Abstract

Keywords

1. Introduction

COVID-19 is a pandemic, with a rapid global spread of infection since January 2020 [1]. Four studies have demonstrated that hydroxychloroquine sulfate (HCQ) inhibits SARS-CoV-2 in vitro [[2], [3], [4], [5]]. One study has demonstrated that the combination of HCQ and azithromycin (AZ) inhibits SARS-CoV-2 in vitro [6].

Several clinical studies addressing the efficacy of HCQ were conducted in COVID-19 patients leading to contradictory results. Three studies showed a favourable effect [[7], [8], [9]]. A Chinese randomized control trial (RCT) conducted in 62 COVID-19 patients showed a significantly shortened body temperature recovery time, cough remission time and a larger proportion of improved pneumonia as assessed by CT scan in patients treated with 400 mg HCQ per day during five days (N = 31) than in controls (N = 31) [7]. Another Chinese RCT conducted in 150 COVID-19 patients showed significant favourable differences between patients treated with 1200 mg HCQ/day for three days, then 800 mg/day for two to three weeks (N = 75) and controls (N = 75) regarding alleviation of symptoms and decrease of C-reactive protein concentration [8]. An Iranian study conducted in a cohort of 100 COVID-19 patients treated with 200 mg HCQ twice daily (400 mg single dose when combined with administration of lopinavir/ritonavir) concluded that HCQ improved the clinical outcome of the patients [9].

A Chinese RCT conducted in 30 COVID-19 patients showed no significant differences between patients treated with 400 mg HCQ per day during five days (N = 15) and controls (N = 15) regarding pharyngeal carriage of viral RNA at day 7 [10]. A French study conducted in 181 COVID-19 patients with relatively severe illness did not show any difference between 84 patients treated with 600 mg HCQ/day and 97 controls regarding transfer to ICU and death [11]. Finally, a retrospective analysis of data from patients hospitalized with confirmed SARS-CoV-2 infection in all United States Veterans Health Administration medical centers found no evidence that, before ventilation, the use of HCQ either with or without AZ, reduced the need for subsequent mechanical ventilation [12]. None of these studies was perfect. In the Chinese and Iranian studies, patients received multiple additional treatments including antivirals.

A preliminary French non-randomized clinical trial conducted in 36 COVID-19 patients showed a significant reduction in viral nasopharyngeal carriage at day 6 in patients treated with HCQ at 600 mg per day during 10 days (N = 20, 70% testing negative), compared to untreated controls (N = 16, 12.5% testing negative). In addition, of the twenty patients who were treated with HCQ, six received AZ for five days (for the purpose of preventing bacterial super-infection) and all (100%) were virologically cured at day 6, compared to 57.1% of the remaining 14 patients [13]. This synergistic effect is the rationale to use the combination HCQ and AZ.

We recently reported on 80 patients using a combination of 200 mg HCQ three times daily for ten days plus AZ (500 mg on day 1 followed by 250 mg daily for the next four days) with good clinical and virological outcomes [14]. AZ has been shown to be active in vitro against Zika and Ebola viruses [[15], [16], [17]] and more recently against SARS-CoV-2 [5].

In a recent international survey conducted among at least 7500 physicians across 30 countries, most of the questioned physicians considered that HCQ and AZ are the two most effective treatments among available therapies for COVID-19 [18]. Here, we report a retrospective evaluation of 1061 new COVID-19 patients, treated for at least 3 days with HCQ+AZ from the time of diagnosis and a follow up of at least nine days. Outcomes were death, clinical worsening and viral shedding persistence.

2. Materials & methods

2.1. Patients and study design (Fig. 1)

The study was conducted at Assistance Publique-Hôpitaux de Marseille (AP-HM), Southern France in the Institut Hospitalo-Universitaire (IHU) Méditerranée Infection (https://www.mediterranee-infection.com/). We have set up early unrestricted massive PCR screening for patients suspect of COVID-19 and for asymptomatic contacts of confirmed cases. Data was collected on patients included from March 3rd to March 31st. Individuals with PCR-documented SARS-CoV-2 RNA from a nasopharyngeal sample [19], were proposed HCQ+AZ early treatment, as standard care, whether or not they had symptoms, with treatment initiation at our day-care hospital (inpatients) or at our infectious disease units (inpatients) when required. Patients initially treated in the day-care hospital or discharged from conventional hospitalization wards before day 10 were followed in the day-care hospital (ambulatory follow-up as outpatients). Patients were also referred to the IHU from other health care facilities. Patients with at least three days of treatment and nine days of follow-up are described in this analysis. Demographics, chronic conditions and concomitant medications were documented. The patients described in previous studies [13,14] were not included in the present work. On April 18th, a new evaluation of data was done to update fatal cases and case fatality rates.

2.2. Clinical and radiological classification and follow-up

Details are available from our previous studies [13,14]. Briefly, patients were grouped according to clinical presentation at admission (upper respiratory tract infections or lower respiratory tract infections symptoms) and severity was assessed using the national early warning score (NEWS) for COVID-19 patients at admission and during follow-up [20]. We defined three risk categories for clinical deterioration: low score (NEWS 0–4), medium score (NEWS 5–6), and high score (NEWS≥7). The time between the onset of symptoms and treatment was documented. Patients underwent an unenhanced chest low-dose computed tomography (LDCT). The need for oxygen therapy, transfer to the intensive care unit (ICU), death, and overall length of stay in hospital (for in-patients) were documented. Virological follow-up included ≥1 test(s) done systematically on days 2, 6 and 10. Patients with persistent positive PCR on day 10 were proposed further testing every 4 days until the test became negative.

2.3. COVID-19 treatment and outcomes

Patients with no contraindications [13,14] were proposed a combination of 200 mg of oral HCQ, three times daily for ten days combined with five days of AZ (500 mg on day 1 followed by 250 mg daily for the next four days). Therapy was not supervised. No children <14 years, pregnant women or patients with G6PD deficiency (based on patient's declaration only) were included. The systematic pre-therapy workup included serum electrolyte analysis, and an electrocardiogram with corrected QT measurement (Bazett's formula). A specific inclusion protocol and follow-up for torsade de pointes risk was designed. Any drug, being used by the patient, with the potential to prolong the QT interval and non-vital potassium-depleting drugs (diuretics prescribed for high blood pressure) were systematically stopped. When potassium-depleting drugs could not be stopped or in case of documented hypokalaemia at admission, potassium supplementation was provided and HCQ was administered only when the potassium level was normalized. Close serum electrolyte analysis monitoring was performed in patients with low serum potassium levels at baseline. An electrocardiogram was routinely performed 48 h after the start of treatment. Treatment with HCQ was discontinued when the corrected QT interval (QTc, Bazett's formula) was > 500ms and the risk-benefit ratio of HCQ+AZ treatment was estimated by the infectious disease specialist and agreed with the cardiologist, at between 460 and 500ms. The indications for this control ECG were restricted after an initial workup in 848 ECG from 424 patients (at day 0 and day 2 for each patient) showing that all contraindicative repolarization abnormalities had been detected on the first ECG.

HCQ dosage was performed as previously described [14,21] and a concentration of > 0.1 μg/mL was considered in the therapeutic range [22]. Broad spectrum antibiotics (ceftriaxone or ertapenem) were added for patients with pneumonia and NEWS score ≥ 5. Symptomatic treatments, including notably oxygen, were added as needed. The primary outcomes were i) an aggressive clinical course requiring oxygen therapy, transfer to the ICU or death after at least three days of treatment, and prolonged hospitalization (10 days or more), and ii) contagiousness as assessed by PCR and culture.

2.4. Additional investigations on patients with treatment failure

Patients with clinical or virological failures were accurately characterized and a close clinical and viral follow-up was performed overtime. We defined a group with poor clinical outcome (PClinO) by either death or transfer to ICU or hospitalization for 10 days or more and a group with poor virological outcome (PVirO) was defined by viral shedding persistence at day 10. Finally, individuals who belonged neither to the PClinO group nor the PVirO group were attributed to a group with a good outcome (GO). Factors associated with clinical failure were identified by comparing the PClinO to the GO group and factors associated with virological failure were identified by comparing the PVirO group to the GO group. We performed additional tests on patients with atypical evolution including late SARS-CoV-2 cultures on Vero E6 cells, as previously described [23], and broad-spectrum detection of other viruses by multiplex PCR [19] in respiratory samples. In addition, cDNA was reverse transcribed directly from total viral SARS-CoV-2 RNA rhinopharyngeal samples following the manufacturer's recommendations. cDNAs were purified by using Agencourt AMPure beads (Beckman Coulter, Villepinte, France). Genomic DNA was extracted using the EZ1 biorobot with the EZ1 DNA tissue kit (Qiagen, Hilden, Germany) and then sequenced on a MiSeq sequencer (Illumina Inc, San Diego, CA,USA) with the Nextera Mate-Pair sample prep and Nextera XT Paired End kits (Illumina Inc., San Diego, CA, USA). The SARS-CoV-2 genomes were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/) or are available at EMBL-EBI under the BioProject: PRJEB37693. Phylogenetic reconstruction was performed using NEXSTRAIN (https://nextstrain.org/) and GISAID (Global Initiative; https://www.gisaid.org/) [24].

2.5. Statistical methods

Continuous and categorical variables were presented as mean (std), median, min-max and n (%), respectively. We used the Student t-test, Mann-Whitney U test, Chi-square test, or Fisher's exact test to compare differences between the three groups (GO, PVirO, and PClinO) where appropriate. The GO group was chosen as the reference group for statistical testing (PVirO vs. GO and PClinO vs. GO respectively). To explore risk factors associated with the PVirO and PClinO groups, we also performed multivariable analyses using logistic regression models. All variables significant at p < .01 in univariate analyses were introduced in the initial multivariate model. A stepwise approach was then used to assess the iteration of variables and to control potential confounders (both values of significance level for entry and stay were set at 0.05.) A two-sided alpha of less than 0.05 was considered statistically significant. All analyses were carried out using SAS 9.4 statistical software (SAS Institute, Cary, NC).

2.6. Ethics statement

Data presented herein were collected retrospectively from routine care using the electronic health recording system of the hospital according to the MR-004 reference methodology for the processing of personal data. Accessibility to data is protected according to European General Data Protection Regulation No 2016/679. The non-interventional retrospective nature of the study has been approved by our institutional review board committee (Mediterranée Infection N°: 2020–13). At the time the study was conducted, HCQ was approved for COVID-19 as a hospital delivery only, in France. For all patients, the prescription of HCQ+AZ was made during either complete hospitalization or at day-care hospital by one of the practicing physicians, independently of the investigator, after collegial decision based on the most recent scientific data available and after assessment of the benefit/harm ratio of the treatment in accordance with the provisions of the Code of Ethics (Article R. 4127-8 of the Public Health Code). No supplementary monitoring or diagnostic procedures were added to normal clinical practice allowing surveillance and management of patient (monitoring of HCQ levels and SARS-CoV-2 viral load surveillance and ECG).

3. Results

3.1. Participants