A reliable diagnostic assay is crucial to early detect new COVID-19 cases and limit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission. Since the onset of the COVID-19 pandemic, the World Health Organization has published several diagnostic molecular approaches developed by referral laboratories, including Charité (Germany), HKU (Hong Kong), China CDC (China), US CDC (United States), and Institut Pasteur, Paris (France). We aimed to compare the sensitivity and specificity of these different RT-PCR assays using SARS-CoV-2 cell culture supernatants and clinical respiratory samples. Overall, the different RT-PCR assays performed well for SARS-CoV-2 detection and were all specific except the N Charité (Germany), and N2 US CDC (United States) assays. RdRp Institut Pasteur (IP2, IP4), N China CDC, and N1 US CDC were found to be the most sensitive assays. The data presented herein are of prime importance to facilitate the equipment choice of diagnostic laboratories, as well as for the development of marketed tests.

We aimed to investigate the first Omicron cases detected in France in order to assess case characteristics and provide supporting information on the possible impact of this variant on the healthcare system. A standardized questionnaire was used to collect information from confirmed and probable Omicron cases. Median age of 468 investigated cases was 35 years, 376 were symptomatic (89%); 64% were vaccinated with two doses and 7% had received three doses. Loss of smell and taste were reported by 8.3% and 9% of cases, respectively. Seven cases were hospitalized, three of those were unvaccinated (including two with reported precondition). No admissions to intensive care and no deaths were reported. Our results confirm a mild clinical presentation among the first Omicron cases detected in France and highlight the importance for the national COVID-19 surveillance system to quickly detect and adapt to the emergence of a new variant.

Objectives: High viral load in upper respiratory tract specimens observed for Delta cases might contribute to its increased infectivity compared to the other variant. However, it is not yet documented if the Omicron variant’s enhanced infectivity is also related to a higher viral load. Our aim was to determine if the Omicron variant’s spread is also related to higher viral loads compared to the Delta variant. Methods: Nasopharyngeal swabs, 129 (Omicron) and 85 (Delta), from Health Care Workers were collected during December 2021 at the University Hospital of Lyon, France. Cycle threshold (Ct) for the RdRp target of cobas^® 6800 SARS-CoV-2 assay was used as a proxy to evaluate SARS-CoV-2 viral load. Variant identification was performed using a screening panel and confirmed by whole genome sequencing. Results: Herein, we showed that the RT-PCR Ct values in Health Care Workers sampled within 5 days after symptom onset were significantly higher for Omicron cases than Delta cases (21.7 for Delta variant and 23.8 for Omicron variant, p = 0.008). This difference was also observed regarding patient with complete vaccination. Conclusions: This result supports the studies showing that the increased transmissibility of Omicron is related to other mechanisms than higher virus excretion.

Viral metagenomics next-generation sequencing (mNGS) is increasingly being used to characterize the human virome. The impact of viral nucleic extraction on virome profiling has been poorly studied. Here, we aimed to compare the sensitivity and sample and reagent contamination of three extraction methods used for viral mNGS: two automated platforms (eMAG; MagNA Pure 24, MP24) and the manual QIAamp Viral RNA Mini Kit (QIAamp). Clinical respiratory samples (positive for Respiratory Syncytial Virus or Herpes Simplex Virus), one mock sample (including five viruses isolated from respiratory samples), and a no-template control (NTC) were extracted and processed through an mNGS workflow. QIAamp yielded a lower proportion of viral reads for both clinical and mock samples. The sample cross-contamination was higher when using MP24, with up to 36.09% of the viral reads mapping to mock viruses in the NTC (vs. 1.53% and 1.45% for eMAG and QIAamp, respectively). The highest number of viral reads mapping to bacteriophages in the NTC was found with QIAamp, suggesting reagent contamination. Our results highlight the importance of the extraction method choice for accurate virome characterization.

Human Anelloviridae is a highly prevalent viral family, including three main genera—Alphatorquevirus (Torque teno virus, TTV), Betatorquevirus (Torque teno mini virus, TTMV), and Gammatorquevirus (Torque teno midi virus, TTMDV). To date, the characterization of Anelloviridae in the respiratory tract of children with acute respiratory infection (ARI) has been poorly reported and mainly focused on TTV. We performed a metagenomic analysis of eight respiratory samples collected from children with an ARI of unknown etiology (eight samples tested negative with a multiplex PCR assay, out of the 39 samples initially selected based on negative routine diagnostic testing). A total of 19 pediatric respiratory samples that tested positive for respiratory syncytial virus (RSV, n = 13) or influenza virus (n = 6) were also sequenced. Anelloviridae reads were detected in 16/27 samples, including 6/8 negative samples, 7/13 RSV samples and 3/6 influenza samples. For samples with a detection of at least one Anelloviridae genus, TTMV represented 87.1 (66.1–99.2)% of Anelloviridae reads, while TTV and TTMDV represented 0.8 (0.0–9.6)% and 0.7 (0.0–7.1)%, respectively (p < 0.001). Our findings highlight a high prevalence of TTMV in respiratory samples of children with an ARI of unknown etiology, as well as in samples with an RSV or influenza infection. Larger studies are needed to explore the role of TTMV in childhood respiratory diseases.

We report the implementation of a two-step strategy for the identification of SARS-CoV-2 variants carrying the spike deletion H69-V70 (ΔH69/ΔV70). This spike deletion resulted in a S-gene target failure (SGTF) of a three-target RT-PCR assay (TaqPath kit). Whole genome sequencing performed on 37 samples with SGTF revealed several receptor-binding domain mutations co-occurring with ΔH69/ΔV70. More importantly, this strategy enabled the first detection of the variant of concern 202012/01 in France on December 21 ^th 2020. Since September a SARS-CoV-2 spike (S) deletion H69-V70 (ΔH69/ΔV70) has attracted increasing attention. This deletion was detected in the cluster-5 variant identified both in minks and humans in Denmark. This cluster-5 variant carries a receptor binding domain (RBD) mutation Y453F and was associated with reduced susceptibility to neutralizing antibodies to sera from recovered COVID-19 patients [1–3]. The ΔH69/ΔV70 has also co-occurred with two other RBD mutations of increasing interest [4]: N439K that is currently spreading in Europe and might also have reduced susceptibility to SARS-CoV-2 antibodies [5]; and N501Y that is part of the SARS-CoV-2 variant of concern (VOC) 202012/01 recently detected in England [6]. Although the impact of ΔH69/ΔV70 on SARS-CoV-2 pathogenesis is not clear, enhanced surveillance is urgently needed. Herein we report the implementation of a two-step strategy enabling a rapid detection of VOC 202012/01 or other variants carrying ΔH69/ΔV70.