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treated with HCQ+AZ ≥ 3 days at IHU Méditerranée infection Marseille, France with day 0 between March 3 and March 31, 2020.
| Poor virological outcomea | Good outcome | Poor clinical outcomea,b | Total | |
|---|---|---|---|---|
| n (%) | n (%) | n (%) | n (%) | |
| Group size | 47 (4.4%) | 973 (91.7%) | 46 (4.3%) | 1061 (100%) |
| Age (years) | ||||
| Mean (SD) | 47.9 (17.5) | 42.4 (14.7) | 69.2 (14.0) | 43.6 (15.6) |
| Median [Min-Max] | 48.0 [18.0–89.0]* | 42.0 [14.0–86.0] | 69.0 [31.0–95.0]*** | 43.0 [14.0–95.0] |
| Male | 19 (40.4%) | 450 (46.3%) | 23 (50%) | 492 (46.4) |
| Chronic condition(s) and treatment(s) | ||||
| Chronic conditions | ||||
| Cancer | 0 (0.0%) | 21 (2.2%) | 7 (15.2%)*** | 28 (2.6%) |
| Diabetes | 3 (6.4%) | 66 (6.8%) | 9 (19.6%)*** | 78 (7.4%) |
| Coronary artery disease | 2 (4.3%) | 36 (3.7%) | 9 (19.6%)*** | 46 (4.3%) |
| Hypertension | 8 (17%) | 120 (12.3%) | 23 (50.0%)*** | 149 (14%) |
| Chronic respiratory diseases | 8 (17%) | 96 (9.9%) | 8 (17.4%) | 111 (10.5%) |
| Obesity | 1 (2.1%) | 57 (5.9%) | 4 (8.7%) | 62 (5.8%) |
| Comedication(s) | ||||
| Biguanides (metformin) | 1 (2.1%) | 15 (1.5%) | 4 (8.7%)** | 20 (1.9%) |
| Selective beta blocking agents | 6 (12.8%)** | 22 (2.3%) | 9 (19.6%)*** | 34 (3.2%) |
| Dihydropyridine derivatives | 3 (6.4%) | 23 (2.4%) | 8 (17.4%)*** | 34 (3.2%) |
| Angiotensin II receptor blockers | 6 (12.8%)** | 22 (2.3%) | 14 (30.4%)*** | 40 (3.8%) |
| HMG CoA reductase | 4 (8.5%) | 28 (2.9%) | 7 (15.2%)*** | 38 (3.6%) |
| Diuretics | 2 (4.3%) | 28 (2.9%) | 5 (10.9%)* | 35 (3.3%) |
| Time between onset of symptoms and first day of treatment start (days)c | ||||
| Mean (SD) | 4.3 (2.5) | 6.5 (3.9) | 5.9 (4.0) | 6.4 (3.8) |
| Median [Min-Max] | 4.0 [0.0–9.0]*** | 6.0 [0.0–27.0] | 5.0 [0.0–16.0]*** | 6.0 [0.0–27.0] |
| Clinical classification (NEWS score) | ||||
| 0–4 (low) | 43 (91.5%)* | 948 (97.4%) | 19 (41.3%)*** | 1008 (95.0%) |
| 5–6 (medium) | 2 (4.3%) | 14 (1.4%) | 10 (21.7%) | 25 (2.4%) |
| ≥ 7 (high) | 2 (4.3%) | 11 (1.1%) | 17 (37.0%) | 28 (2.6%) |
| Low-dose pulmonary CT-scanner within 72 h of admissiond | ||||
| Normal | 11/37 (29.7%) | 231/642 (36.0%) | 4/39 (10.3%)*** | 245/714 (34.3%) |
| Limited | 23/37 (62.2%) | 277/642 (43.2%) | 10/39 (25.6%) | 307/714 (43.0%) |
| Medium | 3/37 (8.1%) | 123/642 (19.2%) | 20/39 (51.3%) | 146/714 (20.5%) |
| Severe | 0/37 (0.0%) | 11/642 (1.7%) | 5/39 (12.8%) | 16/714 (2.2%) |
| Viral load at inclusion (Ct - nasal)e | ||||
| Mean (SD) | 23.4 (5.1) | 26.8 (4.9) | 25.6 (4.8) | 26.6 (5.0) |
| Median [Min-Max] | 22.1 [14.8–34.0]*** | 27.3 [12.8–34.0] | 25.8 [15.0–33.2] | 27.0 [12.8–34.0] |
| Hydroxychloroquine levels at day 2 (μg/mL)f | ||||
| Mean (SD) | 0.25 (0.17) | 0.26 (0.16) | 0.20 (0.17) | 0.25 (0.16) |
| Median [Min-Max] | 0.19 [0.07–0.70] | 0.22 [0.00–1.01] | 0.15 [0.00–0.75]** | 0.21 [0.00–1.01] |
| Number ≤ 0.1 μg/mL | 4/24 (16.7%) | 15/206 (7.3%) | 12/37 (32.4%)*** | 30/263 (11.4%) |
Poor virological outcome (PVirO): viral shedding persistence at day 10; Poor clinical outcome (PClinO): either death or transfer to intensive care unit (ICU) or hospitalization for 10 days or more; Good outcome: individuals who belonged neither to the PClinO group nor the PVirO group. SD: standard deviation. aFive patients belonged to both the PVirO and PClinO outcome so the sum of frequencies may be above 1061. bIncluding 8 deaths. cData available for 928 patients (56 patients who did not declare any symptom before treatment start were excluded and 77 with missing data), dfor 714 patients, efor 992 patients and ffor 263 patients. On low-dose pulmonary CT-scanner, patients were classified as no involvement (lack of lung involvement (ground glass opacities, consolidation or crazy paving pattern); minimal involvement (subtle ground glass opacities); intermediate involvement (less than 50% of segment involvement in no more than 5 segments) and severe involvement (involvement of more than 5 segments). The denominator was mentioned when the result was not available for all patients. *p < 0.05; **p < 0.01; ***p < 0.001 (Fisher's exact test, Student t-test, Wilcoxon-Mann-Whitney where appropriate; reference group is good outcome).
Successful isolation of virus in cell culture was obtained from 204 patients among 915 tested (22.3%) on nasopharyngeal sample collected before treatment. A total of 973 patients (91.7%) had a good clinical outcome (GO). Among 263 patients tested at day 2, HCQ was low (<0.1 μg/mL) in 30 patients including 3 in which it was undetectable. The vast majority of patients did not report any adverse event that could be attributed to their treatment (97.6%). Twenty five patients reported mild adverse events, and three discontinued their treatment (Table 3).
Table 3. Adverse events.
| Patients without any adverse event | 1036 (97.6%) |
| Patients with adverse events possibly related to the treatmenta | 25 (2.4%) |
| Diarrhea | 12 |
| Abdominal pain | 3 |
| Headache | 3 |
| Nausea | 2 |
| Insomnia | 2 |
| Transient blurred vision | 2 |
| Vomiting | 1 |
| Urticaria | 1 |
| Erythematous and bullous rash | 1 |
| Discontinuation of treatment | 3 (abdominal pain, urticaria, erythematous and bullous rash) |
Some patients reported more than one adverse event.
Nine patients had a QTc prolongation of more than 60 ms from baseline but no patient exceeded 500 ms, which corresponds to the threshold contraindicating treatment. No rhythmic cardiac events or sudden deaths were observed.
3.2. Poor clinical outcome
Forty-six patients (4.3%) were classified into the PClinO group including 10 patients transferred into ICU of whom 2 died, 6 who died in conventional hospital units, and 30 additional patients who were hospitalized for 10 days or more (update April 18th). Their median age (69.0 years; 31–95 years) was significantly higher than that of patients included into the GO group (42.0 years; 14–86, p < .001) (Table 2). Sex ratio (M/F) was 1. When compared with patients in the GO group, PClinO group patients were significantly more likely to report previous hypertension (50%), diabetes (19.6%), coronary artery diseases (19.6%) and cancer (15.2%) (p < .001). In addition, they were more likely to receive beta-blocking agents, dihydropyridine derivatives, angiotensin II receptor blockers, and HMG-CoA reductase inhibitors (p < .001), diuretics (p < .001) and metformin (p < .01). The time between onset of symptoms and the beginning of the treatment was shorter and their NEWS score was less likely to be low than in the GO group patients (Table 2). They were less likely to present with normal CT-scan at admission (p < .001). Interestingly, the mean HCQ dosage at day 2 (0.20 μg/ml (0.17)) was significantly lower than in the GO group (p < .01, Table 2) with 12/37 tested cases with a dosage lower than 0.100 μg/mL (p < .001), and 3 without detectable HCQ. However, upon multivariate analysis, only older age (OR = 1.11: 1.07–1.15), selective beta blocking agents (OR = 4.16: 1.19–14.55), angiotensin II receptor blockers (OR = 18.40: 6.28–53.90) and medium and high NEWS scores (OR = 9.48: 3.25–27.66; OR = 10.05: 3.16–32.02, respectively) were significantly associated with the poor clinical outcome (Table 4). When adjusting for hypertension in multivariate analysis, the two variables angiotensin II receptor blockers and selective beta blockers remained statistically associated with PClinO and PVirO. With a model selecting only people with hypertension, these two molecules remained significantly associated to the PClinO but not the PVirO outcome (angiotensin II receptor blockers, point estimate 34.7, 95% CI 4.98–241.6 – selective beta blockers, 26.6, 95%CI 4.81–146.9). Low dose CT scan score revealed pneumonia in 35 PClinO group patients (90%). Three severe patients were treated by anti-IL1 (anakinra) and none of them died. No patients were treated with steroids. High dose preventive or curative anticoagulants were administered for severe patients.
Table 4. Multivariable logistic regressions of variables found statistically different in the univariate analysis.
| Poor virological outcome (versus good outcome) | Poor clinical outcome (versus good outcome) | |||
|---|---|---|---|---|
| OR [95% CI] | p | OR [95% CI] | p | |
| Age (years) | 1.02 [1.00; 1.04] | 0.042 | 1.11 [1.07; 1.15] | <0.0001 |
| Comedication(s) | ||||
| Selective beta blocking agents | 4.57 [1.54; 13.60] | 0.006 | 4.16 [1.19; 14.55] | 0.026 |
| Angiotensin II receptor blockers (ARBs), plain | 3.96 [1.34; 11.68] | 0.013 | 18.40 [6.28; 53.90] | <0.0001 |
| NEWS score | ||||
| 0 – 4 (low) | 1.0 (ref) | 1.0 (ref) | ||
| 5 – 6 (medium) | NS | 9.48 [3.25; 27.66] | 0.043 | |
| ≥ 7 (high) | 10.05 [3.16; 32.02] | 0.040 | ||
| Viral load at inclusion (Ct, nasopharyngeal sample)a | 0.86 [0.81; 0.92] | <0.0001 | NS |
NS: not statistically significant (p > 0.05) after stepwise selection.
a
Missing values (n = 69) were imputed based on the mean value (mean = 26.6, see Table 1).
Regarding specifically the 8 patients who died after having received HCQ+AZ ≥ 3 days, their median age was 79 years (74–95 years) (Table 5). Six patients (75%) reported hypertension and one active cancer. Severity at admission was observed with a NEWS score ranging from 5 to 11 (mean 7.75) and low dose CT scan performed on 4 patients revealed intermediate to severe pneumonia involvement. All deaths resulted from respiratory failure and not from sudden death. All had repeated ECG with none showing torsades de pointe. Finally, mean HCQ dosage at day 2 was 0.162 including one patient with a blood level lower than 0.10 μg/mL. As of 18th of April, 2020, 33 of 46 patients in the PClinO group are now cured. Accordingly, 1048 (98.7%) of patients who received the HCQ+AZ combination are cured so far.
Table 5. Clinical data of eight patients who died from COVID19 infection out of 1,061 treated with hydroxychloroquine or azithromycin for at least three days. Day 0 between March 3rd and March 31, 2020; Follow up regarding fatal issue: April 18th, 2020.
| Age, median (min-max) | 79 (74–95) |
| Chronic condition | |
| Hypertension, N (%) | 6 (75%) |
| Cancer, N (%) | 1 (12.5%) |
| NEWS score, mean (min-max) | 7.75 (5–11) |
| Time between symptoms and hospitalization, mean (min-max) | 5.6 days (2-14) |
| Time between hospitalization and death, mean (min-max) | 16 days (6-26) |
| Day 2-hydroxychloroquine blood level (μg/mL), mean (min-max) | 0.162 (0.071–0.338) |
3.3. Viral clearance
Forty-seven patients, including 5 who were also PClinO, exhibited a persistent nasal viral carriage at completion of treatment. Their sex ratio (M/F) and mean age were 0.68 and 47.9 ± 17.5 years old, respectively. Of the 21 PVirO patients for whom specimens were available after day 10, 20 had negative viral loads by day 15 post onset of treatment (95.2%). In addition, all eleven patients for whom daily culture was attempted were negative by day 10. When compared to GO group patients in this study, PVirO group patients were older, more likely to use selective beta blocking agents and angiotensin II receptor blockers. They also exhibited a significantly higher viral load (p < .01) at diagnosis, were less likely to have a low NEWS score, and they were treated earlier (Table 2). However, in multivariate analysis, time between onset of symptoms and first day of treatment start did not remain significantly associated with viral shedding persistence. The proportion of persistent viral shedders was higher in patients with poor clinical outcome (5/46 (10.8%)) than in patients without good clinical outcome (42/1015 (4.1%)), one-sided mid-p exact test, p = .03).
In order to determine whether virus carriage persistence was associated with another concurrent infection, we tested 8 PVirO individuals using the FTD 21 Plus kit (Fast Track Diagnostics, Luxembourg). Two of these eight patients, sampled on March 21st and 24th 2020, respectively, were positive for bocavirus. All other tested pathogens were negative. Then, in order to determine whether other patients sampled during the same timeframe might have been also infected, we selected among good outcome patients 112 individuals who had been sampled between March 21st and 24th, 2020. None of them were positive for bocavirus or any other viruses (p < .001, Fisher exact test). Whether this co-infection played a role in viral persistence is as-yet unknown. Comparative genomics between viral isolates from 3 non-treatment-responding patients (both PVirO and PClinO), one PClinO patient, one PVirO patient and 10 treatment-responding patients as well as 56 SARS-CoV-2 strains from various geographical origins did not identify any specific viral variant linked to resistance to treatment (Fig. 2).
Fig. 1. Flowchart showing patients included in the analysis.
HCQ, Hydroxychloroquine, AZ, azithromycin.
Fig. 2. Phylogenetic tree of SARS-COV-2 genomes including isolates from five persistent viral shedders and ten treatment-responding patients (green branches). *** =poor clinical outcome and ¶ = poor virological outcome. Phylogenetic reconstruction was performed using NEXSTRAIN (https://nextstrain.org/) and GISAID (Global Initiative; https://www.gisaid.org/) with acknowledgments [24]. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
4. Discussion
The efficacy of the combination of HCQ and AZ against COVID-19 has become a very controversial issue in the medical community, leading to many leaked social media reports and speeches from politicians. Evidence is needed to augment the knowledge of outcomes of patients with COVID-19 who are treated with this drug combination. In our analysis, which is not a RCT but which relates the real-life experience of physicians treating patients in the context of an emerging pandemic, we report the outcomes of 1061 COVID-19 patients treated with an HCQ+AZ combination from the time of diagnosis. The spectrum of severity of COVID-19 ranges from mild symptoms to severe respiratory distress [1]. We assessed patients who received at least three days of treatment and eight days of follow-up. The majority of patients in our work had relatively mild disease at admission (95%). Under these conditions, the treatment was associated with a low proportion of patients with worsening of the disease, as only 10 patients (0.9%) were transferred to the intensive care unit and a low proportion of death, as only eight (0.75%) patients died (case fatality rate updated April 18th, 2020). It was also associated with a low frequency of persistent viral shedding. In our experience, the treatment was well tolerated with only a low proportion of adverse events (2.4%), all of which were mild with three discontinuations of treatment (0.3%) [25].
Regarding viral shedding persistence, we observed that it was 4.4% at day 10 in treated patients, which is extremely low in comparison to Chinese studies, the largest of which showed that viruses are shed on average for 20 days with extremes of up to 38 days [1]. This may have important consequences in terms of contagiousness of the disease. We did not find any specificity in the genomes of viruses in patients with viral shedding persistence.
We were surprised to find in the PClinO group that HCQ blood levels were lower than therapeutic target in 32.4% cases including two patients without any drug in the blood. We cannot exclude that some of these patients were not adherent with the prescribed treatment since therapy intake was not controlled. We therefore recommend that close control of HCQ blood level be performed in treated patients so that drug dosage could be adapted accordingly.
As already described by others [1,26], we confirm that COVID-19 patients with PClinO are significantly more likely to be elderly patients. Moreover, when COVID-19 patients were treated belatedly and already showing clinical or radiological signs of pneumonia, the prognosis was poorer but genomes of viruses associated with PClinO were not apparently different from those in other patients (Fig. 2). Multivariate analysis showed that selective beta-blocking agents and angiotensin II receptor blockers were independent factors associated with poor clinical and virological outcomes (p < .05).
Our study has some limitations. Because services were overwhelmed, data was incomplete on some patients. CT-scans and serum drug levels were not available for all patients, notably in those admitted out of hours.
As a conclusion, based on our experience, we consider reasonable to follow the recommendations made in Asian countries for the control of COVID-19, notably in Korea and China that consist in early testing as many patients as possible and treating them with available drugs where this strategy has produced much better results than in countries where no active policy has been implemented outside containment. In China, drugs that were recommended were primarily HCQ but also α-interferon, lopinavir, ritonavir and umifenovir [27], in Korea, recommended drugs were lopinavir/ritonavir and chloroquine [28]. In the context of a pandemic with a lethal respiratory virus, we believe that early detection of positive cases and carefully controlled treatment with safe and well-tolerated drugs should be generalized in outpatient medicine, i.e. in individuals with mild symptoms before signs of severity appear. Strict attention should be paid to contraindications and possible interactions with concomitant medication. Finally, there is a need to repurpose existing drugs and evaluate these in controlled trials where possible in the constraints of a pandemic.
Author's Note: Since this analysis was completed, and as of the 29th of April, 2020, two more patients in the PClinO group died resulting in an overall 0.9% case fatality rate (CFR) for these 1061 patients.
Funding
This work was funded by ANR-15-CE36-0004-01 and by ANR “Investissements d'avenir”, Méditerranée infection 10-IAHU-03, and was also supported by Région Provence-Alpes-Côte d'Azur. This work had received financial support from the Mediterranean Infection Foundation.
CRediT authorship contribution statement
Matthieu Million: Conceptualization, Formal analysis, Writing - original draft. Jean-Christophe Lagier: Conceptualization, Formal analysis, Writing - original draft. Philippe Gautret: Formal analysis, Writing - original draft. Philippe Colson: Conceptualization, Formal analysis, Writing - original draft. Pierre-Edouard Fournier: Conceptualization, Formal analysis, Writing - original draft. Morgane Mailhe: Conceptualization, Formal analysis, Writing - original draft. Vera Esteves-Vieira: Conceptualization. Florian Correard: Conceptualization. Audrey Giraud-Gatineau: Conceptualization. Yanis Roussel: Conceptualization. Stéphane Honoré: Conceptualization, Formal analysis. Alexis Jacquier: Conceptualization. Jean-Claude Deharo: Conceptualization. Eric Chabrière: Conceptualization. Anthony Levasseur: Conceptualization. Florence Fenollar: Conceptualization. Jean-Marc Rolain: Conceptualization, Formal analysis. Yolande Obadia: Conceptualization, Formal analysis. Philippe Brouqui: Conceptualization, Formal analysis. Michel Drancourt: Conceptualization, Formal analysis. Bernard La Scola: Conceptualization, Formal analysis. Philippe Parola: Conceptualization, Formal analysis, Writing - original draft. Didier Raoult: Conceptualization, Formal analysis, Writing - original draft.
Declaration of competing interest
The authors declare no competing interests. Funding sources had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation, review, or approval of the manuscript. Our group used widely available generic drugs distributed by many pharmaceutical companies (Supplementary data).
Acknowledgments
We are thankful to Marion Bechet, Pascal Chanez, Véronique Filosa, Marc Gainier, Marion Gouitaa, Marie-Thérèse Jimeno, Cléa Melenotte, Matthieu Bardou, Marc Léone, Jean-Robert Harlé, Veronique Veit, all medical students from Aix Marseille University, all nurses, laboratory staff, administrative, technical and security staff of Assistance Publique-Hôpitaux de Marseille and IHU Méditerranée Infection, all medical doctors volunteers, and the Bataillon des Marins Pompiers de Marseille for their help. We thank the 6 reviewers who helped to substantially improving and clarifying the manuscript with their many comments and suggestions.
Appendix A. Supplementary data
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References
[1]Equal contributors.
