Long-term SARS-CoV-2 infections in immunodeficient patients are an important source of variation for the virus but are understudied. Many case studies have been published which describe one or a small number of long-term infected individuals but no study has combined these sequences into a cohesive dataset. This work aims to rectify this and study the genomics of this patient group through a combination of literature searches as well as identifying new case series directly from the COG-UK dataset. The spike gene receptor binding domain (RBD) and N-terminal domains (NTD) were identified as mutation hotspots. Numerous mutations associated with variants of concern were observed to emerge recurrently. Additionally a mutation in the envelope gene, - T30I was determined to be the most recurrent frequently occurring mutation arising in persistent infections. A high proportion of recurrent mutations in immunodeficient individuals are associated with ACE2 affinity, immune escape, or viral packaging optimisation. There is an apparent selective pressure for mutations which aid intra-host transmission or persistence which are often different to mutations which aid inter-host transmission, although the fact that multiple recurrent de novo mutations are considered defining for variants of concern strongly indicates that this potential source of novel variants should not be discounted.

Rapid, sensitive, accurate and portable diagnostics are a mainstay of modern medicine. Tuberculosis is a disease that has been with us since time immemorial and, despite the fact that it can be treated and cured, it still remains the world's biggest infectious killer, taking the lives of millions annually. There have been important developments in the diagnostic devices for tuberculosis however, these are often prone to error, expensive, lack the necessary sensitivity or accuracy and, crucially, not sufficiently portable and thus not applicable in the remote, rural areas, where they are most needed. Modern solutions have been emerging in the past decade, seeking to overcome many of the inhibiting issues in this field by utilising recent advances in molecular biology, genetics and sequencing or even completely ‘reinventing the wheel’, by developing novel and unprecedented diagnostic techniques. In this mini review, the issues and challenges arising from the historical methods of diagnosing tuberculosis are discussed, followed by outlaying their particular lack of appropriateness for regions of the world where tuberculosis still remains endemic. Subsequently, more recent developments of new methods and technological advancements as ‘modern weapons’ in the battle to defeat this disease and associated challenges are reviewed, and finally an outlook is presented, highlighting the future of the modern solutions under development, which are envisioned to lay the platform for improvements in delivering timely intervention, reduce immense expense and burden on healthcare systems worldwide, while saving millions of lives and eventually, may enable the eradication of this ancient disease.

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.

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 otherwise-healthy individuals; 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.We therefore 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 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 2020-21 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.

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.

Genome sequencing is a powerful tool for identifying SARS-CoV-2 variant lineages, however there can be limitations due to sequence drop-out when used to identify specific key mutations. Recently, Thermo Fisher Scientific have developed genotyping assays to help bridge the gap between testing capacity and sequencing capability to generate real-time genotyping results based on specific variants. Over a 6-week period during the months of April and May 2021, we set out to assess the Thermo Fisher TaqMan Mutation Panel Genotyping Assay, initially for three mutations of concern and then an additional two mutations of concern, against SARS-CoV-2 positive clinical samples and the corresponding COG-UK sequencing data. We demonstrate that genotyping is a powerful in-depth technique for identifying specific mutations, an excellent complement to genome sequencing and has real clinical health value potential allowing laboratories to report and action variants of concern much quicker.