Wellcome Open Research

Western blotting has been widely used for decades, yet figure preparation remains unstandardized, error-prone and difficult to audit. Gelato is a free Fiji/ImageJ plugin that streamlines intuitive figure preparation, logs processing coordinates, provides a straightforward audit platform andenables reproduction from raw images. By making these capabilities simple and accessible, Gelato facilitates robust Western blot reporting, reducing a substantial burden on authors and reviewers while strengthening scientific rigor.

Rationale: Plasma proteins are used as biomarkers of inflammation and injury in acute respiratory distress syndrome (ARDS). Whether they vary with body size before ARDS is not well established. Objectives: To determine whether ARDS biomarkers vary with body mass index in adults without acute illness. Methods: We analyzed 8,680 ambulatory adults in the All of Us Research Program with proteomics covering 5,420 protein assays. We grouped 35 proteins associated with ARDS risk or mortality into five compartments by the pathophysiological role assigned in a published systematic review, registered in advance. We modeled body mass index as a restricted cubic spline and reported the difference in each protein between 35 and 25 kg/m2 in standard deviations, adjusted for age, sex, smoking, thirteen chronic conditions and ten genetic principal components. Each estimate was ranked against all 5,420 protein assays. Results: Median age was 50 years, and median body mass index was 27.2 kg/m2. Associations with body mass index across all assays were predominantly positive with a median of 0.065 standard deviations. All four epithelial proteins were lower at higher body mass index, each below at least 92% of assays. Soluble receptor for advanced glycation end products was lower by 0.350 standard deviations and surfactant protein A2, surfactant protein D and club cell secretory protein 16 were lower by 0.081 to 0.084 standard deviations. Interleukin-6 and E-selectin were 0.534 and 0.465 standard deviations higher, above 99% of assays. Differences across body mass index extended to lung enriched proteins outside the prespecified panel. Compartments accounted for 49% of the variance in the 35 protein estimates, against 10% under random reassignment (P < 0.001). At fixed body mass index, type 2 diabetes, hypertension and dyslipidemia showed smaller differences in ARDS biomarkers and were not associated with lower epithelial proteins. Conclusions: Plasma proteins used as biomarkers of inflammation and injury in ARDS vary with body size even in the absence of acute illness. Differences during ARDS may not be attributable to injury alone and may be misinterpreted in patients with obesity due to antecedent variation.

* There are many ways of analyzing the progress of an epidemic, but when it comes to short term forecasting, it is very hard to beat a simple time series regression model. These are good at allowing for the noise in day to day observations, extracting the trend and projecting it forward. * Our regression models are designed to exploit this, using the daily statistics released by PHE and NHSE. These strongly suggest that the tide has turned and that taking one day with the next, the national figures for deaths from this virus will now fall back noticeably, easing the pressure on the NHS and its staff. * There is still a huge range of uncertainty associated with any forecast. The model is currently predicting a total of 113,000 admissions to UK hospitals by the end of April and that 19,000 people will die from the virus in English hospitals by then. There is a 1 in 20 chance that the mortality figures could flatten out more quickly, with around 1,000 more deaths occurring by the end of April. However, there is the same risk that this figure continues to mount, rising to a total of 24,000 by the end of the month. On current trends, the number of deaths in the UK is likely to be 10% higher than the number in England. * Longer term, the impact of the virus will depend critically upon the likely relaxation of the current government strategy of suppression.

ObjectivesTo audit implementation of a local protocol for the treatment of vitamin D deficiency (VDD) among patients hospitalized for Coronavirus Disease 2019 (COVID-19), including an assessment of the prevalence of VDD in these patients, and of potential associations with disease severity and fatality. DesignThis was not a study or clinical trial, but rather a retrospective interim audit (Newcastle-upon-Tyne Hospitals Registration No. 10075) of a local clinical care pathway for hospitalized patients with COVID-19-related illness. The Information (Caldicott) Guardian permitted these data to be shared beyond the confines of our institution. SettingA large tertiary academic NHS Foundation Trust in the North East of England, UK, providing care to COVID-19 patients. ParticipantsOne hundred thirty-four hospitalized patients with documented COVID-19 infection. Main outcome measuresAdherence to local investigation and treatment protocol; prevalence of VDD, and relationship of baseline serum 25(OH)D with markers of COVID-19 severity and inpatient fatality versus recovery. Results55.8% of eligible patients received Colecalciferol replacement, albeit not always loaded as rapidly as our protocol suggested, and no cases of new hypercalcaemia occurred following treatment. Patients admitted to ITU were younger than those managed on medical wards (61.1 years {+/-} 11.8 vs. 76.4 years {+/-} 14.9, p<0.001), with greater prevalence of hypertension, and higher baseline respiratory rate, National Early Warning Score-2 and C-reactive protein level. While mean serum 25(OH)D levels were comparable [i.e. ITU: 33.5 nmol/L {+/-} 16.8 vs. Non-ITU: 48.1 nmol/L {+/-} 38.2, mean difference for Ln-transformed-25(OH)D: 0.14, 95% Confidence Interval (CI) (-0.15, 0.41), p=0.3], only 19% of ITU patients had 25(OH)D levels greater than 50 nmol/L vs. 39.1% of non-ITU patients (p=0.02). However, we found no association with fatality, potentially due to small sample size, limitations of no-trial data and, potentially, the prompt diagnosis and treatment of VDD. ConclusionsSubject to the inherent limitations of observational (non-trial) audit data, analysed retrospectively, we found that patients requiring ITU admission were more frequently vitamin D deficient than those managed on medical wards, despite being significantly younger. Larger prospective studies and/or clinical trials are needed to elucidate the role of vitamin D as a preventive and/or therapeutic strategy for mitigating the effects of COVID-19 infection in patients with VDD. What is already known on this topicO_LIVitamin D deficiency (VDD) is associated with increased risk for acute respiratory tract infections C_LIO_LIA link between VDD and severity of COVID-19 pathophysiology has been proposed C_LIO_LITwo recent (non-peer-reviewed) studies have reported crude associations between VDD in defined geographic populations and COVID-19 severity and mortality C_LI What this study addsO_LIThese data do not arise from a clinical study; rather from an audit of a local replacement protocol for VDD in COVID-19 inpatients in a large UK centre, which found a significantly higher prevalence of VDD among ITU patients compared to non-ITU patients, despite the ITU patients being significantly younger. C_LIO_LIPrompt treatment of VDD following a local protocol did not result in any adverse events, such as hypercalcaemia. C_LIO_LIWhilst by no means conclusive, these data suggest an important association between VDD and COVID-19 severity; hence our report of interim findings in advance of achieving completed outcomes (fatality vs. recovery) for all patients. C_LIO_LIThere is an urgent need for larger studies exploring vitamin D as a potential preventative measure and/or treatment of Covid-19-related illness among individuals with VDD. C_LI

During the current COVID-19 pandemic, it has been suggested that BAME background patients may be disproportionately affected compared to White but few detailed data are available. We took advantage of near real-time hospital data access and analysis pipelines to look at the impact of ethnicity in 1200 consecutive patients admitted between 1st March 2020 and 12th May 2020 to Kings College Hospital NHS Trust in London (UK). Our key findings are firstly that BAME patients are significantly younger and have different co-morbidity profiles than White individuals. Secondly, there is no significant independent effect of ethnicity on severe outcomes (death or ITU admission) within 14-days of symptom onset, after adjustment for age, sex and comorbidities.

IntroductionSince the start of the pandemic SARS-CoV-2 infection has most commonly been confirmed using reverse transcriptase polymerase chain reaction (RT-PCR), with results translated into a binary positive/negative outcomes. Previous studies have found that there is additional useful information in the level of the Cycle threshold (Ct value) of positive cases. Here we characterise variation in Ct values as a proxy for viral loads in more than 3 million test-positive COVID-19 cases in England with the aim of better quantifying the utility of such data. MethodsWe used individual N gene Ct values from symptomatic PCR positive (with Ct value less than 30) Pillar 2 cases in England who self-reported the date of symptom onset, and for whom age, reinfection status, variant status, and the number of vaccines received was available. Those with a positive test result more than 6 days after their reported symptom onset were excluded to mitigate the potential impact of recall bias. We used a generalised additive model, to estimate Ct values empirical mean Ct values for each strata of interest independently as well as to predict Ct values using a model that adjusted for a range of demographic and epidemiological covariates jointly. We present empirical Ct values and compare them to predicted mean Ct values. ResultsWe found that mean Ct values varied by vaccine status, and reinfection status with the number of vaccine doses having little apparent effect. Modelling Ct values as a smooth function of time since onset and other variables struggled to reproduce the individual variation in the data but did match the population-level variation over time relatively well with this being apparently dominated by large differences between variants. Other variation over time was also captured to some degree though their remained several periods where the model could not capture the empirical means with a potential explanation being epidemic phase bias. ConclusionsAnalysing a large dataset of routine Ct values from symptomatic COVID-19 cases in England we found variation based on time since symptom onset, vaccine status, age, and variant. Ct values were highest 1-3 days after symptom onset and differed most due to variant status. We found no clear correlation between previously estimated differences in intrinsic transmissibility and Ct values indicating that this is potentially mediated at least partly by factors other than viral load as estimated using Ct values. We found evidence that a model adjusting for a range of covariates could explain some of the population-level variation over time but systematically underestimated Ct values when incidence was increasing, and overestimated them when incidence was decreasing. This indicates the utility of Ct values from this data source as a tool for surveillance, potentially avoiding some of the biases of aggregated positive counts.

Confirmed Case Data have been widely cited during the current COVID-19 pandemic as an estimate of the spread of the SARS-CoV-2 virus. However, their central role in media, official reports and decision-making may be undeserved and misleading. Previously published Infection Fatality Rates were weighted by age structure in the 50 countries with more reported deaths to obtain country-specific rates. For each country, the number of infections up to the Infection Date (23 days ago = Incubation Period + Onset to Death period) and the present percentage of immune population were estimated using Infection Fatality Rate, the number of reported deaths (which is less prone to undersampling), and projecting back to Infection Date. We then estimated a Detection Index for each country as the percentage of estimated infections that confirmed cases represent. Assuming that detection remains constant after Infection Date, we estimated the number of deaths and the estimated percentage of the population of each country expected to be immune up to 23 days into the future. Estimated Infection Fatality Rates are higher in Europe. In most countries, confirmed cases currently represent less than 30% of estimated infections on Infection Date, and this value decreases with time. Countries with flat curves throughout the pandemic show the lowest immunity percentages and these values seem unlikely to change in the near future, suggesting that they remain vulnerable to new outbreaks. Estimates for some countries with low Infection Fatality Rates suggest a still steep increase in the number of casualties in the next three weeks. Countries that did not control initial outbreaks seem to have reached higher immunity percentages, although mostly still under 5%. We provide the code to monitor the trajectories of these estimates in 178 countries throughout the COVID-19 pandemic.

Background: Sexually transmitted infections (STIs) and vaginal dysbiosis during pregnancy are associated with adverse pregnancy outcomes. Mycoplasma genitalium is the most recent STI implicated but evidence remains limited. The objectives of this study were to investigate 1) the association between M. genitalium infection during pregnancy and gestational age at delivery, preterm birth, miscarriage or stillbirth, and low birth weight and 2) the interaction with vaginal dysbiosis. Methods: We conducted a prospective cohort study in East London, South Africa. We enrolled pregnant women at gestational age <27 weeks, confirmed by ultrasound. We tested vaginal samples using nucleic acid amplification tests for M. genitalium, Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, other genital mycoplasmas and Candida spp. We defined vaginal dysbiosis using Gram-stain criteria as a Nugent score 4-10. We used quantile regression to compare the outcome in women with and without M. genitalium across the gestational age distribution, adjusting for prespecified sociodemographic and clinical characteristics and co-occurring organisms. Results: From April 1, 2021 to August 29, 2023, we enrolled 603 women, followed up 584 and obtained pregnancy outcomes for 560 (93%). Median age was 28 years (interquartile range, IQR 24, 33) and 27% of women were living with HIV. M. genitalium was detected in 44/584 (8%, 95% CI 6, 10%) and vaginal dysbiosis in 375/584 (64%) of women. Median gestational age at delivery was 39 weeks +0 days (IQR 37+4, 40+1) in women with and 39 weeks +0 days (37+4, 40+0) in those without M. genitalium. In multivariable models, associations were not observed for any adverse birth outcomes. There was no interaction between M. genitalium and vaginal dysbiosis. Discussion: M. genitalium in pregnancy was not associated with earlier gestational age at delivery or with other adverse birth outcomes. These findings do not support routine testing and treatment for M. genitalium in pregnancy.

This study examined the progress of MRC-funded researchers, with a focus on their success in securing subsequent research income. We report here results from compiling details of the research income of researchers that completed an MRC early career award between 2011 and 2015. Depending on the funding scheme 50-59 per cent of researchers spent on average more than 100,000 GBP per year from new grants secured within three years of finishing their MRC award. In each scheme around 30 per cent of researchers secured less than 50,000 GBP per year research funding in the three years following their MRC award. We apply the same approach to an analysis of progression for the MRC cadre of intramurally-supported Programme Leader Track researchers, and a sample of MRC research grant holders. We also supplement our results with a small number of interviews with researchers that held MRC New Investigator Research Grants. We suggest that these data confirm reasonably good progression rates, with MRC early career schemes demonstrably effective in supporting research careers. They confirm there is a bottleneck with some researchers taking several years to establish their research career, and that expansion of the research base will need balanced new investment across schemes and stages and cannot be driven through growth in early career schemes alone.

Microscopes are essential tools for discoveries on a scale invisible to the unaided human eye. The development of immuno-fluorescence followed by molecular biology techniques and fluorescent fusion proteins have revolutionised the use of optical microscopy in bioscience. The quality of the data produced is dependent upon the sample, its preparation and the instrument used. However, instruments can degrade over time without easily visible changes to the produced images and, in turn, negatively impacts results. By testing instruments and doing comparisons between results over time and between different instruments, problems can be highlighted and corrective action can be taken. Using small fluorescent beads the point spread function (PSF) of the microscope can be recorded and the image resolution measured. Beads were prepared in a concentration matched to the field of view size and dried onto coverslips and mounted on slides. The beads were then imaged as 3D Z-stacks of sufficient size to fully enclose the PSF of the system. This data was uploaded to OMERO and processed using OMERO-metrics, an OMERO plugin developed for this purpose. This paper summarizes the development of workflows and protocols to enable this process, presents the results obtained and demonstrates the detection of significant instrument issues.

Long-read RNA sequencing enables isoform-resolved transcriptomics, but library preparation introduces systematic biases that shape biological interpretation. We benchmarked Oxford Nanopores two protocols--PCR-cDNA and direct RNA--using SKMM2 myeloma cells stimulated with interleukin-6 (IL-6) and ERCC synthetic spike-ins. Direct RNA produced longer, higher-quality reads and more high-confidence isoforms, but showed pronounced 5' coverage loss. PCR-cDNA yielded shorter fragments with 3' underrepresentation, detecting more low-abundance transcripts at reduced confidence. Protocol-specific biases had major consequences: differential expression analysis revealed limited overlap in IL-6-responsive genes, and pathway enrichment was broader in direct RNA. At the isoform level, differential transcript usage was almost entirely protocol-specific, with case studies (e.g. RPL22L1, GRB2, RNF220) illustrating concordance and divergence. ERCC controls confirmed these biases as technical rather than biological. Together, our results show that while both methods provide accurate gene-level quantification, transcript-level conclusions depend critically on protocol choice, highlighting the need for careful selection in long-read transcriptomics.

IntroductionInfectious intestinal disease (IID) is a significant public health concern, with an estimated 17 million cases occurring annually in the UK. However, only a small proportion of cases in the community present to primary health care, and those that do may not be tested for the causative pathogen. The third study of IID in the community (IID3 study) will provide updated estimates of the incidence and aetiology of IID in the UK. This protocol describes the laboratory methods employed by the IID3 study in primary diagnostic and reference laboratories. Methods and analysisStool specimens obtained from symptomatic subjects enrolled in two cohorts recruited prospectively through Primary Care in the UK are tested for 21 pathogens using PCR-based methods, in two separate workflows at Liverpool Clinical Laboratories. The first workflow comprises EntericBio DX, Viral and Clostridium difficile assays (Serosep, Crawley, UK); and the second a panel of assays for detection of Clostridium perfringens, Aeromonas spp., Cyclospora cayetanensis and diarrhoeagenic Escherichia coli based on 3base(R) Technology (Genetic Signatures, Newtown, Australia). Specimens testing positive for bacterial pathogens are subjected to culture. Specimens positive for, and isolates of Salmonella, Campylobacter, Yersinia, Vibrio and Shigella species and diarrhoeagenic E. coli are referred to the Gastrointestinal Bacteria Reference Unit, UK Health Security Agency (UKHSA) for whole genome sequencing and antimicrobial susceptibility testing. The parasites Cryptosporidium, Giardia and Cyclospora are referred for genotyping to the national Cryptosporidium Reference Unit, Public Health Wales. Norovirus positive faecal specimens are referred for Sanger Sequencing-based genotyping to the Enteric Virus Unit, UKHSA. Ethics and disseminationFavourable ethical opinion was granted on the 10th of August 2022 by the East Midlands - Nottingham 1 Research Ethics Committee (IRAS ID 314268, REC Reference 22/EM/0130). The results will be disseminated to key stakeholders including the Food Standards Agency, participating general practices and to the academic community through publications and conference presentations. A real-time breakdown of pathogens identified in faecal samples could be viewed openly on the study Dashboard throughout the surveillance period. Article SummaryO_ST_ABSStrengths and limitations of this studyC_ST_ABS- A total of 5439 stool samples underwent molecular testing for a total of 21 organisms at the Liverpool Clinical Laboratories (LCL), with detailed pathogen characterisation at national reference laboratories. - A commercial Genetic Signatures assay was evaluated in a routine diagnostic setting using commercially-provided pathogen controls, reference isolates and clinical samples, enabling the inclusion of Aeromonas and ETEC PCR targets into the diagnostic panel. - Real-time referrals of PCR-reactive samples were made to UK Health Security Agency (UKHSA) regardless of bacterial culture results, and to Public Health Wales (PHW). - Norovirus PCR-reactive samples were referred to the Enteric Virus Unit at UKHSA for genotyping ahead of Norovirus becoming a notifiable infection on the 6th of April 2025, adding valuable information on Norovirus genotypes circulating in the general population. - A Biobank has been created for residual faecal specimens, forming a rich resource for future studies. - A major challenge was the lack of a harmonised UK-wide digital referral and reporting system, resulting in an inflated manual reporting workload for staff at LCL.

Several studies suggest a lower prevalence of smoking than expected among adults with coronavirus disease (COVID-19). We conducted logistic regression analyses of the UK Biobank prospective study of 0.5 million adults followed for an average of 11 years. Compared to women, men were more likely to be tested and to test positive. In sex-stratified analyses, current smokers had higher adjusted Odds Ratios (OR) for being tested (male OR 1.60, 95%CI 1.32-1.95 and female OR 1.50,1.21-.1.86). Current smokers were more slightly more likely than never smokers to test positive for COVID-19. Further examination of smoking as a risk factor for COVID-19 is required. These must take into account reverse causality, where smokers quit to avoid disease as well as prior diseases.

COVID-19 point prevalence PCR community testing allows disease burden estimation. In a sample of London residents, point prevalence decreased from 2.2% (95%CI 1.4;3.5) in early April (reflecting infection around lockdown implementation) to 0.2% (95%CI 0.03-1.6) in early May (reflecting infection 3-5 weeks into lockdown). Extrapolation from reports of confirmed cases suggest that 5-7.6% of total infections were confirmed by testing during this period. These data complement seroprevalence surveys improving the understanding of transmission in London.

Background Despite numerous randomized controlled trials (RCTs) and systematic reviews (SRs), current sepsis guidelines continue to issue only weak recommendations for corticosteroids. We examined the clinical scope, underlying study pools, and mortality conclusions of SRs evaluating corticosteroids for sepsis. Methods We conducted a meta-research study of SRs on corticosteroids in sepsis (2015 to 2025), extracting SR characteristics, mortality results, and included RCTs. Study-pool overlap was assessed using an SRxRCT inclusion matrix, Jaccard similarity (J), and hierarchical clustering. SRs and RCTs were classified according to standardized Population, Intervention, Comparison, Outcome (PICO) profiles. We explored discordance in short-term mortality conclusions among clinically comparable SRs and potential associations with study-pool composition, target populations, and methodological characteristics. Results Forty-two SRs including 121 unique RCTs were identified. More than half of pairwise SR comparisons shared no RCTs, and only three pairs showed high overlap (J>0.8). SRs addressing similar intervention and target population profiles frequently relied on different study pools. Among 38 SRs with short-term mortality meta-analyses, 15 (39%) reported benefit and 23 (61%) no evidence of effect. Discordance occurred exclusively among SRs evaluating broad, non-specific corticosteroid strategies; conclusions were consistent for hydrocortisone plus fludrocortisone (benefit) and hydrocortisone, ascorbic acid, and thiamine (no evidence of effect). SRs including sepsis +/- shock populations more frequently reported benefit than those restricted to septic shock (62% vs 22%), although estimates were imprecise. No single methodological or clinical factor consistently explained discordance. Conclusions SRs addressing apparently similar clinical questions frequently synthesized different underlying evidence bases and reported discordant conclusions. Guideline developers should therefore consider not only methodological quality and reported PICO, but also whether the RCTs included in an SR adequately represent the intended clinical question. Clinically coherent evidence syntheses may improve the interpretability of pooled treatment effects and support more targeted corticosteroid therapy in sepsis.

Superbugs: A Pop-up Science Shop was a public engagement event in the school summer holidays of 2019, organised by members of Cardiff Universitys School of Medicine. We transformed an empty retail unit in the centre of Wales largest shopping centre into an interactive and immersive microbiology experience. We aimed to facilitate opportunities for two-way dialogue to impart positive impact on the awareness of antibiotic resistance, whilst concurrently evaluating the efficacy of an engagement strategy focused on the utilisation of public spaces to attract public demographics diverse to those who would normally engage with STEM outreach. Over the course of 14 days, we welcomed 6,566 visitors, with 67% attending as part of the natural footfall of the shopping centre. We created 1,625 young Antibiotic Resistance Champions, located in over 200 schools. Through a multi-lateral evaluation strategy, we were able to collect quantitative and qualitative feedback on the success of our delivery model, and the impact on our stakeholders. Herein, we will discuss the evolution of Superbugs from concept, planning and design, to the logistics of delivering an engagement event of this scale. We will focus in particular on the learning outcomes of the project, and how this will shape the future of our Superbugs project, and engagement events beyond. Key MessagesO_LICreating a multi-disciplinary core team is essential to the success of large-scale engagement events as well as the support and development of large numbers of colleagues/volunteers C_LIO_LIUtilising themes of exhibition and gameplay alongside strong fear-empowerment messages is an impactful way to confer positive influence and behaviour around antimicrobial resistance (AMR) and the use of antibiotics C_LIO_LI Pop-up shop is an effective mode of delivery to capture diverse public demographics far beyond those who would traditionally engage with scientific outreach and science engagement. C_LI

Background FluSurvey is a participatory surveillance system, through which participants report weekly respiratory symptoms, healthcare use and social contacts. To support rapid public health assessment we developed a respiratory self-swabbing feature. We aimed to assess the feasibility of self-swabbing among symptomatic FluSurvey participants. Methods FluSurvey self-swabbing was deployed in England between January-March 2026. Adults reporting recent Acute Respiratory Infection symptoms were eligible for a self-swabbing Covid-19, RSV and Influenza PCR test which was returned to the laboratory by post. Participants were sent optional feedback surveys. We described swabbing interest, timeliness and participant demographics. We evaluated participant feedback to improve the process. Results There were 234 eligible illnesses during the study period; 45.7% (107/234) requested test kits and 93.5%(100/107) test kits were returned. Of these, 97.0%(97/100) had valid results; median participant age was 61.74 years (IQR:47.4-67.4), 68.4% were female and 90.0% were of white ethnicity. All nine regions in England were represented. Timeliness was high; the median time from symptom onset to specimen date was 4.0 days (IQR:3.0-5.0). Of participants completing feedback surveys, 96.7%(59/61) would consider participating in self-swabbing again. Conclusions We developed a tool to test individuals, often not presenting to healthcare, for respiratory viruses using an established community-based surveillance system. This tool can be rapidly deployed and adapted to public health need, to assess community-based emerging respiratory or pandemic infections. The high return rate and positive feedback suggest continued engagement of the cohort in respiratory virus swabbing.

The COVID-19 pandemic has spread to almost every country in the world since it started in China in late 2019. Controlling the pandemic requires a multifaceted approach including whole genome sequencing to support public health interventions at local and national levels. One of the most widely used methods for sequencing is the ARTIC protocol, a tiling PCR approach followed by Oxford Nanopore sequencing (ONT) of up to 96 samples at a time. There is a need, however, for a flexible, platform agnostic, method that can provide multiple throughput options depending on changing requirements as the pandemic peaks and troughs. Here we present CoronaHiT, a method capable of multiplexing up to 96 small genomes on a single MinION flowcell or >384 genomes on Illumina NextSeq, using transposase mediated addition of adapters and PCR based addition of barcodes to ARTIC PCR products. We demonstrate the method by sequencing 95 and 59 SARS-CoV-2 genomes for routine and rapid outbreak response runs, respectively, on Nanopore and Illumina platforms and compare to the standard ARTIC LoCost nanopore method. Of the 154 samples sequenced using the three approaches, genomes with [&ge;] 90% coverage (GISAID criteria) were generated for 64.3% of samples for ARTIC LoCost, 71.4% for CoronaHiT-ONT, and 76.6% for CoronaHiT-Illumina and have almost identical clustering on a maximum likelihood tree. In conclusion, we demonstrate that CoronaHiT can multiplex up to 96 SARS-CoV-2 genomes per MinION flowcell and that Illumina sequencing can be performed on the same libraries, which will allow significantly higher throughput. CoronaHiT provides increased coverage for higher Ct samples, thereby increasing the number of high quality genomes that pass the GISAID QC threshold. This protocol will aid the rapid expansion of SARS-CoV-2 genome sequencing globally, to help control the pandemic.

Objectives To characterise COVID-19 inpatients with and without bacterial/fungal preceding, co- and secondary infections, and describe the distribution of organisms associated with COVID-19 and antibiotic use. Methods We linked ISARIC/WHO CCP-UK data from hospitals in England (admissions 01/01/2020-18/10/2021) with UK Health Security Agency infections datasets of COVID-19, and bacterial- and fungal-positive specimens using a multi-step hierarchical algorithm. We retained those aged >18 years and categorised COVID-19-associated infection as preceding (2-28 days prior to SARS-CoV-2 PCR positive date), co-infection ({+/-}1 day) or secondary (2-28 days after). Results During the study period, there were 173,947 hospitalised episodes of COVID-19; 5.8% (n=10,017) with a bacterial or fungal preceding, co- or secondary infection. Preceding and co-infections were rare (preceding: 0.7%; co-infection: 1.5%) while secondary infections were more common (3.5%). Escherichia coli (29.6%) and Staphylococcus aureus (15.2%) were the most identified organisms in blood specimens, while S. aureus (22.9%) and Pseudomonas aeruginosa (20.4%) were most common in lower respiratory tract specimens. The 28-day case fatality rate (CFR) remained static (36%) in those with secondary infection, whereas it decreased between early 2020 and mid-2021 in the other infection groups (37% to 27%). Of those with no positive microbiology, 72.8% received antibiotics, though this decreased between January-June 2020 (77.6%) and April-October 2021 (62.6%). Conclusion Bacterial and fungal infections complicated approximately 1-in-17 hospitalised episodes of COVID-19, with CFR of >25%. Antibiotic prescribing was common in those without firm evidence of bacterial infection. We recommend adhering to NICE guidance which discourages empirical antibiotic use for hospitalised COVID-19 patients.

Estimates of population prevalence and genetic diversity of bloodborne viruses in healthy humans are essential to support population-scale monitoring for transfusion transmission risk. In the UK and globally, blood donations are routinely screened for a limited number of high-consequence pathogens, but the full composition of the plasma virome remains to be characterised. Using a novel quantitative targeted metagenomics sequencing approach, we analysed previously unscreened plasma donations collected by NHS Blood and Transplant in England for all major pathogenic and known commensal human bloodborne viruses, and quantified their viral burden. Here we show that in a representative sample of 5,064 UK blood donors in pools of 24 collected over a one-month period, the virome was dominated by a small number of largely persistent species, representing ~11% (12/106) of previously identified human bloodborne viruses. Anelloviruses (TTV, TTMV and TTMDV) was detected in 89.0% of pools, albeit at low read count inconsistent with measured anellovirus viral loads. In contrast, human pegivirus type 1 (HPgV-1), had estimated population prevalence of 3.7% (95% CI 3.0-4.4%), with high read count and complete genome recovery in around one half of positive pools, consistent with high titre in plasma. Estimated prevalences for less common detections included one species of gemykibovirus (0.12%), hepatitis C virus (genotype 1a, 0.04%) and various polyomaviruses and herpesviruses between 0.04% (parvovirus 4, BK polyomavirus) and 0.41% (human herpesvirus 6). Phylogenetic analyses revealed mixed TTV, TTMV and TTMDV populations and almost exclusively genotype 2 HPgV-1, consistent with known genotype distributions in Europe. Our results provide a baseline for describing the healthy plasma virome in UK blood donors.