Genomic Investigations of Acute Hepatitis of Unknown Aetiology in Children (preprint)

Since the first reports of hepatitis of unknown aetiology occurring in UK children, over 1000 cases have been reported worldwide, including 268 cases in the UK, with the majority younger than 6 years old. Using genomic, proteomic and immunohistochemical methods, we undertook extensive investigation of 28 cases and 136 control subjects. In five cases who underwent liver transplantation, we detected high levels of adeno-associated virus 2 (AAV2) in the explanted livers. AAV2 was also detected at high levels in blood from 10/11 non-transplanted cases. Low levels of Adenovirus (HAdV) and Human Herpesvirus 6B (HHV-6B), both of which enable AAV2 lytic replication, were also found in the five explanted livers and blood from 15/17 and 6/9 respectively, of the 23 non-transplant cases tested. In contrast, AAV2 was detected at low titre in 6/100 whole bloods from child controls from cohorts with presence or absence of hepatitis and/or adenovirus infection. Our data show an association of AAV2 at high titre in blood or liver tissue, with unexplained hepatitis in children infected in the recent HAdV-F41 outbreak. We were unable to find evidence by electron microscopy, immunohistochemistry or proteomics of HAdV or AAV2 viral particles or proteins in explanted livers, suggesting that hepatic pathology is not due to direct lytic infection by either virus. The potential that AAV2, although not previously associated with disease, may, together with HAdV-F41 and/or HHV-6, be causally implicated in the outbreak of unexplained hepatitis, requires further investigation.

Epidemiological analysis of the first 1,000 cases of SARS-CoV-2 lineage BA.1 (B.1.1.529, Omicron) compared to co-circulating Delta, in Wales, United Kingdom (preprint)

Background: The Omicron (lineage B.1.1.529) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in Wales, UK on 3 December 2021. The aim of the study was to describe the first 1000 cases of the Omicron variant by demographic, vaccination status, travel and severe outcome status and compare this to contemporaneous cases of the Delta variant. Methods: Testing, typing and contact tracing data were collected by Public Health Wales and analysis undertaken by the Communicable Disease Surveillance Centre (CDSC). Risk ratios for demographic factors and symptoms were calculated comparing Omicron cases to Delta cases identified over the same time period. Results: By 14th December 2021, 1000 cases of the Omicron variant had been identified in Wales. Of the first 1000, just 3% of cases had a prior history of travel revealing rapid community transmission. A higher proportion of Omicron cases were identified in individuals aged 20-39 and most cases were double vaccinated (65.9%) or boosted (15.7%). Age adjusted analysis also revealed that Omicron cases were less likely to be hospitalised (0.4%) or report symptoms (60.8%). Specifically a significant reduction was observed in the proportion of Omicron cases reporting anosmia (8.9%). Conclusion: Key findings include a lower risk of anosmia and a reduced risk of hospitalisation in the first 1000 Omicron cases compared to co-circulating Delta cases. We also identify that existing measures for travel restrictions to control importations of new variants identified outside the UK did not prevent the rapid ingress of Omicron within Wales.

A large outbreak of COVID-19 in a UK prison, October 2020 to April 2021 (preprint)

Introduction Prisons are susceptible to outbreaks. Control measures focusing on isolation and cohorting negatively affect wellbeing. We present an outbreak of COVID-19 in a large male prison in Wales, UK, 14 October 2020 to 21 April 2021, and discuss control measures. Methods We gathered case-information, including: demographics, staff-residence postcode, resident cell number, work areas/dates, test results, staff interview dates/notes and resident prison-transfer dates. Epidemiological curves were mapped by prison location. Control measures included isolation (exclusion from work or cell-isolation), cohorting (new admissions and work-area groups), asymptomatic testing (case-finding), removal of communal dining and movement restrictions. Facemask use and enhanced hygiene were already in place. Whole genome sequencing (WGS) and interviews determined genetic relationship between cases plausibility of transmission. Results Of 453 cases, 53% (n=242) were staff, most aged 25-34 years (11.5% females, 27.15% males) and symptomatic (64%). Crude attack-rate was higher in staff (29%, 95%CI: 26-64%) than in residents (12%, 95%CI: 9-15%). Conclusions Whole genome sequencing can help differentiate multiple introductions from person-to-person transmission in prisons. It should be introduced alongside asymptomatic testing as soon as possible to control prison outbreaks. Timely epidemiological investigation, including data visualization, allowed dynamic risk assessment and proportionate control measures, minimizing reduction in resident welfare.

Higher Relative Viral Load Excretion Determined by Normalised Threshold Crossing Value in Acute Cases infected with the B.1.1.7 Lineage VOC 202012/01 (Using S gene target failure as a Proxy) When Compared to other Circulating Lineages in Wales (preprint)

Since the emergence of SARS-CoV-2, global monitoring of the virus using whole genome sequencing has identified mutations occurring across the viral genome. Whilst the majority have little impact on the virus, they are used effectively to monitor the movement of the virus globally and to inform locally on transmission chains. In late 2020, a variant of SARS-CoV-2 (B.1.1.7 - VOC 202012/01) was identified in the UK with a distinct constellation of mutations, including in the spike gene that increased transmissibility. A deletion in spike also affected one of the screening qPCR tests being used in the UK outside of Wales, causing a failure to detect the target. This quickly became a surrogate marker for the variant to allow rapid monitoring of the virus as it seeded into new regions of the UK. A screening study using this assay as a proxy marker, was undertaken to understand the prevalence of the variant in Wales. Secondary analysis of a screening qPCR that didn’t target the S gene and also included an endogenous control, was also performed to understand viral load excretion in those infected with the variant compared to other circulating lineages. Using a combination of analytical methods based on the C t values of two gene targets normalised against the endogenous control, there was a difference in the excreted viral load. Those with the variant excreting more virus than those not infected with the variant. Supporting not only increased infectivity but offering a plausible reason why increased transmission was associated with this particular variant. Whilst there are limitations in this study, the method using C t as a proxy for viral load can be used at the population level to determine differences in viral excretion kinetics associated with different variants.

Localised community circulation of SARS-CoV-2 viruses with an increased accumulation of single nucleotide polymorphisms that adversely affect the sensitivity of real-time reverse transcription assays targeting Nucleocapsid protein. (preprint)

Currently the primary method for confirming acute SARS-CoV-2 infection is through the use of molecular assays that target highly conserved regions within the viral genome. Many, if not most of the diagnostic targets currently in use were produced early in the pandemic, using genomes sequenced and shared in early 2020. As viral diversity increases, mutations may arise in diagnostic target sites that have an impact on the performance of diagnostic tests. Here, we report on a local outbreak of SARS-CoV-2 which had gained an additional mutation at position 28890 of the nucleocapsid protein, on a background of pre-existing mutations at positions 28881, 28882, 28883 in one of the main circulating viral lineages in Wales at that time. The impact of this additional mutation had a statistically significant impact on the Ct value reported for the N gene target designed by the Chinese CDC and used in a number of commercial diagnostic products. Further investigation identified that, in viral genomes sequenced from Wales over the summer of 2020, the N gene had a higher rate of mutations in diagnostic target sites than other targets, with 115 issues identified affecting over 10% of all cases sequenced between February and the end of August 2020. In comparison an issue was identified for ORFab, the next most affected target, in less than 1.4% of cases over the same time period. This work emphasises the potential impact that mutations in diagnostic target sites can have on tracking local outbreaks, as well as demonstrating the value of genomics as a routine tool for identifying and explaining potential diagnostic primer issues as part of a laboratory quality management system. This work also indicates that with increasing genomic sequencing data availability, there is a need to re-evaluate the diagnostic targets that are in use for SARS-CoV-2 testing, to better target regions that are now demonstrated to be of lower variability.

A longitudinal comparison of spike and nucleocapsid SARS-CoV-2 antibody responses in a tertiary hospitals laboratory workers with validation of DBS specimen analysis. (preprint)

There is a requirement for easily accessible, high throughput serological testing as part of the SARS-CoV-2 pandemic response. Whilst of limited diagnostic use in an acute individual setting, its use on a population level is key to informing a coherent public health response. As experience of commercial assays increases, so too does knowledge of their precision and limitations. Here we present our experience of these systems thus far. We perform a spot sero-prevalence study amongst staff in a tertiary hospitals clinical microbiology laboratory, before undertaking validation of DBS serological testing as an alternate specimen for analysis. Finally, we characterise the spike and nucleocapsid antibody response over 160 days post a positive PCR test in nine non-hospitalised staff members. Amongst a cohort of 195 staff, 17 tested positive for SARS-CoV-2 antibodies (8.7%). Self-reporting of SARS-CoV2 infection (P=<0.0001) and testing of a household contact (P = 0.027) were significant variables amongst the positive and negative sub-groups. Testing of 28 matched serum and DBS samples demonstrated 96% accuracy between the sample types. A differential rate of decline of SARS-CoV-2 antibodies against nucleocapsid or spike protein was observed. At 4 months post a positive PCR test 7/9 (78%) individuals had detectable antibodies against spike protein, but only 2/9 (22%) had detectable antibodies against nucleocapsid protein. This study reveals a broad agreement amongst commercial platforms tested and suggests the use of DBS as an alternate specimen option to enable widespread population testing for SARS-CoV-2 antibodies. These results suggest potential limitations of these platforms in estimating historical infection. By setting this temporal point of reference for this cohort of non-patient facing laboratory staff, future exposure risks and mitigation strategies can be evaluated more fully.

Dynamics of IgG seroconversion and pathophysiology of COVID-19 infections (preprint)

We report dynamics of seroconversion to SARS-CoV-2 infections detected by IgG ELISA in 177 individuals diagnosed by RT-PCR. Longitudinal analysis identifies 2-8.5% of individuals who do not seroconvert even weeks after infection. They are younger than seroconverters who have increased co-morbidity and higher inflammatory markers such as C-Reactive Protein. Higher antibody responses are associated with non-white ethnicity. Antibody responses do not decline during follow up almost to 2 months. Serological assays increase understanding of disease severity. Their application in regular surveillance will clarify the duration and protective nature of humoral responses to SARS-CoV-2.

Pre-existing and de novo humoral immunity to SARS-CoV-2 in humans (preprint)

Several related human coronaviruses (HCoVs) are endemic in the human population, causing mild respiratory infections1. Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiologic agent of Coronavirus disease 2019 (COVID-19), is a recent zoonotic infection that has quickly reached pandemic proportions2,3. Zoonotic introduction of novel coronaviruses is thought to occur in the absence of pre-existing immunity in the target human population. Using diverse assays for detection of antibodies reactive with the SARS-CoV-2 spike (S) glycoprotein, we demonstrate the presence of pre-existing humoral immunity in uninfected and unexposed humans to the new coronavirus. SARS-CoV-2 S-reactive antibodies were readily detectable by a sensitive flow cytometry-based method in SARS-CoV-2-uninfected individuals and were particularly prevalent in children and adolescents. These were predominantly of the IgG class and targeted the S2 subunit. In contrast, SARS-CoV-2 infection induced higher titres of SARS-CoV-2 S-reactive IgG antibodies, targeting both the S1 and S2 subunits, as well as concomitant IgM and IgA antibodies, lasting throughout the observation period of 6 weeks since symptoms onset. SARS-CoV-2-uninfected donor sera also variably reacted with SARS-CoV-2 S and nucleoprotein (N), but not with the S1 subunit or the receptor binding domain (RBD) of S on standard enzyme immunoassays. Notably, SARS-CoV-2-uninfected donor sera exhibited specific neutralising activity against SARS-CoV-2 and SARS-CoV-2 S pseudotypes, according to levels of SARS-CoV-2 S-binding IgG and with efficiencies comparable to those of COVID-19 patient sera. Distinguishing pre-existing and de novo antibody responses to SARS-CoV-2 will be critical for our understanding of susceptibility to and the natural course of SARS-CoV-2 infection.

Scalable and Resilient SARS-CoV2 testing in an Academic Centre (preprint)

The emergence of the novel coronavirus SARS-CoV-2 has led to a pandemic infecting more than two million people worldwide in less than four months, posing a major threat to healthcare systems. This is compounded by the shortage of available tests causing numerous healthcare workers to unnecessarily self-isolate. We provide a roadmap instructing how a research institute can be repurposed in the midst of this crisis, in collaboration with partner hospitals and an established diagnostic laboratory, harnessing existing expertise in virus handling, robotics, PCR, and data science to derive a rapid, high throughput diagnostic testing pipeline for detecting SARS-CoV-2 in patients with suspected COVID-19. The pipeline is used to detect SARS-CoV-2 from combined nose-throat swabs and endotracheal secretions/ bronchoalveolar lavage fluid. Notably, it relies on a series of in-house buffers for virus inactivation and the extraction of viral RNA, thereby reducing the dependency on commercial suppliers at times of global shortage. We use a commercial RT-PCR assay, from BGI, and results are reported with a bespoke online web application that integrates with the healthcare digital system. This strategy facilitates the remote reporting of thousands of samples a day with a turnaround time of under 24 hours, universally applicable to laboratories worldwide.