources that include it.

Please let me know if you have any questions, and we look forward to receiving your revised manuscript.

Sincerely,

Richard Turner PhD

Consulting editor, PLOS Medicine

plosmedicine@plos.org

-----------------------------------------------------------

Requests from the editors:

Please remove the information on funding and the publishing licence from the abstract page. In the event of publication, information on funding will appear in the article metadata, via entries in the submission form.

Around line 87 (abstract), please add a sentence to mention the intended trial recruitment and explain briefly why it could not be achieved.

At lines 94, 95, 96 & 99 (abstract), we suggest substituting colons for the four semicolons.

Please remove the sentence at line 107 ("Difficulties conducting ...", abstract), as this issue can be fully dealt with in the Discussion (main text).

Please reformat the 'Author summary' so that the three subsections each comprise 3-4 short points of 1-2 sentences each. Please use the active voice (e.g., "We found ...") in one or two points.

Regarding the summary of outcomes at line 218, please include a full list of all outcomes. We note that there are 'other outcomes' specified, including impact on healthcare costs. We ask you to report all outcomes for which data are available in the present paper.

At line 325, noting 'preventitive', we suggest using 'preventive' throughout.

Please adapt the reference call-outs to journal style throughout, e.g., "... equivalent [25,26]." (noting the absence of spaces within the square brackets).

Despite the retraction, please complete the citation for reference 4 with full access details. Please revisit reference 24, which may be missing some information.

Comments from the reviewers:

*** Reviewer #1:

Thank you to the authors for addressing my previous comments well, I have no further issues to raise.

*** Reviewer #2:

I have no further comments, the authors have responded very adequately. It´s a fine study

*** Reviewer #3:

All my comments have been addressed

***

Any attachments provided with reviews can be seen via the following link:

[LINK]

Decision Letter 3

Richard Turner

24 Dec 2023

Dear Dr. Schilling,

Thank you very much for submitting your revised manuscript "Evaluation of hydroxychloroquine or chloroquine for prevention of COVID-19 (COPCOV): a double-blind, randomised, placebo-controlled trial" (PMEDICINE-D-23-02452R3) for consideration at PLOS Medicine.

Your revised paper was evaluated by our academic editor and discussed among the editorial team. In light of our discussions we will not be able to accept the manuscript for publication in its current form, but we would like to again invite you to submit a revised version that fully addresses the remaining comments; we may seek re-review by one or more of the reviewers.

In revising the manuscript for further consideration, your revisions should address the specific points made by the editors. Please also check the guidelines for revised papers at http://journals.plos.org/plosmedicine/s/revising-your-manuscript for any that apply to your paper. In your rebuttal letter you should indicate your response to the reviewers' and editors' comments, the changes you have made in the manuscript, and include either an excerpt of the revised text or the location (eg: page and line number) where each change can be found. Please submit a clean version of the paper as the main article file; a version with changes marked should be uploaded as a marked up manuscript.

In addition, we request that you upload any figures associated with your paper as individual TIF or EPS files with 300dpi resolution at resubmission; please read our figure guidelines for more information on our requirements: http://journals.plos.org/plosmedicine/s/figures. While revising your submission, please upload your figure files to the PACE digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at PLOSMedicine@plos.org.

We hope to receive your revised manuscript early in the new year. Please email us (plosmedicine@plos.org) if you have any questions or concerns.

We ask every co-author listed on the manuscript to fill in a contributing author statement, making sure to declare all competing interests. If any of the co-authors have not filled in the statement, we will remind them to do so when the paper is revised. If all statements are not completed in a timely fashion this could hold up the re-review process. If new competing interests are declared later in the revision process, this may also hold up the submission. Should there be a problem getting one of your co-authors to fill in a statement we will be in contact. YOU MUST NOT ADD OR REMOVE AUTHORS UNLESS YOU HAVE ALERTED THE EDITOR HANDLING THE MANUSCRIPT TO THE CHANGE AND THEY SPECIFICALLY HAVE AGREED TO IT. You can see our competing interests policy here: http://journals.plos.org/plosmedicine/s/competing-interests.

Please use the following link to submit the revised manuscript:

https://www.editorialmanager.com/pmedicine/

Your article can be found in the "Submissions Needing Revision" folder.

Please let me know if you have any questions, and we look forward to receiving your re-revised manuscript.

Sincerely,

Richard Turner PhD

Consulting editor, PLOS Medicine

plosmedicine@plos.org

-----------------------------------------------------------

Requests from the editors:

In the 'Financial disclosure' section (submission form), the information about the CC-BY licence is superfluous and can be removed, as this licence is standard at PLOS Medicine.

Please remove the quoted secondary outcome findings from the abstract, retaining the primary outcome findings, results of the prespecified meta-analysis and information about safety.

At line 152 (Introduction), please adapt the wording to "a false claim".

At line 163 (Introduction) please adapt the text to state that the trials did not individually provide evidence of benefit.

Noting the information around line 288 (Results) regarding confirmed COVID-19 infection, we ask you to add a supplementary table listing causes of respiratory illness.

At line 330 (Discussion) we feel that "... evidence of moderate preventive efficacy" is an overstatement and ask you to amend the wording to "... some evidence of preventive efficacy" or similar.

At line 331, we ask you to adapt the text to "... an apparent 15% reduction", noting the equivocal p value.

At line 332 we suggest "efficacy" rather than "effect", as elsewhere in the text.

Again at line 332, we ask you to revisit "protective effect is consistent with previous smaller studies". We believe that the previous studies did not provide evidence of significant benefit, and we ask you to adapt the wording to indicate this clearly.

At line 360, we suggest removing "outright fraud", although we are aware that this is not an unfounded statement.

At line 374, we ask you to remove the sentence "This must not be allowed ...".

At line 389, we ask you to remove the word "strong", which seems an overstatement based on the numbers quoted.

At line 397, please remove the phrase "and not impeded".

At line 403, please remove the information about study funding, which is present in the financial disclosures which will appear in the article metadata in the event of publication.

Please edit reference 4: we believe that this can be truncated prior to "Epub ahead of print" so as to retain the essential information.

***

Decision Letter 4

Richard Turner

15 Feb 2024

Dear Dr. Schilling,

Thank you very much for submitting your revised manuscript "Evaluation of hydroxychloroquine or chloroquine for prevention of COVID-19 (COPCOV): A double-blind, randomised, placebo-controlled trial" (PMEDICINE-D-23-02452R4) for consideration at PLOS Medicine.

Following from the recent email exchange, your provisional revised text was discussed among the editorial team, yielding our further specific requests (below). We would now like to invite you to submit a further revised version for consideration in which these points have been fully addressed. In your rebuttal letter you should indicate your response to the reviewers' and editors' comments, the changes you have made in the manuscript, and include either an excerpt of the revised text or the location (eg: page and line number) where each change can be found. Please submit a clean version of the paper as the main article file; a version with changes marked should be uploaded as a marked up manuscript.

We hope to receive your revised manuscript within one week. Please email us (plosmedicine@plos.org) if you have any questions or concerns.

Please use the following link to submit the revised manuscript:

https://www.editorialmanager.com/pmedicine/

Your article can be found in the "Submissions Needing Revision" folder.

We suggest a further email exchange if you would like to discuss any specific point(s) prior to resubmission, and otherwise look forward to receiving your revised manuscript.

Sincerely,

Richard Turner PhD,

Consulting editor, PLOS Medicine

plosmedicine@plos.org

-----------------------------------------------------------

Requests from the editors:

1. Line 78: We suggest abbreviating HCQ here (rather than at line 83).

2. Line 79: Please adapt the text to “Previous randomised controlled trials did not show benefit of HCQ against COVID-19 and, although meta-analysis suggested clinical benefit, guidelines recommend against its use."

3. Line 86: Please list the secondary and tertiary outcomes after the primary endpoint, adding “(not reported here)” where appropriate.

4. Line 88: Please adapt the text to “… because of protracted delays owing to controversies regarding efficacy and adverse events with HCQ use, vaccine rollout in some countries, and other factors. Between 29 April 2020 …”.

5. Line 94: We ask you to adapt the text to “… was similar in the HCQ/CQ compared with the placebo arms: ...”.

6. At line 97, please move the p value inside the brackets for consistency.

7. At line 101, please remove “pre-exposure” (redundant given “prophylaxis” later in the sentence).

8. At line 103, please remove “very” (prior to “well tolerated”), which we feel is unnecessary.

9. Line 109: please adapt the text to “In this large placebo-controlled, double-blind randomised trial, HCQ and CQ were safe and well tolerated in COVID-19 chemoprevention, and there was evidence of moderate protective benefit in a meta-analysis of evidence from this trial and similar RCTs.”

10. At line 118, please adapt the text to: “HCQ proved ineffective in the treatment of hospitalised patients, and individual RCTs testing COVID-19 prophylaxis did not show benefit of HCQ. However, a meta-analysis of trial data suggested some efficacy in preventing COVID-19.”

11. At line 121, please remove “In contrast”.

12. At line 129, please adapt the text to: “We found that HCQ and CQ were well tolerated and safe in prophylaxis. There was some evidence for protection against symptomatic COVID-19, and an apparent reduction in workdays lost to illness.”

13. At line 147, please remove “verging on panic”. You may wish to adapt the sentence to “… global concern about the projected consequences of the developing pandemic for large-scale human ill-health and mortality” or similar.

14. At line 149, please define the abbreviations “CQ” and “HCQ”, assuming that you plan to use these throughout the text.

15. At line 153, please remove reference 4 and instead cite https://www.nature.com/articles/d41586-020-01695-w or a similar article discussing the situation.

16. At line 154, we ask you to amend the text to “… regulatory decisions.”

17. At line 160, regarding “… recommended widely”, please add a reference as we are not clear what kind of recommendations are being discussed.

18. At line 162, please add a comma after “Despite this”.

19. At line 164/5, please run the sentence on (“… underpowered to demonstrate benefit [23], but the evidence ...”.

20. Regarding the presentation around line 220, we note that the clinicaltrials.gov page (https://classic.clinicaltrials.gov/ct2/show/NCT04303507?term=copcov&draw=2&rank=1) currently lists one primary outcome, four secondary outcomes and four “other outcomes”. We suggest editing the page to match the definitive version of the trial protocol.

21. After line 230, please list the endpoints in continuous text rather than bulleted text.

22. At line 336, please report the RR and 95% CI (as at line 102) rather than “reduced by 20%” and the like.

23. Line 344: We ask you to adapt the text to “… was able to provide evidence relevant to possible protective efficacy. For the trial’s primary endpoint (incidence of symptomatic laboratory-confirmed COVID-19), the non-significant finding in favour of HCQ/CQ treatment is consistent with the aggregated results of previous smaller studies [10,11,14-22], all but one of which had a lower incidence of symptomatic COVID-19 in the HCQ treatment arm (Figure 3). Incorporating the COPCOV trial data in a prespecified meta-analysis of pre-exposure RCTs, with little heterogeneity across the studies and no evidence of publication bias, suggests a moderate protective benefit (RR 0.80, 95% CI 0.71-0.91).”

24. At line 351, please remove “41%”.

25. At line 352, please remove “approximate 25%”.

26. Line 372: do you mean the meta-analysis reported in this paper? Please adapt the wording to clarify if so.

27. At line 375, we ask you to adapt the wording to “Bureaucracy, politicisation and heated debate obstructed the undertaking of clinical trials needed to provide objective evidence early in the pandemic.”

28. At line 385, please adapt the text to “… despite the seriousness of the pandemic, there were major obstacles to the conduct of urgently needed clinical research, particularly in low-resource settings.”.

29. At line 408, please adapt the text to “… was safe and well-tolerated, and combined with data from other similar trials provided some evidence that laboratory-confirmed symptomatic COVID-19 might be reduced.”

30. Please reformat table 2 to indicate the ranking of the endpoints.

***

Decision Letter 5

Richard Turner

23 May 2024

Dear Dr. Schilling,

Thank you very much for re-submitting your manuscript "Evaluation of hydroxychloroquine or chloroquine for prevention of COVID-19 (COPCOV): a double-blind, randomised, placebo-controlled trial" (PMEDICINE-D-23-02452R5) for consideration at PLOS Medicine.

With apologies for the tardy reply, I have discussed the paper with editorial colleagues and I am pleased to tell you that, provided the remaining editorial and production issues are fully dealt with, we expect to be able to accept the paper for publication in the journal.

The remaining issues that need to be addressed are listed at the end of this email. Any accompanying reviewer attachments can be seen via the link below. Please take these into account before resubmitting your manuscript:

[LINK]

***Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.***

In revising the manuscript for further consideration here, please ensure you address the specific points made by each reviewer and the editors. In your rebuttal letter you should indicate your response to the reviewers' and editors' comments and the changes you have made in the manuscript. Please submit a clean version of the paper as the main article file. A version with changes marked must also be uploaded as a marked up manuscript file.

Please also check the guidelines for revised papers at http://journals.plos.org/plosmedicine/s/revising-your-manuscript for any that apply to your paper. If you haven't already, we ask that you provide a short, non-technical Author Summary of your research to make findings accessible to a wide audience that includes both scientists and non-scientists. The Author Summary should immediately follow the Abstract in your revised manuscript. This text is subject to editorial change and should be distinct from the scientific abstract.

We hope to receive your revised manuscript within 1 week. Please email us (plosmedicine@plos.org) if you have any questions or concerns.

We ask every co-author listed on the manuscript to fill in a contributing author statement. If any of the co-authors have not filled in the statement, we will remind them to do so when the paper is revised. If all statements are not completed in a timely fashion this could hold up the re-review process. Should there be a problem getting one of your co-authors to fill in a statement we will be in contact. YOU MUST NOT ADD OR REMOVE AUTHORS UNLESS YOU HAVE ALERTED THE EDITOR HANDLING THE MANUSCRIPT TO THE CHANGE AND THEY SPECIFICALLY HAVE AGREED TO IT.

Please ensure that the paper adheres to the PLOS Data Availability Policy (see http://journals.plos.org/plosmedicine/s/data-availability), which requires that all data underlying the study's findings be provided in a repository or as Supporting Information. For data residing with a third party, authors are required to provide instructions with contact information for obtaining the data. PLOS journals do not allow statements supported by "data not shown" or "unpublished results." For such statements, authors must provide supporting data or cite public sources that include it.

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript.

Please note, when your manuscript is accepted, an uncorrected proof of your manuscript will be published online ahead of the final version, unless you've already opted out via the online submission form. If, for any reason, you do not want an earlier version of your manuscript published online or are unsure if you have already indicated as such, please let the journal staff know immediately at plosmedicine@plos.org.

Please let me know if you have any questions in the meantime, and we look forward to receiving the revised manuscript.   

Sincerely,

Richard Turner, PhD

Consulting Editor, PLOS Medicine

plosmedicine@plos.org

------------------------------------------------------------

Requests from Editors:

Should that be "HCQ" at line 85 (abstract)?

At line 107, please make that "... provided a moderate protective benefit ...".

At line 121 (author summary), please indicate the abbreviations for CQ and HCQ and use these abbreviations thereafter in the author summary.

At line 164, alongside "to indicate a lack of efficacy", do you mean for CQ/HCQ or for the various agents mentioned earlier in the sentence more generally? You may wish to add a few words to clarify.

Again in the introduction (main text) HCQ can be used from line 161 onwards.

Please include a completed CONSERVE checklist as a supplementary file, if relevant.

***

Decision Letter 6

Richard Turner

14 Jun 2024

Dear Dr Schilling, 

On behalf of my colleagues, I am pleased to inform you that we have agreed to publish your manuscript "Evaluation of hydroxychloroquine or chloroquine for the prevention of COVID-19 (COPCOV): a double-blind, randomised, placebo-controlled trial" (PMEDICINE-D-23-02452R6) in PLOS Medicine.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. Please be aware that it may take several days for you to receive this email; during this time no action is required by you. Once you have received these formatting requests, please note that your manuscript will not be scheduled for publication until you have made the required changes.

In the meantime, please log into Editorial Manager at http://www.editorialmanager.com/pmedicine/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production process. 

PRESS

We frequently collaborate with press offices. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximise its impact. If the press office is planning to promote your findings, we would be grateful if they could coordinate with medicinepress@plos.org. If you have not yet opted out of the early version process, we ask that you notify us immediately of any press plans so that we may do so on your behalf.

We also ask that you take this opportunity to read our Embargo Policy regarding the discussion, promotion and media coverage of work that is yet to be published by PLOS. As your manuscript is not yet published, it is bound by the conditions of our Embargo Policy. Please be aware that this policy is in place both to ensure that any press coverage of your article is fully substantiated and to provide a direct link between such coverage and the published work. For full details of our Embargo Policy, please visit http://www.plos.org/about/media-inquiries/embargo-policy/.

Thank you again for submitting to PLOS Medicine. We look forward to publishing your paper. 

Sincerely, 

Richard Turner, PhD 

Consulting Editor, PLOS Medicine

plosmedicine@plos.org

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 CONSERVE Checklists. CONSERVE Checklists.

    (DOCX)

    pmed.1004428.s001.docx (19.7KB, docx)
    S1 CONSORT Checklist. CONSORT 2010 checklist of information to include when reporting a randomised trial.

    (DOCX)

    pmed.1004428.s002.docx (62.1KB, docx)
    S1 Protocol. Chloroquine/hydroxychloroquine prevention of coronavirus disease (COVID-19) in the healthcare setting; a randomised, placebo-controlled prophylaxis study (COPCOV).

    (DOCX)

    pmed.1004428.s003.docx (183.3KB, docx)
    S1 RiskOfBias Assessment. Risk of Bias Assessment.

    (XLSX)

    pmed.1004428.s004.xlsx (12.5KB, xlsx)
    S1 Statistical Analysis Plan. Statistical Analysis Plan.

    (PDF)

    pmed.1004428.s005.pdf (1.3MB, pdf)
    S1 Text. Membership of COPCOV Collaborative Group.

    (XLSX)

    pmed.1004428.s006.xlsx (15KB, xlsx)
    S1 Appendix. Table A1.

    List of COPCOV study sites. Table A2. Baseline characteristics in the COPCOV trial (Per Protocol Analysis). Table A3. Outcomes of Chloroquine/Hydroxychloroquine and Placebo Pre-exposure Prophylaxis against COVID-19 in the COPCOV study (Per Protocol Analysis). Table A4. Summary of Serious Adverse Events in the COPCOV study. Table A5. Primary and secondary outcomes of Chloroquine/Hydroxychloroquine Therapy for Pre-exposure Prophylaxis against COVID-19 (missing outcomes treated as not having had COVID-19 during the study period) ITT–Results presented as “Risk differences.” Table A6. Outcomes of Chloroquine/Hydroxychloroquine and Placebo Pre-exposure Prophylaxis against COVID-19 in the COPCOV study (removing cases for which the SEAC judged that a study endpoint could not be determined). Table A7. Summary characteristics of previously published pre-exposure prophylaxis studies considered for meta-analysis. Table A8. Listing of causes of PCR-confirmed respiratory illness. Fig A1. Atlas showing those countries in which investigators were contacted to enquire whether they would be interested in, and able to join the COPCOV study. Fig A2. Atlas showing the location of the COPCOV trial sites which recruited participants, the 4-aminoquinoline tested, and the approximate numbers recruited. Fig A3. Graph showing cumulative enrollment over time (per week) by country. Fig A4. Funnel plot showing the 4-aminoquinoline COVID-19 pre-exposure chemoprevention RCTs included in the prespecified meta-analysis, and the relationship between point estimate risk ratios for the primary outcome and the corresponding standard errors. Fig A5. Meta-analysis of 4-aminoquinoline COVID-19 pre-exposure chemoprevention RCTs based on individual study primary endpoints using Risk Of Bias tool (RoB 2). Fig A6. Meta-analysis of the safety and tolerability outcomes in COVID-19 chemoprevention RCTs using the same methodology as reported in the WHO living guideline [24]. Fig A7. Meta-analysis of adverse events leading to treatment discontinuation reported in double-blind, placebo-controlled, 4-aminoquinoline COVID-19 pre-exposure chemoprevention RCTs. Fig A8. Graph showing cumulative loss to follow-up (LTFU) for Hydroxychloroquine/ Chloroquine arms and Placebo arm.

    (DOCX)

    pmed.1004428.s007.docx (5.6MB, docx)
    Attachment

    Submitted filename: PLOS Med reviews_Final.docx

    pmed.1004428.s008.docx (58.2KB, docx)
    Attachment

    Submitted filename: COPCOV PLOS MED response to editors and reviewers 06_12_23.docx

    pmed.1004428.s009.docx (14.8KB, docx)
    Attachment

    Submitted filename: Response to editors comments 08_01_24.docx

    pmed.1004428.s010.docx (15.1KB, docx)
    Attachment

    Submitted filename: Response to editors comments 08_01_24.docx

    pmed.1004428.s011.docx (15.1KB, docx)
    Attachment

    Submitted filename: Response to requests from Editors.docx

    pmed.1004428.s012.docx (13.8KB, docx)

    Data Availability Statement

    The data underlying the results presented in the study are available from https://github.com/jwatowatson/COPCOV.

    Articles from PLOS Medicine are provided here courtesy of PLOS

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