Introduction

The coronavirus disease 19 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARSCoV-2), has led to over 530 million cases and over 6.2 million deaths worldwide, including over 78 million cases and over 960,000 deaths in the United States [1]. The mainstay of SARSCoV-2 prevention throughout the pandemic focused on infection control measures, such as physical distancing, enhanced hand hygiene, and use of protective equipment. The development and implementation of effective COVID-19 vaccines significantly advanced COVID-19 prevention capacity, although breakthrough infections occur and vaccine rollout worldwide has been hindered by vaccine hesitancy and/or supply and distribution challenges. Multiple therapeutics exist for COVID-19, including immunomodulatory, antiviral, and monoclonal antibody agents; however, only one monoclonal antibody (tixagevimib/cilgavimab) is currently licensed as pre-exposure prophylaxis. Both the supply of tixagevimib/cilgavimab and the population eligible to receive it are limited. Additional effective pre-exposure prophylactic medications against SARS-CoV-2 would mitigate the risk of infection, especially if future SARS-CoV-2 variants capable of eluding vaccine-induced immunity and escaping neutralization by monoclonal antibodies become widespread. Effective preexposure prophylaxis would benefit individuals at high risk of exposure or severe disease in addition to those with decreased vaccine responsiveness.

Hydroxychloroquine is approved by the U.S. Food and Drug Administration for the treatment and prevention of malaria and the treatment of rheumatoid arthritis, systemic lupus erythematosus and chronic discoid lupus erythematosus. Interest in using hydroxychloroquine for SARS-CoV-2 infection developed after hydroxychloroquine and its relative, chloroquine, were demonstrated to inhibit SARS-CoV-2 in vitro [2, 3]. Multiple mechanisms for the inhibition of SARS-CoV-2 by hydroxychloroquine have been proposed, including impairment of spike protein binding to cellular gangliosides, blockade of virus transport from early endosomes to lysosomes, and increased intraorganellar pH [2, 4-7]. Additionally, hydroxychloroquine has immunomodulatory effects that could modify COVID-19 disease by dampening the hyperinflammatory state induced by SARS-CoV-2 infection [7,8]. The U.S. Food and Drug administration issued an emergency use authorization (EUA) for hydroxychloroquine on 28 March 2020, but revoked the EUA on 15 June 2020 based on lack of clinical benefit during treatment of patients hospitalized with COVID-19. The majority of studies of COVID-19 treatment with hydroxychloroquine, either alone or combined with azithromycin, have not demonstrated improvement in clinical outcomes [9-19].

In vitro studies demonstrate that inhibition of SARSCoV-2 by hydroxychloroquine was greater when the drug was added prior to viral infection compared to post-infection [3]. This raised questions as to whether hydroxychloroquine pre-exposure prophylaxis could decrease SARS-CoV-2 infection. Clinical trials of hydroxychloroquine pre-exposure prophylaxis to date have not demonstrated a difference in SARS-CoV-2 infection [20-22]. Here we present the results of a nonrandomized, open-label clinical trial [Clinicaltrials.gov NCT04354870] of hydroxychloroquine pre-exposure prophylaxis among health care workers (HCWs) at high risk of occupational SARS-CoV-2 exposure in the New York University Langone Health System (NYULHS) initiated during the first 2020 wave of the COVID-19 pandemic in New York City, prior to COVID-19 vaccine availability, to determine the efficacy of pre-exposure prophylactic hydroxychloroquine for the prevention of SARS-CoV-2 infection and symptomatic COVID-19.

Methods

Study Design

To evaluate whether hydroxychloroquine pre-exposure prophylaxis reduced the incidence of SARS-CoV-2, we conducted a nonrandomized, open-label clinical trial (ClinicalTrials.gov NCT04354870) among HCWs in the NYULHS. The primary outcome was seroconversion to SARS-CoV-2. Secondary outcomes included COVID-19 illness severity, incidence of symptomatic and asymptomatic SARS-CoV-2 infection, and hydroxychloroquine tolerability/adverse events. All study visits occurred remotely due to the ongoing COVID-19 pandemic. Following a combined screening and enrollment visit, study visits were conducted every 30 days (+/- 7 days) to complete four study visits. The study was reviewed and approved by the New York University Grossman School of Medicine (NYUSOM) institutional review board (IRB) prior to study initiation.

Study Arm Assignments

Participants chose whether they were included in the hydroxychloroquine arm or no hydroxychloroquine arm of the study. Verbal assessment of whether an eligible participant elected to take or declined hydroxychloroquine was obtained at enrollment. Participants were allowed to switch between study arms by contacting the study team or indicating a change in hydroxychloroquine use via digital questionnaire. Primary and secondary outcome analyses were performed based on original study arm assignment using Independent Samples T-test with Welch’s correction (GraphPad Prism 9.2).

Participants

This study recruited adults ages 18 years and older employed as HCWs in the NYULHS who provided informed consent for study participation and were involved in aerosol generating procedures on confirmed COVID-19 patients using appropriate personal protective equipment (PPE), provided direct bedside care to confirmed COVID-19 patients using appropriate PPE for three or more shifts in a 7-day period, and/or provided direct care to suspected COVID-19 patients in the emergency department or another inpatient unit using appropriate PPE for three or more shifts in a 7-day period. Participants were excluded from the hydroxychloroquine arm if they had any of the following: diagnosed with COVID-19; known hypersensitivity to hydroxychloroquine or chloroquine; stage 4 or higher chronic kidney disease, retinal disease, congenital prolonged QTc interval syndrome, or torsade de pontes; or concomitant use of amiodarone, digoxin, flecainide, procainamide, or propafenone.

Sample Size

An anticipated sample size of 350 participants was calculated to have 80% power to detect a 15% difference in seroconversion between the study arms assuming a two-sided type I error rate of 0.05 and a seroconversion rate of 35% in the no hydroxychloroquine arm. We did not know in advance how many study participants would decide to take hydroxychloroquine, but estimated twothirds of participants would elect to take hydroxychloroquine.

Informed Consent, Screening, and Demographics

Interested individuals were prescreened by telephone. Following prescreening, individuals who appeared eligible were emailed electronic consent forms using the Research Electronic Data Capture (REDCap) system. A study coordinator reviewed the electronic consent form with the participant by phone and answered any questions about the study during the screening/ enrollment visit. Participants consented via electronic signature while in verbal communication with the study team, after which demographics, employment information, and medical history were collected to verify eligibility. Additional medication and symptom questionnaires were distributed to participants via REDCap.

Hydroxychloroquine Dispensing

Prescriptions for oral hydroxychloroquine tablets (600mg on day 1 followed by 200mg daily thereafter) were provided to an NYU-based pharmacy which dispensed the study medication from a dedicated study supply for participants who opted to take hydroxychloroquine. Study medication was either picked up by participants in-person or mailed directly to participants. 30day prescriptions were prescribed and filled in a similar manner at study day 30 and 60 for participants who remained in the hydroxychloroquine study arm.

Digital Questionnaires

Individualized links to online REDCap questionnaires were emailed to participants at each study visit (screening/ enrollment and days 30, 60, and 90 following enrollment). Links provided access to questionnaires regarding any potential adverse events, dried blood spot (DBS) card completion, medication use (hydroxychloroquine and concomitant medications), and potential COVID-19 symptoms. Questionnaire responses were connected to individual participants only by study-assigned identification numbers. Any participant reporting a potential adverse event was subsequently contacted by a study investigator to collect additional data.

Dried Blood Spot Collection

DBS cards were self-collected by participants at enrollment, and days 30, 60, and 90 after enrollment. Participants were provided with four DBS collection kits and written instructions for collecting and returning DBS cards via designated drop-off collection boxes located at each participating NYULHS facility. Each collection kit contained: written collection instructions, alcohol pads, lancets, gauze, bandages, pre-labeled DBS cards and sealable foil bags containing desiccant, and envelopes for dropoff. In brief, participants were instructed to wash their hands, clean the tip of their finger with an alcohol pad, and deploy a contactactivated lancet to elicit a drop of blood. The first drop of blood was wiped away and subsequent drops were lightly dabbed onto the DBS card until all five spots on the card were filled, after which a bandage was applied to the finger. The DBS card was allowed to air dry at room temperature for 3 hours and then sealed in the foil bag containing desiccant, which was placed in a biosafety hazardlabeled envelope and deposited at one of the designated drop-off sites within 2 weeks after collection.

Dried Blood Spot Elution

A 6 mm hole-puncher was used to remove dried blood spots from the card. Each punched out spot was placed in a well of a 24well plate containing 300 µl phosphate buffered saline (PBS) with 0.05% Tween 20 (PBS-T) and 0.08% sodium azide and shaken for 4 hours at room temperature or overnight at 4ºC. Eluates were transferred to microcentrifuge tubes and centrifuged for 2 minutes at 10,500 g prior to use.

Dried Blood Spot ELISA Validation

IgM and IgG antibodies to SARS-CoV-2 S1 protein in sera and DBS collected simultaneously from NYULHS employees with or without a known history of SARS-CoV-2 infection, who provided informed consent to participant in a separate NYUSOM IRB-approved protocol for healthy volunteers, were assessed using an in-house developed SARS-CoV-2 S1 ELISA. Dried blood spots eluates and sera were heat inactivated at 56°C for 1 hour prior to use.

SARS-CoV-2 S1 ELISA

96-well ELISA plates were coated with 1 µg/ml SARSCoV-2 S1 protein (Sino Biological Inc. 40591-V08H) diluted in PBS and incubated overnight at 4°C. Plates were washed four times with PBS-T and blocked with PBS-T containing 4% non-fat milk and 5% whey for 1 hour prior to adding sera or DBS eluates. For DBS ELISA validation and controls, sera samples were diluted to a 1:50 concentration while DBS eluates were not diluted.

Plates were washed four times with PBS-T after a 2-hour incubation period at room temperature. Horseradish-peroxidase conjugated goat-anti human IgG and IgM (Southern BioTech 2040-05 and 2020-05) was added in 1:2000 and 1:1000 dilutions, respectively. After incubating for 1 hour plates were washed four times with PBS-T and developed with TMB Peroxidase Substrate 3,3′,5,5′-Tetramethylbenzidine (Thermo Scientific 34029) for 5 minutes, after which the reaction was stopped with 1M sulfuric acid or 1N hydrochloric acid. The optical density (OD) was determined by measuring the absorbance at 450 nm on a Synergy 4 (BioTek) plate reader. Heat inactivated pooled sera from hospitalized individuals with COVID-19 served as positive controls and sera collected from healthy volunteers prior to the COVID-19 pandemic served as negative controls. All control samples were obtained from research protocols approved by the NYUSOM IRB.

Results

Trial Participation

A total of 156 participants were screened for the trial, of which one hundred and thirty participants enrolled between 29 April 2020–15 May 2020. Enrollment was closed on 21 July 2020 due to lack of further interest from potential participants. Eightythree participants elected to take hydroxychloroquine and 47 declined hydroxychloroquine (Figure 1). Participants who elected to take hydroxychloroquine were more likely to be male (p=0.02) and had a median older age (p=0.001) compared to participants who declined hydroxychloroquine (Table 1). No significant differences in race (p=0.91) or ethnicity (p=0.21) were reported among individuals who elected to take or declined hydroxychloroquine. Of the one hundred and thirty enrolled participants, thirty-nine (30%) formally left the study, of which twenty-three (17.7%) were participant-initiated and sixteen (12.3%) were withdrawn by study investigators. Only ten participants (12%) who originally elected to take hydroxychloroquine remained on the medication at day 90.

Discussion

Among health care workers at high risk of occupational SARSCoV-2 exposure who elected to take oral hydroxychloroquine, 600 mg once followed by 200 mg daily, for up to 90 days, we did not observe a statistically significant decrease in SARSCoV-2 seroconversion associated with hydroxychloroquine use. These findings are consistent with those observed among other trials of hydroxychloroquine pre-exposure prophylaxis for HCWs and retrospective analyses of SARS-CoV-2 incidence among individuals taking hydroxychloroquine for other medical conditions [20-22, 24-26].

Our methodology differed from previous studies in the dosing of hydroxychloroquine and the use of dried blood spots to assess for seroconversion with an in-house developed SARSCoV-2 S1 ELISA. Dried blood spots were selected for use in this study due limitations in the availability of commercial serological SARS-CoV-2 testing at the time of study initiation and to facilitate self-collection of samples at home, thereby decreasing the risk of SARS-CoV-2 exposure by eliminating the need for travel to study visits. Although receipt of a COVID-19 vaccine could impact the interpretation of seroconversion of the S1 SARS-CoV-2 ELISA, this would not have affected the outcomes of this study as it was conducted in its entirety in 2020 prior to the availability of COVID-19 vaccines to the NYULHS HCW population.

The incidence of adverse events in the hydroxychloroquine arm in this study (9.6% of hydroxychloroquine users) was much lower than that reported (31%-45%) among other preexposure prophylaxis studies of HCWs [20-22]. We do not anticipate the lower adverse event rate is attributable to the dosing selected, but could be impacted by our high rates of hydroxychloroquine discontinuation (88.0%) over the course of the study. We identify multiple factors that may have contributed to the high rate of hydroxychloroquine discontinuation in this study, including adverse events attributed to hydroxychloroquine, the U.S. Food and Drug administration revoking the EUA for hydroxychloroquine for COVID-19 treatment [27], and negative media coverage of hydroxychloroquine during the study period.

This study has several limitations. The study was underpowered to detect the effectiveness of hydroxychloroquine to reduce SARS-CoV-2 seroconversion. This study was initiated in April 2020, during the peak of the first wave of the pandemic in New York City [28]. Initial estimates of anticipated seroconversion rates during the study period proved to be overestimates as the incidence of new COVID-19 cases significantly declined in New York City in May and June, 2020, and remained low for the remainder of the study period [28]. Adoption of universal PPE use for all patient care encounters in the NYULHS during this period, including fit-tested N95 masks or equivalent, may have contributed to lower than expected seroconversion rates. Additionally, high rates of hydroxychloroquine discontinuation further decreased the ability of the study to detect differences in seroconversion between hydroxychloroquine preexposure prophylaxis users and non-users.

Conclusions

Although our study was underpowered, our results suggest that pre-exposure prophylaxis with oral hydroxychloroquine, 600 mg once followed by 200 mg daily for up to 90 days, was safe but did not reduce seroconversion to SARS-CoV-2 among health care workers at high risk of occupational exposure to SARS-CoV-2 or reduce symptomatic presentation at the time of seroconversion. Further adequately-powered randomized controlled trials in areas with high SARS-CoV-2 circulation would be needed to adequately assess the effectiveness of off-label hydroxychloroquine for the prevention of SARS-CoV-2 infection.

Funding and Acknowledgements

The New York University Langone Health System provided funding for this study. This study was conducted using clinical support provided the NYU-H+H Clinical and Translational Science Institute (CTSI), which is supported by the National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, through Grant Award Number UL1TR001445. We additionally wish to acknowledge technological support for electronic consenting for this study provided by the NYU Division of Advanced Research Technologies and the NYUSOM IRB, as well as efforts provided by Marina Godina, Mahnoor Ali, Laura Frye, Tori Rodrick, and Spandan Pandya.

Conflicts of Interest Statement

The authors report the following potential conflicts of interest:

MJM: Laboratory research and clinical trials contract funding for vaccines or monoclonal antibodies to SARS-CoV-2 with Lilly, Pfizer, and Sanofi Pasteur; personal fees for Scientific Advisory Board service from Merck & Co, Meissa Vaccines, Inc. and Pfizer.

VNR: Clinical trials contract funding for vaccines to SARS-CoV-2 with Sanofi Pasteur and Pfizer, and laboratory research funding related to Ebola vaccine from Merck & Co.

Data Sharing Statement

Individual level data from participants will not be shared to protect the privacy of the participating health care workers.

Authorship Statement

Drs. Belmont and Raabe were responsible for the design and conduct of the hydroxychloroquine trial. Drs. Samanovic, Mulligan, and Lai were responsible for the design and conduct of the dried blood spot validation study. Dr. Belli was responsible for the design and conduct of statistical components for the dried blood spot study. Drs. Fleming, Lai, and Samanovic generated data. All authors contributed to the analysis and interpretation of the data, drafting the article and revising it for important intellectual content, and approved of the final version.

References

1. World Health Organization (2022) WHO coronavirus (COVID-19) [Internet] 2022 June 7 [cited 2022 8 June]. https://covid19. who.int/
2. Vincent MJ, Bergeron E, Benjannet S, Erickson BR, Rollinet PE, et al., (2005) Chloroquine is a potent inhibitor of SARS coronavirus infection and spread. Virol J 2:69.
3. Yao X, Ye F, Zhang M, Cui C, Huang B, et al., (2020) In vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Clin Infect Dis 71:732-739.
4. Fantini, J, Di Scala C, Chahinian H, Yahi N (2020) Structural and molecular modelling studies reveal a new mechanism of action of chloroquine and hydroxychloroquine against SARS-CoV-2 infection. Int J Antimicrob Agents 55:105960.
5. Liu J, Cao R, Xu M, Wang X, Zhang H, et al., (2020) Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARSCoV-2 infection in vitro. Cell Discov 6:16.
6. Richard SA, Kampo S, Hechavarria ME, Sackey M, Buunaaim ADB, et al., (2020) Elucidating the pivotal immunomodulatory and antiinflammatory potentials of chloroquine and hydroxychloroquine. J Immunol Res 2020:4582612.
7. Vitte J, Michel M, Mezouar S, Diallo AB, Boumaza A, et al., (2020) Immune modulation as a therapeutic option during the SARS-CoV-2 outbreak: The case for antimalarial aminoquinolines. Front Immunol 11:2159.
8. Lucchesi A, Silimbani P, Musuraca G, Cerchione C, Martinelli G, et al., (2021) Clinical and biological data on the use of hydroxychloroquine against SARS-CoV-2 could support the role of the nlrp3 inflammasome in the pathogenesis of respiratory disease. J Med Virol 93:124-126.
9. Sivapalan P, Ulrik CP, Lapperre TS, Bojesen RD, Eklöf J, et al., (2022) Azithromycin and hydroxychloroquine in hospitalised patients with confirmed COVID-19-a randomised double-blinded placebo-controlled Eur Respir J 59:2100752.
10. Brown SM, Peltan I, Kumar N, Leither L, Webb BJ, et al., (2020) Hydroxychloroquine vs. Azithromycin for hospitalized patients with COVID-19 (HAHPS): Results of a randomized, active comparator trial. Ann Am Thorac Soc 18:590-597.
11. Schwartz I, Boesen ME, Cerchiaro G, Doram C, Edwards BD, et , (2021) Assessing the efficacy and safety of hydroxychloroquine as outpatient treatment of COVID-19: A randomized controlled trial. CMAJ Open 9:E693-e702.
12. Sands K, Wenzel R, McLean L, Korwek K, Roach J, et al., (2021) No clinical benefit in mortality associated with hydroxychloroquine treatment in patients with COVID-19. Int J Infect Dis 104:34-40.
13. Ulrich RJ, Troxel AB, Carmody E, Eapen J, Bäcker M, et al., (2020) Treating COVID-19 with hydroxychloroquine (TEACH): A multicenter, double-blind randomized controlled trial in hospitalized patients. Open Forum Infect Dis 7: ofaa446.
14. Geleris J, Sun Y, Platt J, Zucker J, Baldwin M, et al., (2020) Observational study of hydroxychloroquine in hospitalized patients with COVID-19. N Engl J Med 382:2411-2418.
15. Ip A, Berry DA, Hansen E, Goy AH, Pecora AL, et al., (2020) Hydroxychloroquine and tocilizumab therapy in COVID-19 patients-an observational study. PLoS One 15: e0237693.
16. Magagnoli J, Narendran S, Pereira F, Cummings TH, Hardin JW, et , (2020) Outcomes of hydroxychloroquine usage in united states veterans hospitalized with COVID-19. Med (N Y) 1:114-127.e3.
17. Mahévas M, Tran V-T, Roumier M, Chabrol A, Paule R, et al., (2020) Clinical efficacy of hydroxychloroquine in patients with COVID-19 pneumonia who require oxygen: Observational comparative study using routine care data. BMJ 369:m1844.
18. Rosenberg ES, Dufort EM, Udo T, Wilberschied LA, Kumar J, et , (2020) Association of treatment with hydroxychloroquine or azithromycin with in-hospital mortality in patients with COVID-19 in new york state. JAMA 323:2493-2502.
19. Self WH, Semler MW, Leither LM, Casey JD, Angus DC, et al., (2020) Effect of hydroxychloroquine on clinical status at 14 days in hospitalized patients with COVID-19: A randomized clinical trial. JAMA 324:2165-2176.
20. Abella BS, Jolkovsky EL, Biney BT, Uspal JE, Hyman MC, et al., (2021) Efficacy and safety of hydroxychloroquine vs placebo for preexposure SARS-CoV-2 prophylaxis among health care workers: A randomized clinical trial. JAMA Intern Med 181:195-202.
21. Grau-Pujol B, Camprubí-Ferrer D, Marti-Soler H, Fernández-Pardos M, Carreras-Abad C, et al., (2021) Pre-exposure prophylaxis with hydroxychloroquine for COVID-19: A double-blind, placebo-controlled randomized clinical trial. Trials 22:808.
22. Rajasingham R, Bangdiwala AS, Nicol MR, Skipper CP, Pastick KA, et al., (2021) Hydroxychloroquine as pre-exposure prophylaxis for coronavirus disease 2019 (COVID-19) in healthcare workers: A randomized trial. Clin Infect Dis 72:e835-e843.
23. Stadlbauer D, Tan J, Jiang K, Hernandez MM, Fabre S, et al., (2021) Repeated cross-sectional sero-monitoring of SARS-CoV-2 in new york Nature 590:146-150.
24. Sjöwall J, Azharuddin M, Frodlund M, Zhang Y, Sandner L, et al., (2021) SARS-CoV-2 antibody isotypes in systemic lupus erythematosus patients prior to vaccination: Associations with disease activity, antinuclear antibodies, and immunomodulatory drugs during the first year of the pandemic. Front Immunol 12:724047.
25. Jung SY, Kim MS, Kim MC, Choi SH, Chung JW, et al., (2021) Effect of hydroxychloroquine pre-exposure on infection with SARSCoV-2 in rheumatic disease patients: A population-based cohort study. Clin Microbiol Infect 27:611-617.
26. Macías J, González-Moreno P, Sánchez-García E, Morillo-Verdugo R, Pérez-Venegas JJ, et al., (2021) Similar incidence of coronavirus disease 2019 (COVID-19) in patients with rheumatic diseases with and without hydroxychloroquine therapy. PLoS One 16:e0249036.
27. S. Food and Drug Administration (2020) Coronavirus (COVID-19) update: FDA revokes emergency use authorization for chloroquine and hydroxychloroquine. [Internet] 2020 2020 Jun 15 [cited 2021 Nov 26]; Available from: https://www.fda.gov/news-events/pressannouncements/coronavirus-COVID-19-update-fda-revokesemergency-use-authorization-chloroquine-and
28. NYC Health (2021) COVID-19: Data. [Internet] 2021 [cited 2021 Nov 26]; Available from: https://www1.nyc.gov/site/doh/covid/COVID-19data-totals.page.