The landscape of somatic mutation in normal colorectal epithelial cells.

The colorectal adenoma-carcinoma sequence has provided a paradigmatic framework for understanding the successive somatic genetic changes and consequent clonal expansions that lead to cancer1. However, our understanding of the earliest phases of colorectal neoplastic changes-which may occur in morphologically normal tissue-is comparatively limited, as for most cancer types. Here we use whole-genome sequencing to analyse hundreds of normal crypts from 42 individuals. Signatures of multiple mutational processes were revealed; some of these were ubiquitous and continuous, whereas others were only found in some individuals, in some crypts or during certain periods of life. Probable driver mutations were present in around 1% of normal colorectal crypts in middle-aged individuals, indicating that adenomas and carcinomas are rare outcomes of a pervasive process of neoplastic change across morphologically normal colorectal epithelium. Colorectal cancers exhibit substantially increased mutational burdens relative to normal cells. Sequencing normal colorectal cells provides quantitative insights into the genomic and clonal evolution of cancer.

Routine Metagenomics Service for ICU Patients with Respiratory Infection.

Rationale: Respiratory metagenomics (RMg) needs evaluation in a pilot service setting to determine utility and inform implementation into routine clinical practice. Objectives: Feasibility, performance, and clinical impacts on antimicrobial prescribing and infection control were recorded during a pilot RMg service. Methods: RMg was performed on 128 samples from 87 patients with suspected lower respiratory tract infection (LRTI) on two general and one specialist respiratory ICUs at Guy's and St Thomas' NHS Foundation Trust, London. Measurements and Main Results: During the first 15 weeks, RMg provided same-day results for 110 samples (86%), with a median turnaround time of 6.7 hours (interquartile range = 6.1-7.5 h). RMg was 93% sensitive and 81% specific for clinically relevant pathogens compared with routine testing. Forty-eight percent of RMg results informed antimicrobial prescribing changes (22% escalation; 26% deescalation) with escalation based on speciation in 20 out of 24 cases and detection of acquired-resistance genes in 4 out of 24 cases. Fastidious or unexpected organisms were reported in 21 samples, including anaerobes (n = 12), Mycobacterium tuberculosis, Tropheryma whipplei, cytomegalovirus, and Legionella pneumophila ST1326, which was subsequently isolated from the bedside water outlet. Application to consecutive severe community-acquired LRTI cases identified Staphylococcus aureus (two with SCCmec and three with luk F/S virulence determinants), Streptococcus pyogenes (emm1-M1uk clone), S. dysgalactiae subspecies equisimilis (STG62647A), and Aspergillus fumigatus with multiple treatments and public health impacts. Conclusions: This pilot study illustrates the potential of RMg testing to provide benefits for antimicrobial treatment, infection control, and public health when provided in a real-world critical care setting. Multicenter studies are now required to inform future translation into routine service.

Real-Time Whole Genome Sequencing to Guide Patient-Tailored Therapy of Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

The management of coronavirus disease 2019 has become more complex due to the expansion of available therapies. The presence of severe acute respiratory syndrome coronavirus 2 variants and mutations further complicates treatment due to their differing susceptibilities to therapies. Here we outline the use of real-time whole genome sequencing to detect persistent infection, evaluate for mutations confering resistance to treatments, and guide treatment decisions.

Variovorax durovernensis sp. nov., a novel species isolated from an infected prosthetic aortic graft in a human.

A novel bacterial strain, GSTT-20T was isolated from an infected, prosthetic endovascular graft explanted from a shepherd in London, United Kingdom. This strain was an aerobic, catalase-positive, oxidase-negative, Gram-stain-negative, motile, curved rod. It grew on blood agar, chocolate agar and MacConkey agar incubated at 37 °C in an aerobic environment after 48 h, appearing as yellow, mucoid colonies. Analysis of the complete 16S rRNA gene sequence showed closest similarity to Variovorax paradoxus with 99.6 % identity and Variovorax boronicumulans with 99.5 % identity. Phylogenetic analysis of the 16S rRNA gene sequence and phylogenomic analysis of single nucleotide polymorphisms within 1530 core genes showed GSTT-20T forms a distinct lineage in the genus Variovorax of the family Comamonadaceae. In silico DNA-DNA hybridization assays against GSTT-20T were estimated at 32.1 % for V. boronicumulans and 31.9 % for V. paradoxus. Genome similarity based on average nucleotide identity was 87.50 % when comparing GSTT-20T to V. paradoxus. Based on these results, the strain represented a novel species for which the name Variovorax durovernensis sp. nov. was proposed. The type strain is GSTT-20T (NCTC 14621T=CECT 30390T).

Somatic mutations reveal asymmetric cellular dynamics in the early human embryo.

Somatic cells acquire mutations throughout the course of an individual's life. Mutations occurring early in embryogenesis are often present in a substantial proportion of, but not all, cells in postnatal humans and thus have particular characteristics and effects. Depending on their location in the genome and the proportion of cells they are present in, these mosaic mutations can cause a wide range of genetic disease syndromes and predispose carriers to cancer. They have a high chance of being transmitted to offspring as de novo germline mutations and, in principle, can provide insights into early human embryonic cell lineages and their contributions to adult tissues. Although it is known that gross chromosomal abnormalities are remarkably common in early human embryos, our understanding of early embryonic somatic mutations is very limited. Here we use whole-genome sequences of normal blood from 241 adults to identify 163 early embryonic mutations. We estimate that approximately three base substitution mutations occur per cell per cell-doubling event in early human embryogenesis and these are mainly attributable to two known mutational signatures. We used the mutations to reconstruct developmental lineages of adult cells and demonstrate that the two daughter cells of many early embryonic cell-doubling events contribute asymmetrically to adult blood at an approximately 2:1 ratio. This study therefore provides insights into the mutation rates, mutational processes and developmental outcomes of cell dynamics that operate during early human embryogenesis.

Analysis of pir gene expression across the Plasmodium life cycle.

BACKGROUND: Plasmodium interspersed repeat (pir) is the largest multigene family in the genomes of most Plasmodium species. A variety of functions for the PIR proteins which they encode have been proposed, including antigenic variation, immune evasion, sequestration and rosetting. However, direct evidence for these is lacking. The repetitive nature of the family has made it difficult to determine function experimentally. However, there has been some success in using gene expression studies to suggest roles for some members in virulence and chronic infection. METHODS: Here pir gene expression was examined across the life cycle of Plasmodium berghei using publicly available RNAseq data-sets, and at high resolution in the intraerythrocytic development cycle using new data from Plasmodium chabaudi. RESULTS: Expression of pir genes is greatest in stages of the parasite which invade and reside in red blood cells. The marked exception is that liver merozoites and male gametocytes produce a very large number of pir gene transcripts, notably compared to female gametocytes, which produce relatively few. Within the asexual blood stages different subfamilies peak at different times, suggesting further functional distinctions. Representing a subfamily of its own, the highly conserved ancestral pir gene warrants further investigation due to its potential tractability for functional investigation. It is highly transcribed in multiple life cycle stages and across most studied Plasmodium species and thus is likely to play an important role in parasite biology. CONCLUSIONS: The identification of distinct expression patterns for different pir genes and subfamilies is likely to provide a basis for the design of future experiments to uncover their function.

Unified metagenomic method for rapid detection of microorganisms in clinical samples.

BACKGROUND: Clinical metagenomics involves the genomic sequencing of all microorganisms in clinical samples ideally after depletion of human DNA to increase sensitivity and reduce turnaround times. Current human DNA depletion methods preferentially preserve either DNA or RNA containing microbes, but not both simultaneously. Here we describe and present data using a practical and rapid mechanical host-depletion method allowing simultaneous detection of RNA and DNA microorganisms linked with nanopore sequencing. METHODS: The human cells from respiratory samples are lysed mechanically using 1.4 mm zirconium-silicate spheres and the human DNA is depleted using a nonspecific endonuclease. The RNA is converted to dsDNA to allow the simultaneous sequencing of DNA and RNA. RESULTS: The method decreases human DNA concentration by a median of eight Ct values while detecting a broad range of RNA & DNA viruses, bacteria, including atypical pathogens (Legionella, Chlamydia, Mycoplasma) and fungi (Candida, Pneumocystis, Aspergillus). The first automated reports are generated after 30 min sequencing from a 7 h end-to-end workflow. Sensitivity and specificity for bacterial detection are 90% and 100%, respectively, and viral detection are 92% and 100% after 2 h of sequencing. Prospective validation on 33 consecutive lower respiratory tract samples from ventilated patients with suspected pneumonia shows 60% concordance with routine testing, detection of additional pathogens in 21% of samples and pathogen genomic assembly achieve for 42% of viruses and 33% of bacteria. CONCLUSIONS: Although further workflow refinement and validation on samples containing a broader range of pathogens is required, it holds promise as a clinically deployable workflow suitable for evaluation in routine microbiology laboratories.

Metagenomics is the analysis of genetic material from microbes such as bacteria and viruses in a sample. There are limitations with existing metagenomics methods, such as not being able to detect the full range of microbes present in a sample. This paper introduces an approach that identifies multiple types of microbes. This is accomplished through the mechanical disruption of human cells, which allows for an effective depletion of human genetic material. Our method demonstrates encouraging preliminary results within a 7 h process, achieving good sensitivity for the detection of bacteria and viruses. We demonstrate the identification of relevant microbes in samples from patients with respiratory infections. This technique holds promise for adoption in clinical settings, potentially enhancing our ability to diagnose respiratory infections quickly.

Feasibility and clinical utility of local rapid Nanopore influenza A virus whole genome sequencing for integrated outbreak management, genotypic resistance detection and timely surveillance.

Rapid respiratory viral whole genome sequencing (WGS) in a clinical setting can inform real-time outbreak and patient treatment decisions, but the feasibility and clinical utility of influenza A virus (IAV) WGS using Nanopore technology has not been demonstrated. A 24 h turnaround Nanopore IAV WGS protocol was performed on 128 reverse transcriptase PCR IAV-positive nasopharyngeal samples taken over seven weeks of the 2022-2023 winter influenza season, including 25 from patients with nosocomial IAV infections and 102 from patients attending the Emergency Department. WGS results were reviewed collectively alongside clinical details for interpretation and reported to clinical teams. All eight segments of the IAV genome were recovered for 97/128 samples (75.8 %) and the haemagglutinin gene for 117/128 samples (91.4 %). Infection prevention and control identified nosocomial IAV infections in 19 patients across five wards. IAV WGS revealed two separate clusters on one ward and excluded transmission across different wards with contemporaneous outbreaks. IAV WGS also identified neuraminidase inhibitor resistance in a persistently infected patient and excluded avian influenza in a sample taken from an immunosuppressed patient with a history of travel to Singapore which had failed PCR subtyping. Accurate IAV genomes can be generated in 24 h using a Nanopore protocol accessible to any laboratory with SARS-CoV-2 Nanopore sequencing capacity. In addition to replicating reference laboratory surveillance results, IAV WGS can identify antiviral resistance and exclude avian influenza. IAV WGS also informs management of nosocomial outbreaks, though molecular and clinical epidemiology were concordant in this study, limiting the impact on decision-making.

A framework for real-time monitoring, analysis and adaptive sampling of viral amplicon nanopore sequencing.

The ongoing SARS-CoV-2 pandemic demonstrates the utility of real-time sequence analysis in monitoring and surveillance of pathogens. However, cost-effective sequencing requires that samples be PCR amplified and multiplexed via barcoding onto a single flow cell, resulting in challenges with maximising and balancing coverage for each sample. To address this, we developed a real-time analysis pipeline to maximise flow cell performance and optimise sequencing time and costs for any amplicon based sequencing. We extended our nanopore analysis platform MinoTour to incorporate ARTIC network bioinformatics analysis pipelines. MinoTour predicts which samples will reach sufficient coverage for downstream analysis and runs the ARTIC networks Medaka pipeline once sufficient coverage has been reached. We show that stopping a viral sequencing run earlier, at the point that sufficient data has become available, has no negative effect on subsequent down-stream analysis. A separate tool, SwordFish, is used to automate adaptive sampling on Nanopore sequencers during the sequencing run. This enables normalisation of coverage both within (amplicons) and between samples (barcodes) on barcoded sequencing runs. We show that this process enriches under-represented samples and amplicons in a library as well as reducing the time taken to obtain complete genomes without affecting the consensus sequence.

The ongoing Streptococcus pyogenes (Group A Streptococcus) outbreak in London, United Kingdom, in December 2022: a molecular epidemiology study.

OBJECTIVES: Epidemiological and whole-genome sequencing analysis of an ongoing outbreak of Streptococcus pyogenes (Group A Streptococcus) in London (United Kingdom). METHODS: Prospective identification of Group A Streptococcus cases from a diagnostic laboratory serving central and south London between 27 November and 10 December 2022. Case notes were reviewed and isolates were retrieved. Case numbers were compared with the previous 5 years. Whole-genome sequencing was performed with long-read, nanopore technology for emm typing and identification of superantigen genes. Associations of pathogen-related factors with an invasive disease were assessed by single-variable and multi-variable logistic regression. RESULTS: Case numbers began increasing in October 2022 from a baseline of 2.0 cases per day, and in December 2022, the average daily case numbers reached 10.8 cases per day, four-fold the number usually seen in winter. A total of 113 cases were identified during the prospective study period. Three quarters (86/113, 76%) were paediatric cases, including 2 deaths. Of 113 cases, 11 (10%) were invasive. In total, 56 isolates were successfully sequenced, including 10 of 11 (91%) invasive isolates. The emm12 (33/56, 59%) and emm1 (9/56, 16%) types were predominant, with 7 of 9 (78%) emm1 isolates being from the M1uk clone. The majority of invasive isolates had superantigen genes spea (7/10, 70%) and spej (8/10, 80%), whereas, in non-invasive isolates, these superantigen genes were found less frequently (spea: 5/46, 11% and spej: 7/46, 15%). By multivariable analysis of pathogen-related factors, spea (OR 8.9, CI 1.4-57, p 0.020) and spej (OR 12, CI 1.8-78, p 0.011) were associated with invasive disease. CONCLUSIONS: emm12 and emm1 types predominate in the ongoing outbreak, which mainly affects children. In this outbreak, the spea and spej superantigen genes are associated with the severity of presentation.

Tissue macrophages and interferon-gamma signalling control blood-stage Plasmodium chabaudi infections derived from mosquito-transmitted parasites.

Natural infection with Plasmodium parasites, the causative agents of malaria, occurs via mosquito vectors. However, most of our knowledge of the immune response to the blood stages of Plasmodium is from infections initiated by injection of serially blood-passaged infected red blood cells, resulting in an incomplete life cycle in the mammalian host. Vector transmission of the rodent malaria parasite, Plasmodium chabaudi chabaudi AS has been shown to give rise to a more attenuated blood-stage infection in C57Bl/6J mice, when compared to infections initiated with serially blood-passaged P. chabaudi-infected red blood cells. In mouse models, the host immune response induced by parasites derived from natural mosquito transmission is likely to more closely resemble the immune responses to Plasmodium infections in humans. It is therefore important to determine how the host response differs between the two types of infections. As the spleen is considered to be a major contributor to the protective host response to P. chabaudi, we carried out a comparative transcriptomic analysis of the splenic response to recently mosquito-transmitted and serially blood-passaged parasites in C57Bl/6J mice. The attenuated infection arising from recently mosquito-transmitted parasites is characterised by an earlier and stronger myeloid- and IFNγ-related response. Analyses of spleen lysates from the two infections similarly showed stronger or earlier inflammatory cytokine and chemokine production in the recently mosquito-transmitted blood-stage infections. Furthermore, tissue macrophages, including red pulp macrophages, and IFNγ-signalling in myeloid cells, are required for the early control of P. chabaudi recently mosquito-transmitted parasites, thus contributing to the attenuation of mosquito-transmitted infections. The molecules responsible for this early activation response to recently-transmitted blood-stage parasites in mice would be important to identify, as they may help to elucidate the nature of the initial interactions between blood-stage parasites and the host immune system in naturally transmitted malaria.

Evaluating the potential for respiratory metagenomics to improve treatment of secondary infection and detection of nosocomial transmission on expanded COVID-19 intensive care units.

BACKGROUND: Clinical metagenomics (CMg) has the potential to be translated from a research tool into routine service to improve antimicrobial treatment and infection control decisions. The SARS-CoV-2 pandemic provides added impetus to realise these benefits, given the increased risk of secondary infection and nosocomial transmission of multi-drug-resistant (MDR) pathogens linked with the expansion of critical care capacity. METHODS: CMg using nanopore sequencing was evaluated in a proof-of-concept study on 43 respiratory samples from 34 intubated patients across seven intensive care units (ICUs) over a 9-week period during the first COVID-19 pandemic wave. RESULTS: An 8-h CMg workflow was 92% sensitive (95% CI, 75-99%) and 82% specific (95% CI, 57-96%) for bacterial identification based on culture-positive and culture-negative samples, respectively. CMg sequencing reported the presence or absence of ß-lactam-resistant genes carried by Enterobacterales that would modify the initial guideline-recommended antibiotics in every case. CMg was also 100% concordant with quantitative PCR for detecting Aspergillus fumigatus from 4 positive and 39 negative samples. Molecular typing using 24-h sequencing data identified an MDR-K. pneumoniae ST307 outbreak involving 4 patients and an MDR-C. striatum outbreak involving 14 patients across three ICUs. CONCLUSION: CMg testing provides accurate pathogen detection and antibiotic resistance prediction in a same-day laboratory workflow, with assembled genomes available the next day for genomic surveillance. The provision of this technology in a service setting could fundamentally change the multi-disciplinary team approach to managing ICU infections. The potential to improve the initial targeted treatment and rapidly detect unsuspected outbreaks of MDR-pathogens justifies further expedited clinical assessment of CMg.