Exploiting Bacteriophages to Tackle Clostridium difficile Infection

Clostridium difficile infection (CDI) currently affects around 20,000 people each year, in healthcare institutions and in the community, and will often follow disruption of the gut microbiome. Current treatment strategies call for the use of further antibiotics, of which there is a limited choice. There is a need for additional remedial and prophylactic solutions with greater specificity and low levels of toxicity and resistance. This thesis describes the pathogenesis of CDI, the current treatment strategies and navigates the growing body of studies investigating the potential use of phage. The project involved extensive screening including faecal samples and environmental sources in an attempt to identify novel phages of C. difficile and documents efforts to improve the therapeutic capacity of a selected phage, ФCD27, by mutagenesis. No exclusively lytic phages were isolated or obtained following mutagenesis with ethylmethane sulphonate, hydroxylamine or sodium pyrophosphate. Batch fermentation models of CDI showed that a prophylactic approach to phage therapy of CDI offers a higher efficacy than a remedial regime. A continuous model of CDI in a colon model was successfully produced and demonstrated variable efficacy rates from no apparent decrease in the burden of C. difficile to a reduction to below the limit of detection by culture, with no detrimental effect on commensal microbiota. The lysogenic capacity of ФCD27 appeared to prevent clearance of C. difficile in the models, but some strains containing the prophage exhibited reduced toxin production phenotypically. A possible mechanism of this altered phenotype included the action of ФCD27 repressor proteins on the promoter regions of C. diffiicle toxin genes or regulatory elements, but affinity of a candidate repressor, ORF44, to PaLoc constituents was not demonstrated. Studies have also demonstrated the ability of ФCD27 to prevent outgrowth of germinating C. difficile spores, thus potential as an environmental decontaminant. iii The findings of the project and the future prospects of phage therapy as an agent against CDI are discussed

Norovirus prevalence and estimated viral load in symptomatic and asymptomatic children from rural communities of Vhembe district, South Africa

Background: Human Norovirus (NoV) is recognized as a major etiological agent of sporadic acute gastroenteritis worldwide. Objectives: This study describes the clinical features associated with Human NoV occurrence in children and determines the prevalence and estimated viral burden of NoV in symptomatic and asymptomatic children in rural South Africa. Study design: Between July 2014 and April 2015, outpatient children under 5 years of age from rural communities of Vhembe district, South Africa, were enrolled for the study. A total of 303 stool specimens were collected from those with diarrhea (n=253) and without (n=50) diarrhea. NoVs were identified using real-time one-step RT-PCR. Results: One hundred and four (41.1%) NoVs were detected (62[59.6%] GII, 16[15.4%] GI, and 26[25%] mixed GI/GII) in cases and 18 (36%) including 9(50%) GII, 2(11.1%) GI and 7(38.9%) mixed GI/GII in controls. NoV detection rates in symptomatic and asymptomatic children (OR = 1.24; 95% CI 0.66 – 2.33) were not significantly different. Comparison of the median CT values for NoV in symptomatic and asymptomatic children revealed significant statistical difference of estimated GII viral load from both groups, with a much higher viral burden in symptomatic children. Conclusions: Though not proven predictive of diarrhea disease in this study, the high detection rate of NoV reflects the substantial exposure of children from rural communities to enteric pathogens possibly due to poor sanitation and hygiene practices. The results suggest that the difference between asymptomatic and symptomatic children with NoV may be at the level of the viral load of NoV genogroups involved

The role of viral genomics in understanding COVID-19 outbreaks in long-term care facilities.

Funder: Biotechnology and Biological Sciences Research CouncilWe reviewed all genomic epidemiology studies on COVID-19 in long-term care facilities (LTCFs) that had been published to date. We found that staff and residents were usually infected with identical, or near identical, SARS-CoV-2 genomes. Outbreaks usually involved one predominant cluster, and the same lineages persisted in LTCFs despite infection control measures. Outbreaks were most commonly due to single or few introductions followed by a spread rather than a series of seeding events from the community into LTCFs. The sequencing of samples taken consecutively from the same individuals at the same facilities showed the persistence of the same genome sequence, indicating that the sequencing technique was robust over time. When combined with local epidemiology, genomics allowed probable transmission sources to be better characterised. The transmission between LTCFs was detected in multiple studies. The mortality rate among residents was high in all facilities, regardless of the lineage. Bioinformatics methods were inadequate in a third of the studies reviewed, and reproducing the analyses was difficult because sequencing data were not available in many facilities

CoronaHiT: high-throughput sequencing of SARS-CoV-2 genomes.

We present CoronaHiT, a platform and throughput flexible method for sequencing SARS-CoV-2 genomes (≤ 96 on MinION or > 96 on Illumina NextSeq) depending on changing requirements experienced during the pandemic. CoronaHiT uses transposase-based library preparation of ARTIC PCR products. Method performance was demonstrated by sequencing 2 plates containing 95 and 59 SARS-CoV-2 genomes on nanopore and Illumina platforms and comparing to the ARTIC LoCost nanopore method. Of the 154 samples sequenced using all 3 methods, ≥ 90% genome coverage was obtained for 64.3% using ARTIC LoCost, 71.4% using CoronaHiT-ONT and 76.6% using CoronaHiT-Illumina, with almost identical clustering on a maximum likelihood tree. This protocol will aid the rapid expansion of SARS-CoV-2 genome sequencing globally.The sequencing costs were funded by the COVID-19 Genomics UK (COG-UK) Consortium which is supported by funding from the Medical Research Council (MRC) part of UK Research & Innovation (UKRI), the National Institute of Health Research (NIHR) and Genome Research Limited, operating as the Wellcome Sanger Institute

Are differential consumption patterns in health-related behaviours an explanation for persistent and widening social inequalities in health in England?

During the last two decades, differential consumption patterns in health-related behaviours have increasingly been highlighted as playing an important role in explaining persistent and widening health inequalities. This period has also seen government public health policies in England place a greater emphasis on changing ‘lifestyle’ behaviours, in an attempt to tackle social inequalities in health. The aim of this study was to empirically examine the variation in health-related behaviour in relation to socio-economic position, in the English adult population, to determine the nature of this relationship and whether it has changed over time

Genetic characterisation of Norovirus strains in outpatient children from rural communities of Vhembe district / South Africa, 2014-2015

Background: Norovirus (NoV) is now the 24 most common causes of both outbreaks and sporadic non-bacterial gastroenteritis worldwide. However, data supporting the role of NoV in diarrheal disease are limited in the African continent. Objectives: This study investigates the distribution of NoV genotypes circulating in outpatient children from rural communities of Vhembe district / South Africa. Study design: Stool specimens were collected from children under five years of age with diarrhea, and controls without diarrhea, between July 2014 and April 2015. NoV positive samples, detected previously by Realtime PCR, were analysed using conventional RT-PCR targeting the partial capsid and polymerase genes. Nucleotide sequencing methods were performed to genotype the strains. Results: The sequence analyses demonstrated multiple NoV genotypes including GI.4 (13.8%), GI.5 (6.9%), GII.14 (6.9%), GII.4 (31%), GII.6 (3.4%), GII.P15 (3.4%), GII.P21 (3.4%) and GII.Pe (31%). The most prevalent NoV genotypes were GII.4 Sydney 2012 variants (n=7) among the capsid genotypes, GII.Pe (n=9) among the polymerase genotypes and GII.Pe/GII.4 Sydney 2012 (n=8) putative recombinants among the RdRp/Capsid genotypes. Two unassigned GII.4 variants were found. Conclusions: The findings highlighted NoV genetic diversity and revealed continuous pandemic spread and predominance of GII.Pe/GII.4 Sydney 2012, indicative of increased NoV activity. An unusual RdRp genotype GII.P15 and two unassigned GII.4 variants were also identified from rural settings of the Vhembe district/South Africa. NoV surveillance is warranted to help to inform investigations into NoV evolution and disease burden, and to support on-going vaccine development programmes

Large-scale sequencing of SARS-CoV-2 genomes from one region allows detailed epidemiology and enables local outbreak management.

The COVID-19 pandemic has spread rapidly throughout the world. In the UK, the initial peak was in April 2020; in the county of Norfolk (UK) and surrounding areas, which has a stable, low-density population, over 3200 cases were reported between March and August 2020. As part of the activities of the national COVID-19 Genomics Consortium (COG-UK) we undertook whole genome sequencing of the SARS-CoV-2 genomes present in positive clinical samples from the Norfolk region. These samples were collected by four major hospitals, multiple minor hospitals, care facilities and community organizations within Norfolk and surrounding areas. We combined clinical metadata with the sequencing data from regional SARS-CoV-2 genomes to understand the origins, genetic variation, transmission and expansion (spread) of the virus within the region and provide context nationally. Data were fed back into the national effort for pandemic management, whilst simultaneously being used to assist local outbreak analyses. Overall, 1565 positive samples (172 per 100 000 population) from 1376 cases were evaluated; for 140 cases between two and six samples were available providing longitudinal data. This represented 42.6 % of all positive samples identified by hospital testing in the region and encompassed those with clinical need, and health and care workers and their families. In total, 1035 cases had genome sequences of sufficient quality to provide phylogenetic lineages. These genomes belonged to 26 distinct global lineages, indicating that there were multiple separate introductions into the region. Furthermore, 100 genetically distinct UK lineages were detected demonstrating local evolution, at a rate of ~2 SNPs per month, and multiple co-occurring lineages as the pandemic progressed. Our analysis: identified a discrete sublineage associated with six care facilities; found no evidence of reinfection in longitudinal samples; ruled out a nosocomial outbreak; identified 16 lineages in key workers which were not in patients, indicating infection control measures were effective; and found the D614G spike protein mutation which is linked to increased transmissibility dominates the samples and rapidly confirmed relatedness of cases in an outbreak at a food processing facility. The large-scale genome sequencing of SARS-CoV-2-positive samples has provided valuable additional data for public health epidemiology in the Norfolk region, and will continue to help identify and untangle hidden transmission chains as the pandemic evolves.The sequencing costs were funded by the COVID-19 Genomics UK (COG-UK) Consortium which is supported by funding from the Medical Research Council (MRC) part of UK Research and Innovation (UKRI), the National Institute of Health Research (NIHR) and Genome Research Limited, operating as the Wellcome Sanger Institute

Evaluating the Effects of SARS-CoV-2 Spike Mutation D614G on Transmissibility and Pathogenicity.

Global dispersal and increasing frequency of the SARS-CoV-2 spike protein variant D614G are suggestive of a selective advantage but may also be due to a random founder effect. We investigate the hypothesis for positive selection of spike D614G in the United Kingdom using more than 25,000 whole genome SARS-CoV-2 sequences. Despite the availability of a large dataset, well represented by both spike 614 variants, not all approaches showed a conclusive signal of positive selection. Population genetic analysis indicates that 614G increases in frequency relative to 614D in a manner consistent with a selective advantage. We do not find any indication that patients infected with the spike 614G variant have higher COVID-19 mortality or clinical severity, but 614G is associated with higher viral load and younger age of patients. Significant differences in growth and size of 614G phylogenetic clusters indicate a need for continued study of this variant

Exponential growth, high prevalence of SARS-CoV-2, and vaccine effectiveness associated with the Delta variant

SARS-CoV-2 infections were rising during early summer 2021 in many countries associated with the Delta variant. We assessed RT-PCR swab-positivity in the REal-time Assessment of Community Transmission-1 (REACT-1) study in England. We observed sustained exponential growth with average doubling time (June-July 2021) of 25 days driven by complete replacement of Alpha variant by Delta, and by high prevalence at younger less-vaccinated ages. Unvaccinated people were three times more likely than double-vaccinated people to test positive. However, after adjusting for age and other variables, vaccine effectiveness for double-vaccinated people was estimated at between ~50% and ~60% during this period in England. Increased social mixing in the presence of Delta had the potential to generate sustained growth in infections, even at high levels of vaccination

Hospital admission and emergency care attendance risk for SARS-CoV-2 delta (B.1.617.2) compared with alpha (B.1.1.7) variants of concern: a cohort study

Background: The SARS-CoV-2 delta (B.1.617.2) variant was first detected in England in March, 2021. It has since rapidly become the predominant lineage, owing to high transmissibility. It is suspected that the delta variant is associated with more severe disease than the previously dominant alpha (B.1.1.7) variant. We aimed to characterise the severity of the delta variant compared with the alpha variant by determining the relative risk of hospital attendance outcomes. Methods: This cohort study was done among all patients with COVID-19 in England between March 29 and May 23, 2021, who were identified as being infected with either the alpha or delta SARS-CoV-2 variant through whole-genome sequencing. Individual-level data on these patients were linked to routine health-care datasets on vaccination, emergency care attendance, hospital admission, and mortality (data from Public Health England's Second Generation Surveillance System and COVID-19-associated deaths dataset; the National Immunisation Management System; and NHS Digital Secondary Uses Services and Emergency Care Data Set). The risk for hospital admission and emergency care attendance were compared between patients with sequencing-confirmed delta and alpha variants for the whole cohort and by vaccination status subgroups. Stratified Cox regression was used to adjust for age, sex, ethnicity, deprivation, recent international travel, area of residence, calendar week, and vaccination status. Findings: Individual-level data on 43 338 COVID-19-positive patients (8682 with the delta variant, 34 656 with the alpha variant; median age 31 years [IQR 17–43]) were included in our analysis. 196 (2·3%) patients with the delta variant versus 764 (2·2%) patients with the alpha variant were admitted to hospital within 14 days after the specimen was taken (adjusted hazard ratio [HR] 2·26 [95% CI 1·32–3·89]). 498 (5·7%) patients with the delta variant versus 1448 (4·2%) patients with the alpha variant were admitted to hospital or attended emergency care within 14 days (adjusted HR 1·45 [1·08–1·95]). Most patients were unvaccinated (32 078 [74·0%] across both groups). The HRs for vaccinated patients with the delta variant versus the alpha variant (adjusted HR for hospital admission 1·94 [95% CI 0·47–8·05] and for hospital admission or emergency care attendance 1·58 [0·69–3·61]) were similar to the HRs for unvaccinated patients (2·32 [1·29–4·16] and 1·43 [1·04–1·97]; p=0·82 for both) but the precision for the vaccinated subgroup was low. Interpretation: This large national study found a higher hospital admission or emergency care attendance risk for patients with COVID-19 infected with the delta variant compared with the alpha variant. Results suggest that outbreaks of the delta variant in unvaccinated populations might lead to a greater burden on health-care services than the alpha variant. Funding: Medical Research Council; UK Research and Innovation; Department of Health and Social Care; and National Institute for Health Research