Early Treatment with Hydroxychloroquine and Azithromycin: A Real-Life Monocentric Retrospective Cohort Study of 30,423 COVID-19 Patients (preprint)

Objective To estimate the comparative effectiveness of combination therapy with hydroxychloroquine (HCQ) and azithromycin for coronavirus disease 2019 (COVID-19)-related death based on a large monocentric cohort independent of investigators putative biases in a real-world setting. Design Retrospective monocentric cohort study, with comprehensive data collection authenticated by an external bailiff and death reports from a national database (French National Death Registry). Setting Institut Hospitalo-Universitaire Mediterranee Infection Center in Marseille, France. Participants All adults older than 18 years with PCR-proven COVID-19 who were treated directly in our centre between 2 March 2020 and 31 December 2021 and did not refuse the use of their data. Interventions HCQ and azithromycin (HCQ-AZ) as a reference treatment were compared to other regimens containing HCQ, ivermectin and azithromycin alone, combined, or none of these three drugs. The effect of vaccination was also evaluated. Main outcome measures 6-week all-cause mortality. Multivariable logistic regression estimated treatment effectiveness with adjustments for age, sex, comorbidities, vaccination, period of infection or virus variant, and outpatient or inpatient care. Results Total 30,423 COVID-19 patients were analysed (86 refused the analysis of their data) including 30,202 with available treatment data, and 535 died (1.77%). All-cause mortality was very low among patients < 50 years (8/15,925 (0.05%)) and among outpatients treated with HCQ-AZ (21 deaths out of 21,135 (0.1%), never exceeding 0.2% regardless of epidemic period). HCQ-AZ treatment was associated with a significantly lower mortality rate than no HCQ-AZ after adjustment for sex, age, period and patient care setting (adjusted OR (aOR) 95% confidence interval (CI) 0.55, 0.45-0.68). The effect was greater among outpatients (71% death protection rate) than among inpatients (45%). In a subset of 16,063 patients with available comorbidities and vaccinations status, obesity (2.01, 1.23-3.29), chronic respiratory disease (2.93, 1.29-6.64), and immunodeficiency (4.01, 1.69-9.50), on the one hand, and vaccination (0.29, 0.12-0.67) and HCQ-AZ treatment (0.47, 0.29-0.76), on the other hand, were independent factors associated with mortality. HCQ, alone or in any association, was associated with significant protection from death among outpatients (0.41, 0.21-0.79) and inpatients (0.59, 0.47-0.73). Conclusions HCQ prescribed early or late protects in part from COVID-19-related death. During pandemic health crises, financial stakes are enormous. Authentication of the data by an independent external judicial officer should be required. Public sharing of anonymized databases, ensuring their verifiability, should be mandatory in this context to avoid fake publications.

Cardiovascular Safety of Hydroxychloroquine-Azithromycin in 424 COVID-19 Patients (preprint)

Background Hydroxychloroquine (HCQ) combined with azithromycin (AZM) has been widely administered to patients with COVID-19 despite scientific controversies. In particular the potential to prolong cardiac repolarisation by using this combination has been discussed. Materials and methods We report a pragmatic and simple safety approach which we implemented in the first patients treated for COVID-19 in our center early 2020. Treatment contraindications were the presence of severe structural or electrical heart disease, baseline corrected QT interval (QTc) >500 ms, hypokalaemia, or other drugs prolonging QTc that could not be interrupted. Electrocardiogram and QTc was evaluated at admission and re-evaluated after 48 hours of the initial prescription. Results Among 424 consecutive adults (mean age 46.3 ± 16.1 years; 216 women). Patients were followed in conventional wards (21.5%) or in a day-care unit (78.5%). A total of 11 patients (2.6%) had contraindications to HCQ-AZ combination. In the remaining 413 treated patients, there were no arrhythmic events in any patient during the 10-day treatment regimen. QTc was slightly but statistically significantly prolonged by 3.75 ± 25.4 ms after two days (p=0.003). Ten patients (2.4%) developed QTc prolongation >60 ms, and none had QTc >500 ms. Conclusions This report do not aim to contribute to knowledge of the efficacy of treating COVID-19 with HCQ-AZ. However, a simple initial assessment of patient medical history, ECG and kalaemia identifies contraindicated patients and enables the safe treatment by HCQ-AZ of COVID-19 patients. QT-prolonging anti-infective drugs can be used safely in acute life-threatening infections, provided that a strict protocol and close collaboration between infectious disease specialists and rhythmologists are followed.

SARS-CoV-2 quasi-species analysis from patients with persistent nasopharyngeal shedding: potential tool to finest genomic watch (preprint)

At the time of a new and unprecedented viral pandemic, many questions are being asked about the genomic evolution of SARS-CoV-2 and the emergence of different variants, leading to therapeutic and immune evasion and survival of this genetically highly labile RNA virus. The nasopharyngeal persistence of infectious virus beyond 17 days proves its constant interaction with the human immune system and increases the intra-individual mutational possibilities. We performed a prospective high-throughput sequencing study (ARTIC Nanopore) of SARS-CoV-2 from so-called "persistent" patients, comparing them with a non-persistent population, and analyzing the quasi-species present in a single sample at time t. Global intra-individual variability in persistent patients was found to be higher than in controls (mean 5.3%, Standard deviation 0.9 versus 4.6% SD 0.3, respectively, p< 0,001). In the detailed analysis, we found a greater difference between persistent and non-persistent patients with non-severe COVID 19, and between the two groups infected with clade 20A. Furthermore, we found minority N501Y and P681H mutation clouds in all patients, with no significant differences found between both groups. The question of the SARS-CoV-2 viral variants’ genesis remains to be further investigated, with the need to prevent new viral propagations and their consequences, and quasi-species analysis could be an important key to watch out.

A 21L/BA.2-21K/BA.1 MixOmicron SARS-CoV-2 hybrid undetected by qPCR that screen for variant in routine diagnosis (preprint)

Among the multiple SARS-CoV-2 variants identified since summer 2020, several have co-circulated, creating opportunities for coinfections and potentially genetic recombinations that are common in coronaviruses. Viral recombinants are indeed beginning to be reported more frequently. Here, we describe a new SARS-CoV-2 recombinant genome that is mostly that of a Omicron 21L/BA.2 variant but with a 3-prime tip originating from a Omicron 21K/BA.1 variant. Two such genomes were obtained in our institute from adults sampled in February 2022 in university hospitals of Marseille, southern France, by next-generation sequencing carried out with the Illumina or Nanopore technologies. The recombination site was located between nucleotides 26,858-27,382. In the two genomic assemblies, mean sequencing depth at mutation-harboring positions was 271 and 1,362 reads and mean prevalence of the majoritary nucleotide was 99.3+/-2.2% and 98.8+/-1.6%, respectively. Phylogeny generated trees with slightly different topologies according to whether genomes were depleted or not of the 3-prime tip. This 3-prime terminal end brought in the Omicron 21L/BA.2 genome a short transposable element of 41 nucleotides named S2m that is present in most SARS-CoV-2 except a few variants among which the Omicron 21L/BA.2 variant and may be involved in virulence. Importantly, this recombinant is not detected by currently used qPCR that screen for variants in routine diagnosis. The present observation emphasizes the need to survey closely the genetic pathways of SARS-CoV-2 variability by whole genome sequencing, and it could contribute to gain a better understanding of factors that lead to observed differences between epidemic potentials of the different variants.

Sequential appearance and isolation of a SARS-CoV-2 recombinant between two major SARS-CoV-2 variants in a chronically infected immunocompromised patient (preprint)

Genetic recombination is a major evolutionary mechanism among RNA viruses, and it is common in coronaviruses, including those infecting humans. A few SARS-CoV-2 recombinants have been reported to date whose genome harbored combinations of mutations from different mutants or variants, but a single patient sample was analyzed, and the virus was not isolated. Here, we report the gradual creation of a hybrid genome of B.1.160 and Alpha variants in a lymphoma patient chronically infected for 14 months, and we isolated the recombinant virus. The hybrid genome was obtained by next-generation sequencing, and recombination sites were confirmed by PCR. This consisted of a parental B.1.160 backbone interspersed with two fragments, including the spike gene, from an Alpha variant. Analysis of seven sequential samples from the patient decoded the recombination steps, including the initial infection with a B.1.160 variant, then a concurrent infection with this variant and an Alpha variant, the generation of hybrid genomes, and eventually the emergence of a predominant recombinant virus isolated at the end of the patient follow-up. This case exemplifies the recombination process of SARS-CoV-2 in real life, and it calls for intensifying genomic surveillance in patients coinfected with different SARS-CoV-2 variants, and more generally with several RNA viruses, as this may lead to the creation of new viruses.

The risk of COVID-19 death is much greater and age-dependent with type I IFN autoantibodies (preprint)

SARS-CoV-2 infection fatality rate (IFR) doubles with every five years of age from childhood onward. Circulating autoantibodies neutralizing IFN-α, IFN-ω, and/or IFN-β are found in ~20% of deceased patients across age groups. In the general population, they are found in ~1% of individuals aged 20-70 years and in >4% of those >70 years old. With a sample of 1,261 deceased patients and 34,159 uninfected individuals, we estimated both IFR and relative risk of death (RRD) across age groups for individuals carrying autoantibodies neutralizing type I IFNs, relative to non-carriers. For autoantibodies neutralizing IFN-α2 or IFN-ω, the RRD was 17.0[95% CI:11.7-24.7] for individuals under 70 years old and 5.8[4.5-7.4] for individuals aged 70 and over, whereas, for autoantibodies neutralizing both molecules, the RRD was 188.3[44.8-774.4] and 7.2[5.0-10.3], respectively. IFRs increased with age, from 0.17%[0.12-0.31] for individuals <40 years old to 26.7%[20.3-35.2] for those ≥80 years old for autoantibodies neutralizing IFN-α2 or IFN-ω, and from 0.84%[0.31-8.28] to 40.5%[27.82-61.20] for the same two age groups, for autoantibodies neutralizing both molecules. Autoantibodies against type I IFNs increase IFRs, and are associated with high RRDs, particularly those neutralizing both IFN-α2 and -ω. Remarkably, IFR increases with age, whereas RRD decreases with age. Autoimmunity to type I IFNs appears to be second only to age among common predictors of COVID-19 death.

The emergence, spread and vanishing of a French SARS-CoV-2 variant exemplifies the fate of RNA virus epidemics and obeys the Black Queen rule (preprint)

Summary The nature and dynamics of mutations associated with the emergence, spread and vanishing of SARS-CoV-2 variants causing successive waves are complex 1-5 . We determined the kinetics of the most common French variant (“Marseille-4”) for 10 months since its onset in July 2020 5 . Here, we analysed and classified into subvariants and lineages 7,453 genomes obtained by next-generation sequencing. We identified two subvariants, Marseille-4A, which contains 22 different lineages of at least 50 genomes, and Marseille-4B. Their average lifetime was 4.1±1.4 months, during which 4.1±2.6 mutations accumulated. Growth rate was 0.079±0.045, varying from 0.010 to 0.173. All the lineages exhibited a “gamma” distribution. Several beneficial mutations at unpredicted sites initiated a new outbreak, while the accumulation of other mutations resulted in more viral heterogenicity, increased diversity and vanishing of the lineages. Marseille-4B emerged when the other Marseille-4 lineages vanished. Its ORF8 gene was knocked out by a stop codon, as reported in several mink lineages and in the alpha variant. This subvariant was associated with increased hospitalization and death rates, suggesting that ORF8 is a nonvirulence gene. We speculate that the observed heterogenicity of a lineage may predict the end of the outbreak.

Analysis of SARS-CoV-2 variants from 24,181 patients exemplifies the role of globalisation and zoonosis in pandemics (preprint)

After the end of the first epidemic episode of SARS-CoV-2 infections, as cases began to rise again during the summer of 2020, we at IHU Mediterranee Infection in Marseille, France, intensified the genomic surveillance of SARS-CoV-2, and described the first viral variants. In this study, we compared the incidence curves of SARS-CoV-2-associated deaths in different countries and reported the classification of SARS-CoV-2 variants detected in our institute, as well as the kinetics and sources of the infections. We used mortality collected from a COVID-19 data repository for 221 countries. Viral variants were defined based on [≥]5 hallmark mutations shared by [≥]30 genomes. SARS-CoV-2 genotype was determined for 24,181 patients using next-generation genome and gene sequencing (in 47% and 11% of cases, respectively) or variant-specific qPCR (in 42% of cases). Sixteen variants were identified by analysing viral genomes from 9,788 SARS-CoV-2-diagnosed patients. Our data show that since the first SARS-CoV-2 epidemic episode in Marseille, importation through travel from abroad was documented for seven of the new variants. In addition, for the B.1.160 variant of Pangolin classification (a.k.a. Marseille-4), we suspect transmission from mink farms. In conclusion, we observed that the successive epidemic peaks of SARS-CoV-2 infections are not linked to rebounds of viral genotypes that are already present but to newly-introduced variants. We thus suggest that border control is the best mean of combating this type of introduction, and that intensive control of mink farms is also necessary to prevent the emergence of new variants generated in this animal reservoir.

The Need for Early Management in Patients With COVID-19 (preprint)

In March 2020, the IHU Méditerranée Infection set up a screening and treatment center for patients with COVID-19, a system that has been ultimately recommended by French public health authorities. The recent publication of the profiles of patients hospitalized in France published by the Directorate for Research, Studies, Evaluation and Statistics gives us the opportunity to measure the impact of this multidisciplinary early management system coupled with screening on mortality at 90 days. Analysis of the data shows that the system established at IHU-MI was associated with lower mortality, taking age and sex into account. Regarding the age-standardized mortality rate, mortality rates were lower than national data regardless of the period of the epidemic. Early management seems to have significantly decreased the mortality rate in the under-60 age group, suggesting the importance of early management, regardless of age. In addition, these patients had pejorative clinical criteria (high NEWS-2 score, ICU visits, oxygen saturation below 95%) requiring hospitalization, and co-morbidities that are now known to be aggravating factors [7]. This reinforces the need to care for all individuals, regardless of age. Early medical care, as part of a system integrating a screening center and a day hospital, may explain the lower mortality rates.

Expression of ACE2 receptor, soluble ACE2, Angiotensin I, Angiotensin II and Angiotensin (1-7), is modulated in COVID-19 patients (preprint)

Although SARS-CoV-2 is primarily a pulmonary-tropic virus, it is nonetheless responsible for multi-organ failure in patients with severe forms of COVID-19, particularly those with hypertension or cardiovascular disease. Infection requires virus binding to the angiotensin I converting enzyme 2 (ACE2) monocarboxypeptidase, a regulator of blood pressure homeostasis through its ability to catalyze the proteolysis of Angiotensin II (AngII) into Ang(1-7). Although assumed, it had not been proven so far whether the SARS-CoV-2 replication in COVID-19 patients could modulate the expression of the ACE2 receptor and/or the AngII plasma levels. We demonstrate here, that in COVID-19 patients the ACE2 mRNA expression is markedly reduced in circulating blood cells. This ACE2 gene dysregulation mainly affects the monocytes which also show a lower expression of membrane ACE2 protein. Moreover, a significant decrease in soluble ACE2 plasma levels is observed in COVID-19 patients, whereas the concentration of sACE2 returns to normal levels in patients recovered from COVID-19. In the plasma of COVID-19 patients, we also found an increase in AngI and AngII. On the other hand, the plasma levels of Ang(1-7) remains almost stable in COVID-19 patients. Despite the Ang(1-7) presence in the plasma of COVID-19 patients it seems insufficient to prevent the effects of massive AngII accumulation. These are the first direct evidence that the SARS-CoV-2 may affect the expression of blood pressure regulators with possible harmful consequences on COVID-19 outcome.

Introduction into the Marseille geographical area of a mild SARS-CoV-2 variant originating from sub-Saharan Africa (preprint)

BACKGROUNDIn Marseille, France, the COVID-19 incidence evolved unusually with several successive epidemic episodes. The second outbreak started in July, was associated with North Africa, and involved travelers and an outbreak on passenger ships. This suggested the involvement of a new viral variant. METHODSWe sequenced the genomes from 916 SARS-CoV-2 strains from COVID-19 patients in our institute. The patients demographic and clinical features were compared according to the infecting viral variant. RESULTSFrom June 26th to August 14th, we identified a new viral variant (Marseille-1). Based on genome sequences (n=89) or specific qPCR (n=53), 142 patients infected with this variant were detected. It is characterized by a combination of 10 mutations located in the nsp2, nsp3, nsp12, S, ORF3a, ORF8 and N/ORF14 genes. We identified Senegal and Gambia, where the virus had been transferred from China and Europe in February-April as the sources of the Marseille-1 variant, which then most likely reached Marseille through Maghreb when French borders reopened. In France, this variant apparently remained almost limited to Marseille. In addition, it was significantly associated with a milder disease compared to clade 20A ancestor strains. CONCLUSIONOur results demonstrate that SARS-CoV-2 can genetically diversify rapidly, its variants can diffuse internationally and cause successive outbreaks.

Genomic diversity and evolution of coronavirus (SARS-CoV-2) in France from 309 COVID-19-infected patients (preprint)

The novel coronavirus (SARS-CoV-2) causes pandemic of viral pneumonia. The evolution and mutational events of the SARS-CoV-2 genomes are critical for controlling virulence, transmissibility, infectivity, severity of symptoms and mortality associated to this infectious disease. We collected and investigated 309 SARS-CoV-2 genomes from patients infected in France. Detailed genome cartography of all mutational events (SNPs, indels) was reported and correlated to clinical features of patients. A comparative analysis between our 309 SARS-CoV-2 genomes from French patients and the reference Wuhan coronavirus genome revealed 315 substitution mutations and six deletion events: ten were in 5/3 UTR, 178 were nonsynonymous, 126 were synonymous and one generated a stop codon. Six different deleted areas were also identified in nine viral variants. In particular, 30 substitution mutations (18 nonsynonymous) and one deletion (21765-21770) concerned the spike S glycoprotein. An average of 7.8 mutational events (+/- 1.7 SD) and a median of 8 (range, 7-9) were reported per viral isolate. Comparative analyses and clustering of specific mutational signatures in 309 genomes disclose several divisions in groups and subgroups combining their geographical and phylogenetic origin. Clinical outcomes of the 309 COVID-19-infected patients were investigated according to the mutational signatures of viral variants. These findings highlight the genome dynamics of the coronavirus 2019-20 and shed light on the mutational landscape and evolution of this virus. Inclusion of the French cohort enabled us to identify 161 novel mutations never reported in SARS-CoV-2 genomes collected worldwide. These results support a global and continuing surveillance of the emerging variants of the coronavirus SARS-CoV-2.

Evaluating the serological status of COVID-19 patients using an indirect immunofluorescent assay, France. (preprint)

An indirect immunofluorescent assay was developed in order to assess the serological status of 888 RT-PCR-confirmed COVID-19 patients (1,302 serum samples) and controls in Marseille, France. Incorporating an inactivated clinical SARS CoV-2 isolate as the antigen, the specificity of the assay was measured as 100% for IgA titre [≥] 1:200; 98.6% for IgM titre [≥] 1:200; and 96.3% for IgG titre [≥] 1:100 after testing a series of negative controls as well as 150 serums collected from patients with non-SARS-CoV-2 Coronavirus infection, non-Coronavirus pneumonia and infections known to elicit false-positive serology. Seroprevalence was then measured at 3% before a five-day evolution up to 47% after more than 15 days of evolution. We observed that the seroprevalence as well as the titre of specific antibodies were both significantly higher in patients with a poor clinical outcome than in patients with a favourable evolution. These data, which have to be integrated into the ongoing understanding of the immunological phase of the infection, suggest that serotherapy may not be a therapeutic option in patients with severe COVID-19 infection. The IFA assay reported here is useful for monitoring SARS-CoV-2 exposure at the individual and population levels.

Hydroxychloroquine and Azithromycin as a treatment of COVID-19: preliminary results of an open-label non-randomized clinical trial (preprint)

Background Chloroquine and Hydroxychloroquine have been found to be efficient on COV-19, and reported to be efficient in Chinese patients infected by this virus. We evaluate the role of Hydroxychloroquine on respiratory viral loads. Patients and methods Patients were included in a single arm protocol to receive 600mg of hydroxychloroquine daily and their viral load in nasal swabs was tested daily. Depending on their clinical presentation azithromycin was added to the treatment. Untreated patients from another center and cases refusing the protocol were included as negative control. Presence and absence of virus at Day-6 was considered the end point. Results Twenty cases were treated in this study and showed a significant reduction of the viral carriage at D-6 compared to controls, and much lower than reported average carrying duration of untreated patients in the literature. Azithromycin added to Hydroxychloroquine was significantly more efficient for virus elimination. Conclusion : Hydroxychloroquine is significantly associated with viral load reduction/disappearance in patients with COVID-19 and its effect is reinforced by Azithromycin.