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.

Meta-analysis of rapid direct-to-PCR assays for the qualitative detection of SARS-CoV-2 (preprint)

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the ensuing COVID-19 pandemic present significant challenges to current diagnostic and therapeutic patient care pathways including whether new in vitro diagnostic tests can accurately identify and rule out current SARS-CoV-2 infection. The gold standard diagnostic test to identify a current SARS-CoV-2 infection is a central laboratory-based molecular assay using reverse transcription polymerase chain reaction (RT-PCR) with very high accuracy of detection but which typically requires 1-2 days turn-around for results. Therefore, rapid RTPCR assays and systems have been developed which can be deployed locally (near-patient or point of care (POC), provide faster results and not impact on already stressed central laboratory capacity. Rapid test results can be returned within the same clinical encounter, facilitating timely decisions that optimise the patient care pathway and support more rapid COVID-19 diagnosis, isolation and contract tracing activities. Direct-to-PCR is an evolution of RT-PCR in which the patient sample is added directly to an amplification reaction without being subjected to prior nucleic acid extraction, purification, or quantification to reduce the time and monetary resources required to process samples. Rapid, direct-to-PCR systems further increase the speed of testing by combining rapid PCR instruments with direct-to-PCR assays, to generate results in less than two hours. This appears to be the first meta-analysis assessing the accuracy of rapid direct-to-PCR in the detection of SARS-CoV-2. In total, 10,957 unique records were identified and screened using a search string evaluation, 420 full-text reports and/or supplemental materials were assessed for inclusion. This resulted in 14 studies reporting 20 datasets with 4593 patient samples (1391 positive) included in the analysis. The overall agreement between the rapid direct RT-PCR and gold standard centralised laboratory RT-PCR was 97.1% with 93.60% positive percent agreement and 98.63% negative percent agreement. The Cohens kappa statistical coefficient k = 0.93, indicating an almost perfect agreement and Youden Index = 0.92. These results indicate that direct-to-PCR assays can perform equivalently to the standard centralised laboratory PCR systems for the detection of SARS-CoV-2. Objectives: To assess the efficacy of rapid direct-to-PCR assays and systems for the detection of SARS-CoV-2 in the hospital, care home and medical research population in England from November 2020 to April 2021. Search methods: Electronic searches of the Cochrane COVID-19 Study Register (which includes daily updates from PubMed and Embase and preprints from medRxiv and bioRxiv) were undertaken on the 30th of April 2020. Please see the PRISMA flow diagram below (figure 2). Selection criteria: Studies of subjects with either suspected SARS-CoV-2 infection, known SARS-CoV-2 infection or known absence of infection, or those who were being screened for infection were included. Commercially available and research use rapid direct-to-PCR assays (without RNA extraction and purification reporting results within two hours) were included in the study. Data collection, extraction and analysis: Studies were screened independently, in duplicate with any disagreements resolved by discussion with a third author. Study characteristics were extracted by one author and checked by a second; extraction of study results and assessments of risk of bias and applicability were undertaken independently in duplicate. Where studies were not publicly available, sites that undertook in-service evaluations of rapid direct-to-PCR system were contacted and asked to supply anonymised datasets. Both reviewers independently performed data extraction and verification and calculated 2x2 contingency tables of the number of true positives, false positives, false negatives and true negatives. They resolved any disagreements by discussion and by review with the third reviewer. Main results: Twenty study cohorts were included (described in 14 study reports, including 4 unpublished reports), reporting results for 4593 samples (1391 with confirmed SARS-CoV-2). Studies were mainly from Europe and North America, and evaluated nine direct-to-PCR assays. Conclusions: This appears to be the first meta-analysis assessing the accuracy of rapid direct-to-PCR in the detection of SARS-CoV-2. In total, 10,957 unique records were identified and screened using a search string evaluation, 420 full-text reports and/or supplemental materials were assessed for inclusion. This resulted in 14 studies reporting 20 datasets with 4593 patient samples (1391 positive) included in the analysis. The overall agreement between the rapid direct RT-PCR and gold standard centralised laboratory RT-PCR was 97.10% with 93.60% positive percent agreement and 98.63% negative percent agreement. The Cohens kappa statistical coefficient k = 0.93, indicating an almost perfect agreement and Youden Index = 0.92. These results show that direct-to-PCR assay perform equivalently to the gold standard centralised laboratory RT-PCR systems for the detection of SARS-CoV-2.

A prospective diagnostic study to measure the accuracy of detection of SARS-CoV-2 Variants Of Concern (VOC) utilising a novel RT-PCR GENotyping algorithm in an In silico Evaluation (VOC-GENIE) (preprint)

Background SARS-CoV-2 variants of concern (VOCs) have been associated with higher rate of transmission, and evasion of immunisation and antibody therapeutics. Variant sequencing is widely utilized in the UK. However, only 0.5% (~650k) of the 133 million cumulative positive cases worldwide were sequenced (in GISAID) on 08 April 2021 with 97% from Europe and North America and only ~0.25% (~320k) were variant sequences. This may be due to the lack of availability, high cost, infrastructure and expert staff required for sequencing. Public health decisions based on a non-randomised sample of 0.5% of the population may be insufficiently powered, and subject to sampling bias and systematic error. In addition, sequencing is rarely available in situ in a clinically relevant timeframe and thus, is not currently compatible with diagnosis and treatment patient care pathways. Therefore, we investigated an alternative approach using polymerase chain reaction (PCR) genotyping to detect the key single nucleotide polymorphisms (SNPs) associated with increased transmission and immune evasion in SARS-CoV-2 variants. Methods We investigated the utility of SARS-CoV-2 SNP detection with a panel of PCR-genotyping assays in a large data set of 640,482 SARS-CoV-2 high quality, full length sequences using a prospective in silico trial design and explored the potential impact of rapid in situ variant testing on the COVID-19 diagnosis and treatment patient pathway. Results Five SNPs were selected by screening the published literature for a reported association with increased transmission and / or immune evasion. 344881 sequences contained one or more of the five SNPs. This algorithm of SNPs was found to be able to identify the four variants of concern (VOCs) and sequences containing the E484K and L452R escape mutations. Interpretation The in silico analysis suggest that the key mutations and variants of SARS-CoV-2 may be reliably detected using a focused algorithm of biologically relevant SNPs. This highlights the potential for rapid in situ PCR genotyping to compliment or replace sequencing or to be utilized instead of sequences in settings where sequencing is not feasible, accessible or affordable. Rapid detection of variants with in situ PCR genotyping may facilitate a more effective COVID-19 diagnosis and treatment patient pathway.

A Prospective Diagnostic Study to Measure the Accuracy of Detection of SARS-CoV-2 Variants Of Concern (VOC) Utilising a Novel RT-PCR GENotyping Algorithm in an in silico Evaluation (VOC-GENIE) (preprint)

Background: SARS-CoV-2 variants of concern (VOCs) have been associated with higher rate of transmission, and evasion of immunisation and antibody therapeutics. Variant sequencing is widely utilized in the UK. However, only 0.5% (~650k) of the 133 million cumulative positive cases worldwide were sequenced (in GISAID) on 08 April 2021 with 97% from Europe and North America and only ~0.25% (~320k) were variant sequences. This may be due to the lack of availability, high cost, infrastructure and expert staff required for sequencing. Public health decisions based on a non-randomised sample of 0.5% of the population may be insufficiently powered, and subject to sampling bias and systematic error. In addition, sequencing is rarely available in situ in a clinically relevant timeframe and thus, is not currently compatible with diagnosis and treatment patient care pathways. Therefore, we investigated an alternative approach using polymerase chain reaction (PCR) genotyping to detect the key single nucleotide polymorphisms (SNPs) associated with increased transmission and immune evasion in SARS-CoV-2 variants.Methods: We investigated the utility of SARS-CoV-2 SNP detection with a panel of PCR-genotyping assays in a large data set of 640,482 SARS-CoV-2 high quality, full length sequences using a prospective in silico trial design and explored the potential impact of rapid in situ variant testing on the COVID-19 diagnosis and treatment patient pathway. Results: Five SNPs were selected by screening the published literature for a reported association with increased transmission and / or immune evasion. 344881 sequences contained one or more of the five SNPs. This algorithm of SNPs was found to be able to identify the four variants of concern (VOCs) and sequences containing the E484K and L452R escape mutations.Interpretation: The in silico analysis suggest that the key mutations and variants of SARS-CoV-2 may be reliably detected using a focused algorithm of biologically relevant SNPs. This highlights the potential for rapid in situ PCR genotyping to compliment or replace sequencing or to be utilized instead of sequences in settings where sequencing is not feasible, accessible or affordable. Rapid detection of variants with in situ PCR genotyping may facilitate a more effective COVID-19 diagnosis and treatment patient pathway. Funding: The study was funded by Primer Design (UK), with kind contributions from all academic partners.Declaration of Interests: Stephen Kidd, Nick Cortes, Nathan Moore, Kate Templeton, Alex Richter and Alice Goring have no conflicting interests. R.A Trevor, Daryl Borley, Paul Oladimeji, Prachi Teltumbde, Seden Grippon, and Aida Sanchez-Bretano are employees of Novacyt group, which is a medical diagnostics company operating in the COVID-19 variant testing field. R.A Trevor has no additional direct conflicts but is a shareholder in a number of un-related private and public companies that do not operate in the COVID-19 or diagnostics field. Joanne Martin has no direct conflicts of interest. She is a principal investigator of a care home trial using Novacyt rapid testing and National Specialty Advisor for Pathology for NHS England and Improvement. She is a director and shareholder of Biomoti a drug delivery company and has a shareholding in Glyconics, a diagnostics company.