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.

Evidence of antibodies against SARS-CoV-2 in wild mustelids from Brittany (France) (preprint)

In the French region of Brittany, mainly in the department of the Cotes d'Armor, during the first semester of 2021, seropositivity for SARS-CoV-2 was detected in five wild mustelids out of 32 animals tested. Anti-SARS-CoV-2 IgG against at least four out of five recombinant viral proteins (S1 receptor binding domain, nucleocapsid, S1 subunit, S2 subunit and spike) were detected using automated western blot technique in three martens (Martes martes) and two badgers (Meles meles). An ELISA test also objectified seropositivities. Although the 171 qPCRs carried out on samples from the 33 mustelids were all negative, these preliminary results (observational study) nevertheless bear witness to infections of unknown origin. The epidemiological surveillance of Covid-19 in wildlife must continue, in particular with the tools of efficient serology.

Emergence in Southern France of a new SARS-CoV-2 variant of probably Cameroonian origin harbouring both substitutions N501Y and E484K in the spike protein (preprint)

SARS-CoV-2 variants have become a major virological, epidemiological and clinical concern, particularly with regard to the risk of escape from vaccine-induced immunity. Here we describe the emergence of a new variant. For twelve SARS-CoV-positive patients living in the same geographical area of southeastern France, qPCR testing that screen for variant-associated mutations showed an atypical combination. The index case returned from a travel in Cameroon. The genomes were obtained by next-generation sequencing with Oxford Nanopore Technologies on GridION instruments within approximately 8 h. Their analysis revealed 46 mutations and 37 deletions resulting in 30 amino acid substitutions and 12 deletions. Fourteen amino acid substitutions, including N501Y and E484K, and 9 deletions are located in the spike protein. This genotype pattern led to create a new Pangolin lineage named B.1.640.2, which is a phylogenetic sister group to the old B.1.640 lineage renamed B.1.640.1. Both lineages differ by 25 nucleotide substitutions and 33 deletions. The mutation set and phylogenetic position of the genomes obtained here indicate based on our previous definition a new variant we named 'IHU'. These data are another example of the unpredictability of the emergence of SARS-CoV-2 variants, and of their introduction in a given geographical area from abroad.

The polarity and specificity of SARS-CoV2 -specific T lymphocyte responses determine disease susceptibility (preprint)

Optimal vaccination and immunotherapy against coronavirus disease COVID-19 relies on the in-depth comprehension of immune responses determining the individual susceptibility to be infected by SARS-CoV-2 and to develop severe disease. We characterized the polarity and specificity of circulating SARS-CoV-2-specific T cell responses against whole virus lysates or 186 unique peptides derived from the SARS-CoV-2 or SARS-CoV-1 ORFeome on 296 cancer-bearing and 86 cancer-free individuals who were either from the pre-COVID-19 era (67 individuals) or contemporary COVID-19-free (237 individuals) or who developed COVID-19 (78 individuals) in 2020/21. The ratio between the prototypic T helper 1 (TH1) cytokine, interleukin-2, and the prototypic T helper 2 (TH2) cytokine, interleukin-5 (IL-5), released from SARS-CoV-2-specific memory T cells measured in early 2020, among SARS-CoV-2-negative persons, was associated with the susceptibility of these individuals to develop PCR-detectable SARS-CoV-2 infection in late 2020 or 2021. Of note, T cells from individuals who recovered after SARS-CoV-2 re-infection spontaneously produced elevated levels of IL-5 and secreted the immunosuppressive TH2 cytokine interleukin-10 in response to SARS-CoV-2 lysate, suggesting that TH2 responses to SARS-CoV-2 are inadequate. Moreover, individuals susceptible to SARS-CoV-2 infection exhibited a deficit in the TH1 peptide repertoire affecting the highly mutated receptor binding domain (RBD) amino acids (331-525) of the spike protein. Finally, current vaccines successfully triggered anti-RBD specific TH1 responses in 88% healthy subjects that were negative prior to immunization. These findings indicate that COVID-19 protection relies on TH1 cell immunity against SARS-CoV-2 S1-RBD which in turn likely drives the phylogenetic escape of the virus. The next generation of COVID-19 vaccines should elicit high-avidity TH1 (rather than TH2)-like T cell responses against the RBD domain of current and emerging viral variants.

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.