First cases of infection with the 21L/BA.2 Omicron variant in Marseille, France.

The SARS-CoV-2 21K/BA.1, 21L/BA.2, and BA.3 Omicron variants have recently emerged worldwide. To date, the 21L/BA.2 Omicron variant has remained very minority globally but became predominant in Denmark instead of the 21K/BA.1 variant. Here, we describe the first cases diagnosed with this variant in south-eastern France. We identified 13 cases using variant-specific qPCR and next-generation sequencing between 28/11/2021 and 31/01/2022, the first two cases being diagnosed in travelers returning from Tanzania. Overall, viral genomes displayed a mean (±standard deviation) number of 65.9 ± 2.5 (range, 61-69) nucleotide substitutions and 31.0 ± 8.3 (27-50) nucleotide deletions, resulting in 49.6 ± 2.2 (45-52) amino acid substitutions (including 28 in the spike protein) and 12.4 ± 1.1 (12-15) amino acid deletions. Phylogeny showed the distribution in three different clusters of these genomes, which were most closely related to genomes from England and South Africa, from Singapore and Nepal, or from France and Denmark. Structural predictions highlighted a significant enlargement and flattening of the surface of the 21L/BA.2 N-terminal domain of the spike protein compared to that of the 21K/BA.1 Omicron variant, which may facilitate initial viral interactions with lipid rafts. Close surveillance is needed at global, country, and center scales to monitor the incidence and clinical outcome of the 21L/BA.2 Omicron variant.

Reinfections with Different SARS-CoV-2 Omicron Subvariants, France.

We describe 188 patients in France who were successively infected with different SARS-CoV-2 Omicron subvariants, including BA.1, BA.2, and BA.5. Time between 2 infections was <90 days for 50 (26.6%) patients and <60 days for 28 (14.9%) patients. This finding suggests that definitions for SARS-CoV-2 reinfection require revision.

Analysis of SARS-CoV-2 Variants From 24,181 Patients Exemplifies the Role of Globalization and Zoonosis in Pandemics.

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 Méditerranée 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 along the whole genome 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 analyzing 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 farm minks. 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.

Culture and identification of a "Deltamicron" SARS-CoV-2 in a three cases cluster in southern France.

Multiple SARS-CoV-2 variants have successively, or concomitantly spread worldwide since the summer of 2020. A few co-infections with different variants were reported and genetic recombinations, common among coronaviruses, were reported or suspected based on co-detection of signature mutations of different variants in a given genome. Here we report three infections in southern France with a Delta 21J_AY.4-Omicron 21K/BA.1 "Deltamicron" recombinant. The hybrid genome harbors signature mutations of the two lineages, supported by a mean sequencing depth of 1163-1421 reads and a mean nucleotide diversity of 0.1%-0.6%. It is composed of the near full-length spike gene (from codons 156-179) of an Omicron 21K/BA.1 variant in a Delta 21J/AY.4 lineage backbone. Importantly, we cultured an isolate of this recombinant and sequenced its genome. It was observed by scanning electron microscopy. As it is misidentified with current variant screening quantitative polymerase chain reaction (qPCR), we designed and implemented for routine diagnosis a specific duplex qPCR. Finally, structural analysis of the recombinant spike suggested its hybrid content could optimize viral binding to the host cell membrane. These findings prompt further studies of the virological, epidemiological, and clinical features of this recombinant.

Characteristics of the first 1119 SARS-CoV-2 Omicron variant cases, in Marseille, France, November-December 2021.

One thousand one hundred and nineteen cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant cases have been diagnosed at the Institut Hospitalo-Universitaire Méditerranée Infection, Marseille, France, between November 28, 2021, and December 31, 2021. Among the 825 patients with known vaccination status, 383 (46.4%) were vaccinated, of whom 91.9% had received at least two doses of the vaccine. Interestingly, 26.3% of cases developed SARS-CoV-2 infection within 21 days following the last dose of vaccine suggesting possible early production of anti-SARS-CoV-2 facilitating antibodies. Twenty-one patients have been hospitalized, one patient required intensive care, and another patient who received a vaccine booster dose died.

SARS-CoV-2 reinfection and COVID-19 severity.

SARS-CoV-2 reinfection rate is low. The relative severity of the first and second episodes of infection remains poorly studied. In this study, we aimed at assessing the frequency of SARS-CoV-2 reinfections and comparing the severity of the first and second episodes of infection. We retrospectively included patients with SARS-CoV-2 positive RT-PCR at least 90 days after clinical recovery from a COVID-19 episode and with at least one negative RT-PCR after the first infection. Whole genome sequencing and variant-specific RT-PCR were performed and clinical symptoms and severity of infection were retrospectively documented from medical files. A total of 209 COVID-19 reinfected patients were identified, accounting for 0.4% of positive cases diagnosed from 19 March 2020 to 24 August 2021. Serology was performed in 64 patients, of whom 39 (60.1%) had antibodies against SARS-CoV-2 when sampled at the early stage of their second infection. Only seven patients (3.4%) were infected twice with the same variant. We observed no differences in clinical presentation, hospitalization rate, and transfer to ICU when comparing the two episodes of infections. Our results suggest that the severity of the second episode of COVID-19 is in the same range as that of the first infection, including patients with antibodies.

Introduction of the SARS-CoV-2 Beta variant from Comoros into the Marseille geographical area.

BACKGROUND: We describe the epidemiology of the first cases diagnosed in our institute of infections with the SARS-CoV-2 Beta variant and how this variant was imported to Marseille. METHODS: The Beta variant was identified based on analyses of sequences of viral genomes or of a spike gene fragment obtained by next-generation sequencing using Illumina technology, or by a real-time reverse-transcription-PCR (qPCR) specific of the Beta variant. RESULTS: The first patient diagnosed as infected with the SARS-CoV-2 Beta variant was sampled on January 15, 2021. Twenty-nine patients were diagnosed in January 2021 (two weeks). Fifteen (52%) patients were of Comorian nationality. Eight (28%) had travelled abroad, including six who had returned from Comoros. Phylogeny based on SARS-CoV-2 genomes from 11 of these patients and their best BLAST hits from the GISAID database showed that seven patients, including the four returning from Comoros, were clustered with 27 other genomes from GISAID that included the six first Beta variant genomes described in Comoros in January 2021. CONCLUSIONS: Our analyses highlight that, as for the case of other SARS-CoV-2 variants that have been diagnosed in Marseille, the Beta variant was imported to Marseille through travel from abroad. It had limited spread in our geographical area.

SARS-CoV-2 Vaccination and Protection Against Clinical Disease: A Retrospective Study, Bouches-du-Rhône District, Southern France, 2021.

From January 18th to August 13th, 2021, 13,804 unvaccinated and 1,156 patients who had received at least one COVID-19 vaccine dose were tested qPCR-positive for SARS-CoV-2 in our center. Among vaccinated patients, 949, 205 and 2 had received a single, two or three vaccine doses, respectively. Most patients (80.3%) had received the Pfizer-BioNTech vaccine. The SARS-CoV-2 variants infecting vaccinated patients varied over time, reflecting those circulating in the Marseille area, with a predominance of the Marseille-4/20A.EU2 variant from weeks 3 to 6, of the Alpha/20I variant from weeks 7 to 25, and of the Delta/21A variant from week 26. SARS-CoV-2 infection was significantly more likely to occur in the first 13 days post-vaccine injection in those who received a single dose (48.9%) than two doses (27.4%, p< 10-3). Among 161 patients considered as fully vaccinated, i.e., >14 days after the completion of the vaccinal scheme (one dose for Johnson and Johnson and two doses for Pfizer/BioNTech, Moderna and Sputnik vaccines), 10 (6.2%) required hospitalization and four (2.5%) died. Risks of complications increased with age in a nonlinear pattern, with a first breakpoint at 54, 33, and 53 years for death, transfer to ICU, and hospitalization, respectively. Among patients infected by the Delta/21A or Alpha/20I variants, partial or complete vaccination exhibited a protective effect with a risk divided by 3.1 for mortality in patients ≥ 55 years, by 2.8 for ICU transfer in patients ≥ 34 years, and by 1.8 for hospitalization in patients ≥ 54 years. Compared to partial vaccination, complete vaccination provided an even stronger protective effect, confirming effectiveness to prevent severe forms of COVID-19.

Isolation of 4000 SARS-CoV-2 shows that contagiousness is associated with viral load, not vaccine or symptomatic status.

Culture inoculation of 6722 nasopharyngeal samples since February 2020 allowed isolation of 3637 SARS-CoV-2 and confirmed that isolation rate is correlated to viral load, regardless symptomatology or vaccination status. Moreover, the delta variant is associated with higher viral loads and therefore higher rates of viral isolation, explaining its greater contagiousness.

First evidence of human-to-dog transmission of SARS-CoV-2 B.1.160 variant in France.

Since the start of the coronavirus disease of 2019 (COVID-19) pandemic, several episodes of human-to-animal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission have been described in different countries. The role of pets, especially domestic dogs, in the COVID-19 epidemiology is highly questionable and needs further investigation. In this study, we report a case of COVID-19 in a French dog living in close contact with its owners who were COVID-19 patients. The dog presented rhinitis and was sampled 1 week after its owners (a man and a woman) were tested positive for COVID-19. The nasal swabs for the dog tested remained positive for SARS-CoV-2 by reverse transcription quantitative real-time PCR (RT-qPCR) 1 month following the first diagnosis. Specific anti-SARS-CoV-2 antibodies were detectable 12 days after the first diagnosis and persisted for at least 5 months as tested using enzyme-linked immunoassay (ELISA) and automated western blotting. The whole-genome sequences from the dog and its owners were 99%-100% identical (with the man and the woman's sequences, respectively) and matched the B.1.160 variant of concern (Marseille-4 variant), the most widespread in France at the time the dog was infected. This study documents the first detection of B.1.160 in pets (a dog) in France, and the first canine genome recovery of the B.1.160 variant of global concern. Moreover, given the enhanced infectivity and transmissibility of the Marseille-4 variant for humans, this case also highlights the risk that pets may potentially play a significant role in SARS-CoV-2 outbreaks and may transmit the infection to humans. We have evidence of human-to-dog transmission of the Marseille-4 variant since the owners were first to be infected. Finally, owners and veterinarians must be vigilent for canine COVID-19 when dogs are presented with respiratory clinical signs.

Monitoring the Circulation of SARS-CoV-2 Variants by Genomic Analysis of Wastewater in Marseille, South-East France.

The monitoring of SARS-CoV-2 RNA in sewage has been proposed as a simple and unbiased means of assessing epidemic evolution and the efficiency of the COVID-19 control measures. The past year has been marked by the emergence of variants that have led to a succession of epidemic waves. It thus appears that monitoring the presence of SARS-CoV-2 in wastewater alone is insufficient, and it may be important in the future to also monitor the evolution of these variants. We used a real-time RT-PCR screening test for variants in the wastewater of our city to assess the effectiveness of direct SARS-CoV-2 sequencing from the same wastewater. We compared the genome sequencing results obtained over the large RS network and the smaller B7 network with the different distributions of the variants observed by RT-PCR screening. The prevalence of the "UK variant" in the RS and B7 networks was estimated to be 70% and 8% using RT-PCR screening compared to 95% and 64% using genome sequencing, respectively. The latter values were close to the epidemiology observed in patients of the corresponding area, which were 91% and 58%, respectively. Genome sequencing in sewage identified SARS-CoV-2 of lineage B.1.525 in B7 at 27% (37% in patients), whereas it was completely missed by RT-PCR. We thus determined that direct sequencing makes it possible to observe, in wastewater, a distribution of the variants comparable to that revealed by genomic monitoring in patients and that this method is more accurate than RT-PCR. It also shows that, rather than a single large sample, it would be preferable to analyse several targeted samples if we want to more appropriately assess the geographical distribution of the different variants. In conclusion, this work supports the wider surveillance of SARS-CoV-2 variants in wastewater by genome sequencing and targeting small areas on the condition of having a sequencing capacity and, when this is not the case, to developing more precise screening tests based on the multiplexed detection of the mutations of interest.

Whole Genome Sequencing of SARS-CoV-2 Strains in COVID-19 Patients From Djibouti Shows Novel Mutations and Clades Replacing Over Time.

Since the start of COVID-19 pandemic the Republic of Djibouti, in the horn of Africa, has experienced two epidemic waves of the virus between April and August 2020 and between February and May 2021. By May 2021, COVID-19 had affected 1.18% of the Djiboutian population and caused 152 deaths. Djibouti hosts several foreign military bases which makes it a potential hot-spot for the introduction of different SARS-CoV-2 strains. We genotyped fifty three viruses that have spread during the two epidemic waves. Next, using spike sequencing of twenty-eight strains and whole genome sequencing of thirteen strains, we found that Nexstrain clades 20A and 20B with a typically European D614G substitution in the spike and a frequent P2633L substitution in nsp16 were the dominant viruses during the first epidemic wave, while the clade 20H South African variants spread during the second wave characterized by an increase in the number of severe forms of COVID-19.

Implementation of an in-house real-time reverse transcription-PCR assay for the rapid detection of the SARS-CoV-2 Marseille-4 variant.

INTRODUCTION: The SARS-CoV-2 pandemic has been associated with the occurrence since summer 2020 of several viral variants that overlapped or succeeded each other in time. Those of current concern harbor mutations within the spike receptor binding domain (RBD) that may be associated with viral escape to immune responses. In our geographical area a viral variant we named Marseille-4 harbors a S477 N substitution in this RBD. MATERIALS AND METHODS: We aimed to implement an in-house one-step real-time reverse transcription-PCR (qPCR) assay with a hydrolysis probe that specifically detects the SARS-CoV-2 Marseille-4 variant. RESULTS: All 6 cDNA samples from Marseille-4 variant strains identified in our institute by genome next-generation sequencing (NGS) tested positive using our Marseille-4 specific qPCR, whereas all 32 cDNA samples from other variants tested negative. In addition, 39/42 (93 %) respiratory samples identified by NGS as containing a Marseille-4 variant strain and 0/26 samples identified as containing non-Marseille-4 variant strains were positive. Finally, 2018/3960 (51%) patients SARS-CoV-2-diagnosed in our institute, 10/277 (3.6 %) respiratory samples collected in Algeria, and none of 207 respiratory samples collected in Senegal, Morocco, or Lebanon tested positive using our Marseille-4 specific qPCR. DISCUSSION: Our in-house qPCR system was found reliable to detect specifically the Marseille-4 variant and allowed estimating it is involved in about half of our SARS-CoV-2 diagnoses since December 2020. Such approach allows the real-time surveillance of SARS-CoV-2 variants, which is warranted to monitor and assess their epidemiological and clinical characterics based on comprehensive sets of data.