Lateral flow devices (LFDs) are quickly being implemented for use in large-scale population surveillance programs for SARS-CoV-2 infection in the United Kingdom. These programs have been piloted in city-wide screening in the city of Liverpool and are now being rolled out to support care home visits and the return home of University students for the Christmas break. Here, we present data on the performance of LFDs to test almost 8,000 students at the University of Birmingham between December 2 and December 9, 2020. The performance is validated against almost 800 samples using PCR performed in the University Pillar 2 testing lab and theoretically validated on thousands of Pillar 2 PCR testing results performed on low-prevalence care home testing samples. Our data show that LFDs do not detect infections presenting with PCR Ct values over 29 to 30 as determined using the Thermo Fisher TaqPath asssay. This may be of particular importance in detecting individuals that are either at the early, or late stages of infection, and reinforces the need for frequent, recurrent testing.

Hepatitis B virus (HBV) infection is of global importance with over 2 billion people exposed to the virus during their lifetime and at risk of progressive liver disease, cirrhosis and hepatocellular carcinoma. HBV is a member of the Hepadnaviridae family that replicates via episomal copies of a covalently closed circular DNA (cccDNA) genome. The chromatinization of this small viral genome, with overlapping open reading frames and regulatory elements, suggests an important role for epigenetic pathways to regulate viral transcription. The chromatin‐organising transcriptional insulator protein, CCCTC‐binding factor (CTCF), has been reported to regulate transcription in a diverse range of viruses. We identified two conserved CTCF binding sites in the HBV genome within enhancer I and chromatin immunoprecipitation (ChIP) analysis demonstrated an enrichment of CTCF binding to integrated or episomal copies of the viral genome. siRNA knock‐down of CTCF results in a significant increase in pre‐genomic RNA levels in de novo infected HepG2 cells and those supporting episomal HBV DNA replication. Furthermore, mutation of these sites in HBV DNA minicircles abrogated CTCF binding and increased pre‐genomic RNA levels, providing evidence of a direct role for CTCF in repressing HBV transcription.

The ubiquitous host protein, CCCTC-binding factor (CTCF), is an essential regulator of cellular transcription and functions to maintain epigenetic boundaries, stabilise chromatin loops and regulate splicing of alternative exons. We have previously demonstrated that CTCF binds to the E2 open reading frame (ORF) of human papillomavirus (HPV) 18 and functions to repress viral oncogene expression in undifferentiated keratinocytes by co-ordinating an epigenetically repressed chromatin loop within HPV episomes. Keratinocyte differentiation disrupts CTCF-dependent chromatin looping of HPV18 episomes promoting induction of enhanced viral oncogene expression. To further characterise CTCF function in HPV transcription control we utilised direct, long-read Nanopore RNA-sequencing which provides information on the structure and abundance of full-length transcripts. Nanopore analysis of primary human keratinocytes containing HPV18 episomes before and after synchronous differentiation allowed quantification of viral transcript species, including the identification of low abundance novel transcripts. Comparison of transcripts produced in wild type HPV18 genome-containing cells to those identified in CTCF-binding deficient genome-containing cells identifies CTCF as a key regulator of differentiation-dependent late promoter activation, required for efficient E1^E4 and L1 protein expression. Furthermore, our data show that CTCF binding at the E2 ORF promotes usage of the downstream weak splice donor (SD) sites SD3165 and SD3284, to the dominant E4 splice acceptor site at nucleotide 3434. These findings demonstrate that in the HPV life cycle both early and late virus transcription programmes are facilitated by recruitment of CTCF to the E2 ORF.

Lateral flow devices are quickly being implemented for use in large scale population surveillance programs for SARS-CoV-2 infection in the United Kingdom. These programs have been piloted in city wide screening in the city of Liverpool, and are now being rolled out to support care home visits and the return home of University students for the Christmas break. Here we present data on the performance of Lateral Flow devices to test almost 8,000 students at the University of Birmingham between December 2nd and December 9th 2020. The performance is validated against almost 800 samples using PCR performed in the University Pillar 2 testing lab, and theoretically validated on thousands of Pillar 2 PCR testing results performed on low-prevalence care home testing samples. Our data shows that Lateral Flow Devices do not detect infections presenting with PCR Ct values over 29-30, meaning that only 3.2% (95% CI 0.6% to 15.6%) of total cases in the student population were detected, but that as many of 85% of cases tested in the Pillar 2 PCR lab would have been detected theoretically

Genome sequencing is a powerful tool for identifying SARS-CoV-2 variant lineages; however, there can be limitations due to sequence dropout when used to identify specific key mutations. Recently, ThermoFisher Scientific has developed genotyping assays to help bridge the gap between testing capacity and sequencing capability to generate real-time genotyping results based on specific variants.

Previous studies have described RT-LAMP methodology for the rapid detection of SARS-CoV-2 in nasopharyngeal (NP) and oropharyngeal (OP) swab and saliva samples. This study describes the validation of an improved sample preparation method for extraction free RT-LAMP and defines the clinical performance of four different RT-LAMP assay formats for detection of SARS-CoV-2 within a multisite clinical evaluation. Direct RT-LAMP was performed on 559 swabs and 86,760 saliva samples and RNA RT-LAMP on extracted RNA from 12,619 swabs and 12,521 saliva from asymptomatic and symptomatic individuals across healthcare and community settings. For Direct RT-LAMP, overall diagnostic sensitivity (DSe) of 70.35% (95% CI 63.48-76.60%) on swabs and 84.62% (79.50-88.88%) on saliva was observed, with diagnostic specificity (DSp) of 100% (98.98-100.00%) on swabs and 100% (99.72-100.00%) on saliva when compared to RT-qPCR; analysing samples with RT-qPCR ORF1ab C T values of < 25 and < 33, DSe of 100% (96.34-100%) and 77.78% (70.99-83.62%) for swabs were observed, and 99.01% (94.61-99.97%) and 87.61% (82.69-91.54%) for saliva, respectively. For RNA RT-LAMP, overall DSe and DSp were 96.06% (92.88-98.12%) and 99.99% (99.95-100%) for swabs, and 80.65% (73.54-86.54%) and 99.99% (99.95-100%) for saliva, respectively. These findings demonstrate that RT-LAMP is applicable to a variety of use-cases, including frequent, interval-based testing of saliva with Direct RT-LAMP from asymptomatic individuals that may otherwise be missed using symptomatic testing alone.

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