Hydroxychloroquine plus azithromycin early treatment of mild COVID-19 in an outpatient setting:

Hydroxychloroquine plus azithromycin early treatment of mild COVID-19 in an outpatient setting: a randomized, double-blinded, placebo-controlled clinical trial evaluating viral clearance

Background

Hydroxychloroquine has shown a potential to block viral replication of SARS-CoV-2 in some in vitro studies. This randomized, double-blinded, placebo-controlled clinical trial evaluated the efficacy of hydroxychloroquine plus azithromycin (HCQ/AZT) in reducing viral loads in patients with early and mild SARS-CoV-2 infection.

Methods

A single-center randomized placebo-controlled clinical trial was conducted with outpatients with early and mild SARS-CoV-2 infection. Inclusion criteria were: patients aged 18–65 years with symptoms suggestive of COVID-19 for < 5 days, no significant comorbidities, and positive nasopharyngeal/oropharyngeal swab screening tests (POCT-PCR). Randomized patients received either hydroxychloroquine for 7 days plus azithromycin for 5 days or placebo. The primary endpoint was viral clearance within 9 days. Secondary endpoints included viral load reduction, clinical evolution, hospitalization rates, chest computed tomography evolution, and adverse effects.

Results

Of 107 potential trial participants, 84 were enrolled following predetermined criteria. Statistical analyses were performed on an intention-to-treat (N = 84) and per-protocol (PP) basis (N = 70). On the PP analysis, the treatment (N = 36) and placebo (N = 34) groups displayed similar demographic characteristics. At 95% CI, no statistically significant between-group differences were found in viral clearance rates within 9 days following enrolment (P = 0.26).

Conclusions

This randomized, double-blinded, placebo-controlled clinical trial evaluating outpatients with early and mild COVID-19 showed that viral clearance rates within 9 days from enrolment did not change with HCQ/AZT treatment compared with placebo, although no major cardiovascular events were observed in participants without comorbidities. Secondary outcomes were also not significantly improved with HCQ/AZT treatment compared with placebo. These findings do not support the use of HCQ/AZT in this setting.

METHODS

2.1. Study design and participants

This study was designed as a prospective, double-blinded, placebo-controlled, randomized clinical trial by The Consolidated Standards of Reporting Trials (CONSORT) Statement, approved by the local ethics and research committee, and registered at REBEC (30413020.8.0000.0008). Written informed consent was obtained from all subjects and it followed the 1964 Declaration of Helsinki (amended mostly recently in 2008) of the World Medical Association. It was conducted at a single center: Hospital Santa Paula located in São Paulo, Brazil. Participants were enrolled after providing written consent in a hospital emergency room setting. Inclusion criteria were: aged 18–65 years, with mild symptoms suggestive of COVID-19, with an interval from symptom onset to the enrolment of 2–5 days, and detection of viral RNA in nasopharyngeal/oropharyngeal swabs through a real-time reverse-transcription polymerase chain (RT-PCR) reaction screening point-of-care test (POCT-PCR). The clinical picture suggestive of COVID-19 was defined as two or more of the following: cough, fever, shortness of breath, nausea/vomiting, diarrhea, body aches, weakness/fatigue, headache, sore throat, runny nose/congestion, and sudden gustatory or olfactory loss. Exclusion criteria included: known hypersensitivity to HCQ or AZT, pre-existing pulmonary disease, history of immunosuppression, active cancer diagnosis, pregnancy or lactation, history of cardiac abnormalities or QTc prolongation (QTc > 480 ms), known glucose-6-phosphate dehydrogenase (G6PD) deficiency, patients requiring hospital admittance, and patients with inadequate hematological parameters, heart, renal, or liver function.

ClinicalTrials.gov definition of serious adverse events was followed, defined as adverse events that result in death, a life-threatening adverse event, inpatient hospitalization, and persistent or significant incapacity or substantial disruption of the ability to conduct normal functions [18]. Complete information on the inclusion and exclusion criteria is provided in the Supplementary Appendix. All study data were collected and managed using the Research Electronic Data Capture (REDCap) system [19]. All data from the participants were adequately anonymized.

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2.2. Sample size calculation

The sample size was calculated using the survival routine from R software [20]. This sample was calculated considering the survival analysis with the Lachin and Foulkes method (1986) [21] based on the follow-up period, recruitment time, and hazard ratio, comparing control and intervention groups. The following assumptions were considered: study period of 30 days, recruitment period of 30 days, statistical significance level of 5%, 85% power, and 30% of follow-up losses. For the effect size, viral clearance rates after 4 treatment days were assumed based on Gautret et al. [4]: 83.3% in the treatment group and 25% in the control group. The sample size calculation resulted in 42 participants for each study arm (84 total number of participants).

2.3. Molecular testing

At the enrolment visit, nasopharyngeal/oropharyngeal swabs were collected and combined into 3 mL sterile saline for testing by POCT-PCR. An aliquot of 300 µL was submitted to RNA extraction in the Veri-Q PREP M16 equipment using the Viral DNA/RNA Prep Kit – Airway Clinical Sample (Micobiomed, Korea) with an elution volume of 50 µL. Amplification and detection were performed using the Veri-Q PCR 316 real-time thermocycler using the Coronavirus disease 2019 (COVID-19) Detection Kit (Microbiome). Both systems and kits work together as a small footprint integrated system, suitable for simultaneously running 1–6 samples in approximately 2 hours. This kit targets two SARS-CoV-2 genomic regions: the N gene and ORF 3a, in addition to internal control, added at the PCR step. Cycle threshold (Ct) values < 35 in any of the two genes were assigned as positive, while Ct values 35–40 were considered ‘indeterminate’, according to the manufacturer's instructions.

An aliquot from the emergency department day 0 sample and all samples from the subsequent home visits at 3, 6, and 9 days were processed at the molecular biology lab from Diagnósticos da América S.A. (Dasa, Brazil). An aliquot of 200 µL was extracted by the DSP Virus/Pathogen kit in the automated platform QIAsymphony and eluted in 60 µL; 5 µL of the eluate was submitted to RT-PCR with primers and probe from the viral E gene in duplex to the cellular control RNAseP, as described [22], employing TaqMan Fast Virus 1-Step Master Mix (ThermoFisher, Brazil). A Ct value of 35 was adopted as the cut-off. This system is referred to as RT-PCR to distinguish it from the POCT-PCR platform used in the initial screening. The limit of detection was determined as 408 copies/mL by probit analysis using the ACCUPLEX SARS-COV-2 reference material (0505-0126, Seracare, USA).

2.4. Randomisation and double-blinding

Eligible patients were randomly assigned (1:1) to receive either HCQ/AZT or placebo. Patients, treating clinicians, and study personnel was all blinded to study group assignment. An independent pharmacist dispensed all trial medications (or placebo) according to a computer-generated randomization list. The therapeutics and placebo were provided in capsule form and were identical in appearance. Capsules were pre-packed in envelopes and consecutively numbered for each participant according to the randomization schedule. Double-blinding was maintained throughout the trial.

2.5. Interventions

Following randomization, the participants had blood samples collected and were prescribed appropriately. Treatment group participants received two 200 mg HCQ capsules twice a day (bid) for a total course of 7 days (i.e. 28 capsules in total) and one 500 mg AZT capsule taken on day 1, followed by one 250 mg AZT capsule daily for the next 4 days (i.e. six capsules in total). The placebo capsules were formulated to have a similar size, shape, color, and taste as HCQ and AZT capsules, and with an identical dosing regimen. The first dose of medication was given orally to all patients in the emergency department under pharmacist supervision and the remaining doses were ingested at home. As the standard of care, no vitamin or mineral supplements were prescribed. Participants also received instructions regarding social distancing and hygiene to be followed at home and were given 24/7 telephonic access to clinical staff in case of questions, symptom evolution, or adverse effects. At their homes, participants received telephonic check-up calls on days 3, 6, 9, and between 14–21 days following enrolment, when they responded to a symptom and adverse effect questionnaires. Participants also received visits from diagnostic staff on days 3, 6, and 9 who collected blood samples, performed electrocardiograms and collected nasopharyngeal/oropharyngeal swabs for RT-PCR testing. Between days 14–21, participants returned to a diagnostic facility where an additional chest computed tomography was performed followed by the telephonic check-up as before.

All of the nasopharyngeal specimens of the included patients that were collected to perform the study screening enrolment POCT-PCR were also submitted for PCR testing in a central lab to obtain the day-0 viral load, to apply the same molecular method for the primary and secondary virological outcomes. Additionally, enrolment samples were submitted to a molecular assay comprising a panel of 20 respiratory pathogens (FilmArray, BioMerieux, Brazil) to investigate co-infection with other viral and bacterial agents. Complete information of the trial procedures and examinations performed at each key date is provided in the Supplementary Appendix.

DISCUSSION

In this randomized, double-blinded, placebo-controlled clinical trial evaluating outpatients with early and mild COVID-19 treated with HCQ/AZT or placebo, there was no benefit in the treatment arm on primary and secondary outcomes. The study population comprised adult patients aged < 65 years, with no comorbidities and mild symptom onset 2–5 days before enrolment. Viral clearance rates within 9 days from symptom onset were similar in both groups, showing that the HCQ/AZT combination is not effective in reducing viral shedding and thus unlikely to minimize the SARS-CoV-2 rate of transmission.

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