Multiplex MinION sequencing suggests enteric adenovirus F41 genetic diversity comparable to pre-COVID-19 era.

Human adenovirus F41 causes acute gastroenteritis in children, and has recently been associated with an apparent increase in paediatric hepatitis of unknown aetiology in the UK, with further cases reported in multiple countries. Relatively little is known about the genetic diversity of adenovirus F41 in UK children; and it is unclear what, if any, impact the COVID-19 pandemic has had on viral diversity in the UK. Methods that allow F41 to be sequenced from clinical samples without the need for viral culture are required to provide the genomic data to address these questions. Therefore, we evaluated an overlapping-amplicon method of sequencing adenovirus genomes from clinical samples using Oxford Nanopore technology. We applied this method to a small sample of adenovirus-species-F-positive extracts collected as part of standard care in the East of England region in January-May 2022. This method produced genomes with >75 % coverage in 13/22 samples and >50 % coverage in 19/22 samples. We identified two F41 lineages present in paediatric patients in the East of England in 2022. Where F41 genomes from paediatric hepatitis cases were available (n=2), these genomes fell within the diversity of F41 from the UK and continental Europe sequenced before and after the 2020-2021 phase of the COVID-19 pandemic. Our analyses suggest that overlapping amplicon sequencing is an appropriate method for generating F41 genomic data from high-virus-load clinical samples, and currently circulating F41 viral lineages were present in the UK and Europe before the COVID-19 pandemic.

SARS-CoV-2 evolution during treatment of chronic infection.

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for virus infection through the engagement of the human ACE2 protein1 and is a major antibody target. Here we show that chronic infection with SARS-CoV-2 leads to viral evolution and reduced sensitivity to neutralizing antibodies in an immunosuppressed individual treated with convalescent plasma, by generating whole-genome ultra-deep sequences for 23 time points that span 101 days and using in vitro techniques to characterize the mutations revealed by sequencing. There was little change in the overall structure of the viral population after two courses of remdesivir during the first 57 days. However, after convalescent plasma therapy, we observed large, dynamic shifts in the viral population, with the emergence of a dominant viral strain that contained a substitution (D796H) in the S2 subunit and a deletion (ΔH69/ΔV70) in the S1 N-terminal domain of the spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype were reduced in frequency, before returning during a final, unsuccessful course of convalescent plasma treatment. In vitro, the spike double mutant bearing both ΔH69/ΔV70 and D796H conferred modestly decreased sensitivity to convalescent plasma, while maintaining infectivity levels that were similar to the wild-type virus.The spike substitution mutant D796H appeared to be the main contributor to the decreased susceptibility to neutralizing antibodies, but this mutation resulted in an infectivity defect. The spike deletion mutant ΔH69/ΔV70 had a twofold higher level of infectivity than wild-type SARS-CoV-2, possibly compensating for the reduced infectivity of the D796H mutation. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy, which is associated with the emergence of viral variants that show evidence of reduced susceptibility to neutralizing antibodies in immunosuppressed individuals.

SARS-CoV-2 screening: effectiveness and risk of increasing transmission.

Testing asymptomatic people for SARS-CoV-2 aims to reduce COVID-19 transmission. Screening programmes' effectiveness depends upon testing strategy, sample handling logistics, test sensitivity and individual behaviour, in addition to dynamics of viral transmission. The interaction between these factors is not fully characterized. We investigated the interaction between these factors to determine how to optimize reduction of transmission. We estimate that under idealistic assumptions 70% of transmission may be averted, but under realistic assumptions only 7% may be averted. We show that programmes that overwhelm laboratory capacity or reduce isolation of those with minor symptoms have increased transmission compared with those that do not: programmes need to be designed to avoid these issues, or they will be ineffective or even counter-productive. Our model allows optimal selection of whom to test, quantifies the balance between accuracy and timeliness, and quantifies potential impacts of behavioural interventions. We anticipate our model can be used to understand optimal screening strategies for other infectious diseases with substantially different dynamics.

Specificity and positive predictive value of SARS-CoV-2 nucleic acid amplification testing in a low-prevalence setting.

OBJECTIVES: When the prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is low, many positive test results are false positives. Confirmatory testing reduces overdiagnosis and nosocomial infection and enables real-world estimates of test specificity and positive predictive value. This study estimates these parameters to evaluate the impact of confirmatory testing and to improve clinical diagnosis, epidemiological estimation and interpretation of vaccine trials. METHODS: Over 1 month we took all respiratory samples from our laboratory with a patient's first detection of SARS-CoV-2 RNA (Hologic Aptima SARS-CoV-2 assay or in-house RT-PCR platform), and repeated testing using two platforms. Samples were categorized by source, and by whether clinical details suggested COVID-19 or corroborative testing from another laboratory. We estimated specificity and positive predictive value using approaches based on maximum likelihood. RESULTS: Of 19 597 samples, SARS-CoV-2 RNA was detected in 107; 52 corresponded to first-time detection (0.27% of tests on samples without previous detection). Further testing detected SARS-CoV-2 RNA once or more ('confirmed') in 29 samples (56%), and failed to detect SARS-CoV-2 RNA ('not confirmed') in 23 (44%). Depending upon assumed parameters, point estimates for specificity and positive predictive value were 99.91-99.98% and 61.8-89.8% respectively using the Hologic Aptima SARS-CoV-2 assay, and 97.4-99.1% and 20.1-73.8% respectively using an in-house assay. CONCLUSIONS: Nucleic acid amplification testing for SARS-CoV-2 is highly specific. Nevertheless, when prevalence is low a significant proportion of initially positive results fail to confirm, and confirmatory testing substantially reduces the detection of false positives. Omitting additional testing in samples with higher prior detection probabilities focuses testing where it is clinically impactful and minimizes delay.