SARS-CoV-2 Co-Infections and Recombinations Identified by Long-Read Single-Molecule Real-Time Sequencing.

Co-infection with at least 2 strains of virus is the prerequisite for recombination, one of the means of genetic diversification. Little is known about the prevalence of these events in SARS-CoV-2, partly because it is difficult to detect them. We used long-read PacBio single-molecule real-time (SMRT) sequencing technology to sequence whole genomes and targeted regions for haplotyping. We identified 17 co-infections with SARS-CoV-2 strains belonging to different clades in 6829 samples sequenced between January and October, 2022 (prevalence 0.25%). There were 3 Delta/Omicron co-infections and 14 Omicron/Omicron co-infections (4 cases of 21K/21L, 1 case of 21L/22A, 2 cases of 21L/22B, 4 cases of 22A/22B, 2 cases of 22B/22C and 1 case of 22B/22E). Four of these patients (24%) also harbored recombinant minor haplotypes, including one with a recombinant virus that was selected in the viral quasispecies over the course of his chronic infection. While co-infections remain rare among SARS-CoV-2-infected individuals, long-read SMRT sequencing is a useful tool for detecting them as well as recombinant events, providing the basis for assessing their clinical impact, and a precise indicator of epidemic evolution. IMPORTANCE SARS-CoV-2 variants have been responsible for the successive waves of infection over the 3 years of pandemic. While co-infection followed by recombination is one driver of virus evolution, there have been few reports of co-infections, mainly between Delta and Omicron variants or between the first 2 Omicron variants 21K_BA.1 and 21L_BA.2. The 17 co-infections we detected during 2022 included cases with the recent clades of Omicron 22A, 22B, 22C, and 22E; 24% harbored recombinant variants. This study shows that long-read SMRT sequencing is well suited to SARS-CoV-2 genomic surveillance.

Whole-genome single molecule real-time sequencing of SARS-CoV-2 Omicron.

New variants and genetic mutations of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome can only be identified using accurate sequencing methods. Single molecule real-time (SMRT) sequencing has been used to characterize Alpha and Delta variants, but not Omicron variants harboring numerous mutations in the SARS-CoV-2 genome. This study assesses the performance of a target capture SMRT sequencing protocol for whole genome sequencing (WGS) of SARS-CoV-2 Omicron variants and compared it to that of an amplicon SMRT sequencing protocol optimized for Omicron variants. The failure rate of the target capture protocol (6%) was lower than that of the amplicon protocol (34%, p < 0.001) on our data set, and the median genome coverage with the target capture protocol (98.6% [interquartile range (IQR): 86-99.4]) was greater than that with the amplicon protocol (76.6% [IQR: 66-89.6], [p < 0.001]). The percentages of samples with >95% whole genome coverage were 64% with the target capture protocol and 19% with the amplicon protocol (p < 0.05). The clades of 96 samples determined with both protocols were 93% concordant and the lineages of 59 samples were 100% concordant. Thus, target capture SMRT sequencing appears to be an efficient method for WGS, genotyping and detecting mutations of SARS-CoV-2 Omicron variants.

Whole-genome sequencing of SARS-CoV-2: Comparison of target capture and amplicon single molecule real-time sequencing protocols.

Fast, accurate sequencing methods are needed to identify new variants and genetic mutations of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome. Single-molecule real-time (SMRT) Pacific Biosciences (PacBio) provides long, highly accurate sequences by circular consensus reads. This study compares the performance of a target capture SMRT PacBio protocol for whole-genome sequencing (WGS) of SARS-CoV-2 to that of an amplicon PacBio SMRT sequencing protocol. The median genome coverage was higher (p < 0.05) with the target capture protocol (99.3% [interquartile range, IQR: 96.3-99.5]) than with the amplicon protocol (99.3% [IQR: 69.9-99.3]). The clades of 65 samples determined with both protocols were 100% concordant. After adjusting for Ct values, S gene coverage was higher with the target capture protocol than with the amplicon protocol. After stratification on Ct values, higher S gene coverage with the target capture protocol was observed only for samples with Ct > 17 (p < 0.01). PacBio SMRT sequencing protocols appear to be suitable for WGS, genotyping, and detecting mutations of SARS-CoV-2.

The influence of the nasopharyngeal viral load on the spread of the Omicron BA.2 variant.

We used variant typing PCR to describe the evolution of SARS-CoV-2 Omicron sublineages between December 2021 and mid-March 2022. The selective advantage of the BA.2 variant over BA.1 is not due to greater nasopharyngeal viral loads.

Influence of the Delta Variant and Vaccination on the SARS-CoV-2 Viral Load.

Studies comparing SARS-CoV-2 nasopharyngeal (NP) viral load (VL) according to virus variant and host vaccination status have yielded inconsistent results. We conducted a single center prospective study between July and September 2021 at the drive-through testing center of the Toulouse University Hospital. We compared the NP VL of 3775 patients infected by the Delta (n = 3637) and Alpha (n = 138) variants, respectively. Patient's symptoms and vaccination status (2619 unvaccinated, 636 one dose and 520 two doses) were recorded. SARS-CoV-2 RNA testing and variant screening were assessed by using Thermo Fisher® TaqPath™ COVID-19 and ID solutions® ID™ SARS-CoV-2/VOC evolution Pentaplex assays. Delta SARS-CoV-2 infections were associated with higher VL than Alpha (coef = 0.68; p ≤ 0.01) independently of patient's vaccination status, symptoms, age and sex. This difference was higher for patients diagnosed late after symptom onset (coef = 0.88; p = 0.01) than for those diagnosed early (coef = 0.43; p = 0.03). Infections in vaccinated patients were associated with lower VL (coef = -0.18; p ≤ 0.01) independently of virus variant, symptom, age and sex. Our results suggest that Delta infections could lead to higher VL and for a longer period compared to Alpha infections. By effectively reducing the NP VL, vaccination could allow for limiting viral spread, even with the Delta variant.

Waves of attention: patterns and themes of international antimicrobial resistance reports, 1945-2020.

This article uses quantitative and qualitative approaches to review 75 years of international policy reports on antimicrobial resistance (AMR). Our review of 248 policy reports and expert consultation revealed waves of political attention and repeated reframings of AMR as a policy object. AMR emerged as an object of international policy-making during the 1990s. Until then, AMR was primarily defined as a challenge of human and agricultural domains within the Global North that could be overcome via 'rational' drug use and selective restrictions. While a growing number of reports jointly addressed human and agricultural AMR selection, international organisations (IOs) initially focused on whistleblowing and reviewing data. Since 2000, there has been a marked shift in the ecological and geographic focus of AMR risk scenarios. The Global South and One Health (OH) emerged as foci of AMR reports. Using the deterritorialised language of OH to frame AMR as a Southern risk made global stewardship meaningful to donors and legitimised pressure on low-income and middle-income countries to adopt Northern stewardship and surveillance frameworks. It also enabled IOs to move from whistleblowing to managing governance frameworks for antibiotic stewardship. Although the environmental OH domain remains neglected, realisation of the complexity of necessary interventions has increased the range of topics targeted by international action plans. Investment nonetheless continues to focus on biomedical innovation and tends to leave aside broader socioeconomic issues. Better knowledge of how AMR framings have evolved is key to broadening participation in international stewardship going forward.

Testing individual and pooled saliva samples for sars-cov-2 nucleic acid: a prospective study.

Control of the rapid spread of the SARS-CoV-2 virus requires efficient testing. We collected paired nasopharyngeal swab (NPs) and saliva samples from 303 subjects (52.8% symptomatic) at a drive-through testing center; 18% of whom tested positive. The NPs, salivas and five saliva pools were tested for SARS-CoV-2 RNA using the Aptima™ assay and a laboratory-developed test (LDT) on the Panther-Fusion™ Hologic® platform. The saliva sensitivity was 80% (LDT) and 87.5% (Aptima™) whereas that of NPs was 96.4% in both assays. The pooled saliva sensitivity of 72.7% (LDT) and 75% (Aptima™) was not significantly different of that of individual saliva testing. Saliva specimens appear to be suitable for sensitive non-invasive assays to detect SARS-CoV-2 nucleic acid; pooling them for a single test will improve laboratory throughput.

Evaluation of the Aptima™ transcription-mediated amplification assay (Hologic®) for detecting SARS-CoV-2 in clinical specimens.

BACKGROUND: The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which appeared in late 2019, has been limited by isolating infected individuals. However, identifying such individuals requires accurate diagnostic tools. OBJECTIVE: This study evaluates the capacity of the Aptima™ Transcription-Mediated Amplification (TMA) assay (Hologic® Panther System) to detect the virus in clinical samples. STUDY DESIGN: We compared the Aptima™ assay to two in-house real-time RT-PCR techniques, one running on the Panther Fusion™ module and the other on the MagNA Pure 96 and Light-Cycler 480 instruments. We included a total of 200 respiratory specimens: 100 tested prospectively and 100 retrospectively (25 -ve/75 +ve). RESULTS: The final Cohen's kappa coefficients were: κ = 0.978 between the Aptima™ and Panther Fusion™ assays, κ = 0.945 between the Aptima™ and MagNA/LC480 assays and κ = 0.956 between the MagNA/LC480 and Panther Fusion™ assays. CONCLUSION: These findings indicate that the Aptima™ SARS-CoV-2 TMA assay data agree well with those obtained with our routine methods and that this assay can be used to diagnose coronavirus disease 2019 (COVID-19).