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The Oxford Vaccine Group is an independent multi-disciplinary clinical trials and epidemiology group based at the Centre for Clinical Vaccinology and Tropical Medicine, University of Oxford. OVG works towards the goal of developing new and improved vaccines for the prevention of infection in adults and children.
Absolute quantification of rare gene targets in limited samples using crude lysate and ddPCR
Accurate quantification of rare genes from limited clinical samples is crucial for research purposes but is technically challenging, especially due to nucleic acid extraction. Using the commercially available genomic DNA (gDNA) extraction kits, which mostly include a DNA purification step through silica columns, magnetic beads or ethanol precipitation, are the preferred choice for many researchers. These kits, however, have a minimum cell number requirement for optimal DNA quality and yield. They are not ideal for use for clinical samples with limited cell numbers. Here, we report the development and validation of a novel crude lysate method for preparing DNA for the absolute quantification of rare genes, TRECs in our case, by droplet digital PCR (ddPCR), from infrequent cells, that removes the need for DNA extraction. Multiple optimization steps and analytical validation of this novel assay was performed on PBMCs extracted from the blood of healthy donors. The newly developed assay shows good agreement with standard ddPCR and has high accuracy, specificity, and reproducibility; additionally, it can also be applied to fixed and permeabilized cells. The assay has the potential to be used for quantification of other trace targets from limited cell samples.
Development and validation of the Oxford Benchmark Scale for Rating Vaccine Technologies (OBSRVT), a scale for assessing public attitudes to next-generation vaccine delivery technologies.
Next-generation vaccine delivery technologies may provide significant gains from both a technical and behavioral standpoint, but no scale has yet been developed to assess public attitudes to novel vaccine delivery technologies. We therefore performed a cross-sectional validation study that included 1,001 demographically representative participants from the UK and US to develop and validate a novel scale, the Oxford Benchmark Scale for Rating Vaccine Technologies (OBSRVT). A sample of 500 UK participants was used to perform exploratory factor analysis with categorical variables (using a polychoric correlation matrix) followed by promax oblique factor rotation to develop the initial model. This yielded a 15-item 4-domain scale with domains including acceptance (6 items), effectiveness (4 items), comfort (3 items), and convenience (2 items). This model was tested for robustness on a 501-participant demographically representative sample from the US. A confirmatory factor analysis with a Satorra-Bentler scaled test statistic was performed, which demonstrated adequate goodness of fit statistics including the root mean squared error of approximation (0.057), standardized root mean squared residual (0.053), and comparative fit index (0.938). Reliability as internal consistency was excellent (alpha = 0.92). Convergent validity with the Oxford Needle Experience Scale was supported by an adequate correlation (r = 0.31, p
Etiology and Antimicrobial Resistance of Culture-Positive Infections in Ugandan Infants: A Cohort Study of 7000 Neonates and Infants.
BACKGROUND: Epidemiological evidence about the etiology and antimicrobial resistance of neonatal infections remains limited in low-resource settings. We aimed to describe the etiology of neonatal infections in a prospective observational cohort study conducted at two hospital sites in Kampala, Uganda. METHODS: Babies admitted to either unit with risk factors or signs of sepsis, pneumonia, or meningitis had a blood culture, nasopharyngeal swab, and lumbar puncture (if indicated) collected. Basic demographics were collected, and babies were followed up until discharge or death to determine admission outcome. Blood cultures were processed using the BACTEC system and identification confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Cerebrospinal fluid was processed using standard microbiological testing and swabs were processed using the multiplex real-time polymerase chain reaction assay. Antimicrobial susceptibilities of bacterial isolates to World Health Organization-recommended first-line antibiotics (ampicillin or benzylpenicillin and gentamicin) were assessed using e-tests. RESULTS: A total of 7323 infants with signs or risk factors for sepsis had blood cultures, 2563 had nasopharyngeal swabs, and 23 had lumbar punctures collected. Eleven percent of blood cultures and 8.6% of swabs were positive. Inpatient mortality was 12.1%, with 27.7% case fatality observed among infants with Gram-negative bloodstream infections. Escherichia coli (14.8%), Acinetobacter spp. (10.3%), and Klebsiella spp. (7.6%), were notable contributors to Gram-negative sepsis, whereas Group B Streptococcus was the predominant Gram-positive pathogen identified (13.5%). Almost 60% of Gram-negative pathogens were ampicillin- and gentamicin-resistant. CONCLUSIONS: Our study demonstrates high levels of antimicrobial resistance and inpatient mortality from neonatal sepsis in the first months of life in Uganda. This underscores the pressing need for revised, context-specific antimicrobial treatment guidelines that account for the evolving landscape of antimicrobial resistance in neonatal sepsis.
“Bye-Bye Germs”: Respiratory Tract Infection Prevention—An Education Intervention for Children
Becoming one of the first studies in the field to do so, specially developed educational interventions (Germ’s Journey), designed to teach children about respiratory tract infection prevention, were delivered to 273 pupils aged five to six across five primary schools in the U.K. The intervention aimed to increase understanding of pathogens and respiratory tract illness, transmission and infection prevention, and preparedness for future pandemics due to a lack of such resources for young children at present. To assess the impact of the intervention, children were asked five questions related to knowledge of pathogens, transmission of infection, and infection prevention directly before and after activity-based workshops, as well as one month later. Responses were scored for pupils’ level of knowledge; differences in the frequency of responses between the time points were analysed using Pearson’s chi-squared test. Teachers also took part in semi-structured interviews to evaluate the workshop from the educators’ points of view. Children showed increased knowledge in all three areas immediately after the workshops. This improvement was retained to a lesser or equal extent one month following the learning intervention workshop. The consistent use of teaching resources and interventions such as Germ’s Journey should be implemented in the school curriculum in order to increase understanding and reduce the transmission of respiratory tract illness. Specially designed activity-based workshops using a range of learning skills can help young children to understand the link between pathogens, and infection transmission and control.
IL7 genetic variation and toxicity to immune checkpoint blockade in patients with melanoma.
Treatment with immune checkpoint blockade (ICB) frequently triggers immune-related adverse events (irAEs), causing considerable morbidity. In 214 patients receiving ICB for melanoma, we observed increased severe irAE risk in minor allele carriers of rs16906115, intronic to IL7. We found that rs16906115 forms a B cell-specific expression quantitative trait locus (eQTL) to IL7 in patients. Patients carrying the risk allele demonstrate increased pre-treatment B cell IL7 expression, which independently associates with irAE risk, divergent immunoglobulin expression and more B cell receptor mutations. Consistent with the role of IL-7 in T cell development, risk allele carriers have distinct ICB-induced CD8+ T cell subset responses, skewing of T cell clonality and greater proportional repertoire occupancy by large clones. Finally, analysis of TCGA data suggests that risk allele carriers independently have improved melanoma survival. These observations highlight key roles for B cells and IL-7 in both ICB response and toxicity and clinical outcomes in melanoma.
Relation Between the Dantu Blood Group Variant and Bacteremia in Kenyan Children: A Population-Based Case-Control Study.
BACKGROUND: The Dantu blood group variant protects against Plasmodium falciparum infections, but its wider consequences have not been previously explored. Here, we investigate the impact of Dantu on susceptibility to bacteremia. METHODS: We conducted a case-control study in children presenting with community-acquired bacteremia to Kilifi County Hospital in Kenya between 1998 and 2010. We used logistic regression to test for associations between the Dantu marker single-nucleotide polymorphism rs186873296 A > G and both all-cause and pathogen-specific bacteremia under an additive model. We used date of admission as a proxy measure of malaria transmission intensity, given known differences in malaria prevalence over the course of the study. RESULTS: Dantu was associated with protection from all-cause bacteremia (OR, 0.81; P = .014), the association being greatest in homozygotes (OR, 0.30; P = .013). This protection was shared across the major bacterial pathogens but, notably, was only significant during the era of high malaria transmission pre-2003 (OR, 0.79; P = .023). CONCLUSIONS: Consistent with previous studies showing the indirect impact on bacteremia risk of other malaria-associated red cell variants, our study also shows that Dantu is protective against bacteremia via its effect on malaria risk. Dantu does not appear to be under balancing selection through an increased risk of bacterial infections.
BIRC6 modifies risk of invasive bacterial infection in Kenyan children.
Invasive bacterial disease is a major cause of morbidity and mortality in African children. Despite being caused by diverse pathogens, children with sepsis are clinically indistinguishable from one another. In spite of this, most genetic susceptibility loci for invasive infection that have been discovered to date are pathogen specific and are not therefore suggestive of a shared genetic architecture of bacterial sepsis. Here, we utilise probabilistic diagnostic models to identify children with a high probability of invasive bacterial disease among critically unwell Kenyan children with Plasmodium falciparum parasitaemia. We construct a joint dataset including 1445 bacteraemia cases and 1143 severe malaria cases, and population controls, among critically unwell Kenyan children that have previously been genotyped for human genetic variation. Using these data, we perform a cross-trait genome-wide association study of invasive bacterial infection, weighting cases according to their probability of bacterial disease. In doing so, we identify and validate a novel risk locus for invasive infection secondary to multiple bacterial pathogens, that has no apparent effect on malaria risk. The locus identified modifies splicing of BIRC6 in stimulated monocytes, implicating regulation of apoptosis and autophagy in the pathogenesis of sepsis in Kenyan children.
Natural Killer cells demonstrate distinct eQTL and transcriptome-wide disease associations, highlighting their role in autoimmunity.
Natural Killer cells are innate lymphocytes with central roles in immunosurveillance and are implicated in autoimmune pathogenesis. The degree to which regulatory variants affect Natural Killer cell gene expression is poorly understood. Here we perform expression quantitative trait locus mapping of negatively selected Natural Killer cells from a population of healthy Europeans (n = 245). We find a significant subset of genes demonstrate expression quantitative trait loci specific to Natural Killer cells and these are highly informative of human disease, in particular autoimmunity. A Natural Killer cell transcriptome-wide association study across five common autoimmune diseases identifies further novel associations at 27 genes. In addition to these cis observations, we find novel master-regulatory regions impacting expression of trans gene networks at regions including 19q13.4, the Killer cell Immunoglobulin-like Receptor region, GNLY, MC1R and UVSSA. Our findings provide new insights into the unique biology of Natural Killer cells, demonstrating markedly different expression quantitative trait loci from other immune cells, with implications for disease mechanisms.
Multi-ancestry meta-analysis of host genetic susceptibility to tuberculosis identifies shared genetic architecture.
The heritability of susceptibility to tuberculosis (TB) disease has been well recognized. Over 100 genes have been studied as candidates for TB susceptibility, and several variants were identified by genome-wide association studies (GWAS), but few replicate. We established the International Tuberculosis Host Genetics Consortium to perform a multi-ancestry meta-analysis of GWAS, including 14,153 cases and 19,536 controls of African, Asian, and European ancestry. Our analyses demonstrate a substantial degree of heritability (pooled polygenic h2 = 26.3%, 95% CI 23.7-29.0%) for susceptibility to TB that is shared across ancestries, highlighting an important host genetic influence on disease. We identified one global host genetic correlate for TB at genome-wide significance (p<5 × 10-8) in the human leukocyte antigen (HLA)-II region (rs28383206, p-value=5.2 × 10-9) but failed to replicate variants previously associated with TB susceptibility. These data demonstrate the complex shared genetic architecture of susceptibility to TB and the importance of large-scale GWAS analysis across multiple ancestries experiencing different levels of infection pressure.
Altered IL-6 signalling and risk of tuberculosis: a multi-ancestry mendelian randomisation study.
BACKGROUND: The role of IL-6 responses in human tuberculosis risk is unknown. IL-6 signalling inhibitors, such as tocilizumab, are thought to increase the risk of progression to tuberculosis, and screening for latent Mycobacterium tuberculosis infection before using these drugs is widely recommended. We used single nucleotide polymorphisms (SNPs) in and near the IL-6 receptor gene (IL6R), including the non-synonymous variant, rs2228145, for which the C allele contributes to reduced classic (cis) IL-6 signalling activity, to test the hypothesis that altered IL-6 signalling is causally associated with the risk of developing tuberculosis. METHODS: We performed a meta-analysis of genome-wide association studies (GWAS) published in English from database inception to Jan 1, 2024. GWAS were identified from the European Bioinformatics Institute, MRC Integrative Epidemiology Unit catalogues, and MEDLINE, selecting publicly available studies for which tuberculosis was an outcome and that included the IL6R rs2228145 SNP. Using each study's population-level summary statistics, effect estimates were extracted for each additional copy of the C allele of rs2228145. We used these estimates to perform multi-ancestry, two-sample mendelian randomisation analyses to estimate the causal effect of reduced IL-6 signalling on tuberculosis. Our primary analyses used rs2228145-C as a genetic instrument, weighted on C-reactive protein (CRP) reduction as a measure of the effect on IL-6 signalling. We also took an alternative, ancestry-specific, multiple SNP approach using IL-6 receptor plasma protein as an exposure. Additionally, we compared the effects of rs2228145 in tuberculosis with those in critical COVID-19, rheumatoid arthritis, Crohn's disease, and coronary artery disease using the summary statistics extracted from GWAS. FINDINGS: 17 GWAS were included, collating data for 19 302 individuals with tuberculosis (cases) and 1 019 821 population controls across multiple ancestries. For each additional rs2228145-C allele, the odds of tuberculosis reduced (odds ratio [OR] 0·94 [95% CI 0·92-0·97]; p=6·8 × 10-6). Multi-ancestry mendelian randomisation analyses supported these findings, with decreased odds of tuberculosis associated with readouts of reduced IL-6 signalling (0·52 [0·39-0·69] for each natural log CRP decrease; p=6·8 × 10-6), with weak evidence of heterogeneity (I2=0·315; p=0·11). Ancestry-specific, multiple SNP mendelian randomisation using increase in IL-6 receptor plasma protein as an exposure revealed a similar reduced risk of tuberculosis (OR 0·94 [95% CI 0·93-0·96]; p=2·4 × 10-10). The protective effects on tuberculosis seen with rs2228145-C were similar in size and direction to those observed in critical COVID-19 (0·66 [0·50-0·86]), Crohn's disease (0·57 [0·44-0·74]), and rheumatoid arthritis (0·45 [0·36-0·58]), all of which benefit from the therapeutic effects of IL-6 antagonism. INTERPRETATION: Our findings propose a causal relationship between reduced IL-6 signalling and lower risk of tuberculosis, akin to the effect seen in other IL-6 mediated diseases. This study suggests that IL-6 antagonists do not increase the risk of tuberculosis but rather should be investigated as therapeutic adjuncts in its treatment. FUNDING: UK National Institute for Health and Care Research, Wellcome Trust, EU European Regional Development Fund, the Welsh Government, and UK Research and Innovation.
Intracranial Empyema in Children: A Single-center Retrospective Case Series.
We conducted a retrospective, observational study of 42 children with intracranial empyema admitted to a pediatric neurosurgical center over a 9-year period. Intracranial empyema is rare, but causes significant morbidity and mortality. Twenty-eight cases had neurosurgical source control, more commonly for subdural collections. Streptococcus anginosus group bacteria are important pathogens in subdural empyema, whose isolation predicts more complicated postoperative courses.
Cross-sectional study of IgG antibody levels to invasive nontyphoidal Salmonella lps O-antigen with age in Uganda
Invasive nontyphoidal Salmonella (iNTS) disease is a major cause of deaths among children and HIV-infected individuals in sub-Saharan Africa. Acquisition of IgG to iNTS lipopolysaccharide (LPS) O-antigen in Malawi in early childhood corresponds with a fall in cases of iNTS disease suggesting that vaccines able to induce such antibodies could confer protection. To better understand the acquisition of IgG to iNTS in other African settings, we performed a cross-sectional seroepidemiological study using sera from 1090 Ugandan individuals aged from infancy to old age. Sera were analysed for IgG to LPS O-antigen of S. Typhimurium and S. Enteritidis using an in-house ELISA. Below 18 months of age, most children lacked IgG to both serovars. Thereafter, specific IgG levels increased with age, peaking in adulthood, and did not wane noticeably in old age. There was no clear difference in antibody levels between the sexes and the few HIV-infected individuals in the study did not have obviously different levels from uninfected subjects. While IgG to iNTS is acquired at a younger age in Malawian compared with Ugandan children, it is not clear whether this is due to differences in the populations themselves, their exposure to iNTS, or variations between assays used. In conclusion, there is a need to develop a harmonised method and standards for measuring antibodies to iNTS across studies and to investigate acquisition of such antibodies with age across different sites in sub-Saharan Africa.
Cytokine Profiles during Invasive Nontyphoidal Salmonella Disease Predict Outcome in African Children.
Nontyphoidal Salmonella is a leading cause of sepsis in African children. Cytokine responses are central to the pathophysiology of sepsis and predict sepsis outcome in other settings. In this study, we investigated cytokine responses to invasive nontyphoidal Salmonella (iNTS) disease in Malawian children. We determined serum concentrations of 48 cytokines with multiplexed immunoassays in Malawian children during acute iNTS disease (n = 111) and in convalescence (n = 77). Principal component analysis and logistic regression were used to identify cytokine signatures of acute iNTS disease. We further investigated whether these responses are altered by HIV coinfection or severe malnutrition and whether cytokine responses predict inpatient mortality. Cytokine changes in acute iNTS disease were associated with two distinct cytokine signatures. The first is characterized by increased concentrations of mediators known to be associated with macrophage function, and the second is characterized by raised pro- and anti-inflammatory cytokines typical of responses reported in sepsis secondary to diverse pathogens. These cytokine responses were largely unaltered by either severe malnutrition or HIV coinfection. Children with fatal disease had a distinctive cytokine profile, characterized by raised mediators known to be associated with neutrophil function. In conclusion, cytokine responses to acute iNTS infection in Malawian children are reflective of both the cytokine storm typical of sepsis secondary to diverse pathogens and the intramacrophage replicative niche of NTS. The cytokine profile predictive of fatal disease supports a key role of neutrophils in the pathogenesis of NTS sepsis.