register interest

Dr David Wyllie

Research Area: Microbiology
Technology Exchange: Bioinformatics, Flow cytometry, Transcript profiling and Vaccine production and evaluation
Scientific Themes: Immunology & Infectious Disease
Keywords: Vaccine development and Infection detection and prevention

The main aim of my programme is to develop a vaccine reducing the burden of Staphylococcus aureus disease.  Staphylococcus aureus is a common microorganism which is an important cause of infection in both the community and within hospitals.  It colonises (lives without apparent ill effect to humans) the mucosal surfaces and bowel in about one third of the population, but can also cause serious invasive disease. Infections in pigs, cattle and chickens are also well recognised, and these animal diseases present economic and animal welfare challenges in agriculture.  Some strains are resistant to methicillin (MRSA), and are in many setting a major driver of antibiotic use.

We are developing a S. aureus vaccine based on non-replicating adenoviral and modified Vaccinia Ankara viral vectors which incorporates novel protective antigens we have discovered, as part of the EU-funded BELLEROPHON project.   

We understand rather poorly how the human population responds to S. aureus, and which responses are protective.  This is potentially a problem for S. aureus vaccine development, as vaccines will be given to a partially immune population.  To assist with this goal we are studying how S. aureus and the human immune system interact during carriage and disease, and how this relates to skin disease.  This work is done in partnership with the Royal Navy. 

We also understand poorly how the bacterium responds to the hostile environment of the host during infection. Claudia Lindemann (DPhil student, co-supervised by Dr Daniel Wilson, and supported by the EU Vactrain program) is looking both at how the bacterium responds to the host, using transcriptional profiling during infection.

Finally, I am continuing to study the epidemiology of S. aureus and MRSA at a population level, an ongoing activity in collaboration with Professors Crook and Peto and the Modernising Medical Microbiology initiative.  

Name Department Institution Country
Professor Daniel J Wilson Experimental Medicine Division Oxford University, John Radcliffe Hospital United Kingdom
Dr Christine S Rollier Jenner Institute Oxford University, Centre for Clinical Vaccinology and Tropical Medicine United Kingdom
Professor Derrick Crook Experimental Medicine Division Oxford University, John Radcliffe Hospital United Kingdom
Professor Andrew Pollard Jenner Institute Oxford University, Centre for Clinical Vaccinology and Tropical Medicine United Kingdom
Professor Tim Peto Experimental Medicine Division Oxford University, John Radcliffe Hospital United Kingdom
Prof Jurgen E Schneider (RDM) Cardiovascular Medicine Oxford University, Henry Wellcome Building of Genomic Medicine United Kingdom
Dr Jo Fallowfield Institute of Naval Medicine United Kingdom
Diemen PMV, Leneghan DB, Brian IJ, Miura K, Long CA, Milicic A, Biswas S, Rollier CS, Wyllie DH. 2017. The S. aureus 4-oxalocrotonate tautomerase SAR1376 enhances immune responses when fused to several antigens. Sci Rep, 7 (1), pp. 1745. | Show Abstract | Read more

A persistent goal of vaccine development is the enhancement of the immunogenicity of antigens while maintaining safety. One strategy involves alteration of the presentation of the antigen by combining antigens with a multimeric scaffold. Multi-antigen vaccines are under development, and there are presently far more candidate antigens than antigen scaffolding strategies. This is potentially problematic, since prior immunity to a scaffold may inhibit immune responses to the antigen-scaffold combination. In this study, a series of domains from S. aureus which have been shown to crystallise into multimeric structures have been examined for their scaffolding potential. Of these domains, SAR1376, a 62 amino acid member of the 4-oxalocrotonate tautomerase (4-OT) family, was pro-immunogenic in mice when fused to a range of pathogen antigens from both S. aureus and P. falciparum, and delivered by either DNA vaccination, viral vector vaccines or as protein-in-adjuvant formulations. The adjuvant effect did not depend on enzymatic activity, but was abrogated by mutations disrupting the hexameric structure of the protein. We therefore propose that SAR1376, and perhaps other members of the 4-OT protein family, represent very small domains which can be fused to a wide range of antigens, enhancing immune responses against them.

Votintseva AA, Bradley P, Pankhurst L, Del Ojo Elias C, Loose M, Nilgiriwala K, Chatterjee A, Smith EG, Sanderson N, Walker TM et al. 2017. Same-Day Diagnostic and Surveillance Data for Tuberculosis via Whole-Genome Sequencing of Direct Respiratory Samples. J Clin Microbiol, 55 (5), pp. 1285-1298. | Show Abstract | Read more

Routine full characterization of Mycobacterium tuberculosis is culture based, taking many weeks. Whole-genome sequencing (WGS) can generate antibiotic susceptibility profiles to inform treatment, augmented with strain information for global surveillance; such data could be transformative if provided at or near the point of care. We demonstrate a low-cost method of DNA extraction directly from patient samples for M. tuberculosis WGS. We initially evaluated the method by using the Illumina MiSeq sequencer (40 smear-positive respiratory samples obtained after routine clinical testing and 27 matched liquid cultures). M. tuberculosis was identified in all 39 samples from which DNA was successfully extracted. Sufficient data for antibiotic susceptibility prediction were obtained from 24 (62%) samples; all results were concordant with reference laboratory phenotypes. Phylogenetic placement was concordant between direct and cultured samples. With Illumina MiSeq/MiniSeq, the workflow from patient sample to results can be completed in 44/16 h at a reagent cost of £96/£198 per sample. We then employed a nonspecific PCR-based library preparation method for sequencing on an Oxford Nanopore Technologies MinION sequencer. We applied this to cultured Mycobacterium bovis strain BCG DNA and to combined culture-negative sputum DNA and BCG DNA. For flow cell version R9.4, the estimated turnaround time from patient to identification of BCG, detection of pyrazinamide resistance, and phylogenetic placement was 7.5 h, with full susceptibility results 5 h later. Antibiotic susceptibility predictions were fully concordant. A critical advantage of MinION is the ability to continue sequencing until sufficient coverage is obtained, providing a potential solution to the problem of variable amounts of M. tuberculosis DNA in direct samples.

Dingle KE, Didelot X, Quan TP, Eyre DW, Stoesser N, Golubchik T, Harding RM, Wilson DJ, Griffiths D, Vaughan A et al. 2017. Effects of control interventions on Clostridium difficile infection in England: an observational study. Lancet Infect Dis, 17 (4), pp. 411-421. | Show Abstract | Read more

BACKGROUND: The control of Clostridium difficile infections is an international clinical challenge. The incidence of C difficile in England declined by roughly 80% after 2006, following the implementation of national control policies; we tested two hypotheses to investigate their role in this decline. First, if C difficile infection declines in England were driven by reductions in use of particular antibiotics, then incidence of C difficile infections caused by resistant isolates should decline faster than that caused by susceptible isolates across multiple genotypes. Second, if C difficile infection declines were driven by improvements in hospital infection control, then transmitted (secondary) cases should decline regardless of susceptibility. METHODS: Regional (Oxfordshire and Leeds, UK) and national data for the incidence of C difficile infections and antimicrobial prescribing data (1998-2014) were combined with whole genome sequences from 4045 national and international C difficile isolates. Genotype (multilocus sequence type) and fluoroquinolone susceptibility were determined from whole genome sequences. The incidence of C difficile infections caused by fluoroquinolone-resistant and fluoroquinolone-susceptible isolates was estimated with negative-binomial regression, overall and per genotype. Selection and transmission were investigated with phylogenetic analyses. FINDINGS: National fluoroquinolone and cephalosporin prescribing correlated highly with incidence of C difficile infections (cross-correlations >0·88), by contrast with total antibiotic prescribing (cross-correlations <0·59). Regionally, C difficile decline was driven by elimination of fluoroquinolone-resistant isolates (approximately 67% of Oxfordshire infections in September, 2006, falling to approximately 3% in February, 2013; annual incidence rate ratio 0·52, 95% CI 0·48-0·56 vs fluoroquinolone-susceptible isolates: 1·02, 0·97-1·08). C difficile infections caused by fluoroquinolone-resistant isolates declined in four distinct genotypes (p<0·01). The regions of phylogenies containing fluoroquinolone-resistant isolates were short-branched and geographically structured, consistent with selection and rapid transmission. The importance of fluoroquinolone restriction over infection control was shown by significant declines in inferred secondary (transmitted) cases caused by fluoroquinolone-resistant isolates with or without hospital contact (p<0·0001) versus no change in either group of cases caused by fluoroquinolone-susceptible isolates (p>0·2). INTERPRETATION: Restricting fluoroquinolone prescribing appears to explain the decline in incidence of C difficile infections, above other measures, in Oxfordshire and Leeds, England. Antimicrobial stewardship should be a central component of C difficile infection control programmes. FUNDING: UK Clinical Research Collaboration (Medical Research Council, Wellcome Trust, National Institute for Health Research); NIHR Oxford Biomedical Research Centre; NIHR Health Protection Research Unit on Healthcare Associated Infection and Antimicrobial Resistance (Oxford University in partnership with Public Health England [PHE]), and on Modelling Methodology (Imperial College, London in partnership with PHE); and the Health Innovation Challenge Fund.

Quan TP, Fawcett NJ, Wrightson JM, Finney J, Wyllie D, Jeffery K, Jones N, Shine B, Clarke L, Crook D et al. 2016. Increasing burden of community-acquired pneumonia leading to hospitalisation, 1998-2014. Thorax, 71 (6), pp. 535-542. | Show Abstract | Read more

BACKGROUND: Community-acquired pneumonia (CAP) is a major cause of mortality and morbidity in many countries but few recent large-scale studies have examined trends in its incidence. METHODS: Incidence of CAP leading to hospitalisation in one UK region (Oxfordshire) was calculated over calendar time using routinely collected diagnostic codes, and modelled using piecewise-linear Poisson regression. Further models considered other related diagnoses, typical administrative outcomes, and blood and microbiology test results at admission to determine whether CAP trends could be explained by changes in case-mix, coding practices or admission procedures. RESULTS: CAP increased by 4.2%/year (95% CI 3.6 to 4.8) from 1998 to 2008, and subsequently much faster at 8.8%/year (95% CI 7.8 to 9.7) from 2009 to 2014. Pneumonia-related conditions also increased significantly over this period. Length of stay and 30-day mortality decreased slightly in later years, but the proportions with abnormal neutrophils, urea and C reactive protein (CRP) did not change (p>0.2). The proportion with severely abnormal CRP (>100 mg/L) decreased slightly in later years. Trends were similar in all age groups. Streptococcus pneumoniae was the most common causative organism found; however other organisms, particularly Enterobacteriaceae, increased in incidence over the study period (p<0.001). CONCLUSIONS: Hospitalisations for CAP have been increasing rapidly in Oxfordshire, particularly since 2008. There is little evidence that this is due only to changes in pneumonia coding, an ageing population or patients with substantially less severe disease being admitted more frequently. Healthcare planning to address potential further increases in admissions and consequent antibiotic prescribing should be a priority.

Das S, Lindemann C, Young BC, Muller J, Österreich B, Ternette N, Winkler AC, Paprotka K, Reinhardt R, Förstner KU et al. 2016. Natural mutations in a Staphylococcus aureus virulence regulator attenuate cytotoxicity but permit bacteremia and abscess formation. Proc Natl Acad Sci U S A, 113 (22), pp. E3101-E3110. | Show Abstract | Read more

Staphylococcus aureus is a major bacterial pathogen, which causes severe blood and tissue infections that frequently emerge by autoinfection with asymptomatically carried nose and skin populations. However, recent studies report that bloodstream isolates differ systematically from those found in the nose and skin, exhibiting reduced toxicity toward leukocytes. In two patients, an attenuated toxicity bloodstream infection evolved from an asymptomatically carried high-toxicity nasal strain by loss-of-function mutations in the gene encoding the transcription factor repressor of surface proteins (rsp). Here, we report that rsp knockout mutants lead to global transcriptional and proteomic reprofiling, and they exhibit the greatest signal in a genome-wide screen for genes influencing S. aureus survival in human cells. This effect is likely to be mediated in part via SSR42, a long-noncoding RNA. We show that rsp controls SSR42 expression, is induced by hydrogen peroxide, and is required for normal cytotoxicity and hemolytic activity. Rsp inactivation in laboratory- and bacteremia-derived mutants attenuates toxin production, but up-regulates other immune subversion proteins and reduces lethality during experimental infection. Crucially, inactivation of rsp preserves bacterial dissemination, because it affects neither formation of deep abscesses in mice nor survival in human blood. Thus, we have identified a spontaneously evolving, attenuated-cytotoxicity, nonhemolytic S. aureus phenotype, controlled by a pleiotropic transcriptional regulator/noncoding RNA virulence regulatory system, capable of causing S. aureus bloodstream infections. Such a phenotype could promote deep infection with limited early clinical manifestations, raising concerns that bacterial evolution within the human body may contribute to severe infection.

Whitehouse J, Flaxman A, Rollier C, O'Shea MK, Fallowfield J, Lindsay M, Gunner F, Knox K, Wyllie DH, Yamaguchi Y. 2016. Population variation in anti-S. aureus IgG isotypes influences surface protein A mediated immune subversion. Vaccine, 34 (15), pp. 1792-1799. | Show Abstract | Read more

BACKGROUND: Staphylococcus aureus is a pathogen which causes life-threatening infection, the incidence of which rises during adult life. This, together with the emergence of drug-resistant strains and the expansion of more susceptible elderly populations, represents the rationale for the ongoing development of S. aureus vaccines targeting adult populations. Humoral responses to S. aureus naturally develop early in life, influence susceptibility to infection, and potentially influence the effect of vaccination. Despite this, the nature of pre-existing anti-S. aureus antibodies in healthy adult populations is not fully characterised. METHODS: Immunoglobulin levels against S. aureus surface antigens were measured by a filter membrane enzyme-linked immunosorbent assay using fixed ΔSpA S. aureus as an antigen in serum samples obtained from three clinical cohorts comprising 133 healthy adult volunteers from 19 to 65 years of age. Functional capacity of antibody was also assessed, using antibody-mediated attachment of FITC-stained S. aureus to differentiated HL-60 cells. RESULTS: Wide variation in the concentrations of immunoglobulins recognising S. aureus surface antigens was observed among individuals in all three cohorts. There was a decline of anti-S. aureus IgG1 with age, and a similar trend was observed in IgM, but not in IgA or other IgG sub-classes. Antibody mediated bacterial attachment to cells was associated with IgG1 and IgG3 concentrations in serum. The presence of SpA on the bacterial cell surface reduced antibody-mediated binding of bacteria to phagocytes in serum with low, but not high, levels of naturally occurring anti-S. aureus IgG3 antibodies. CONCLUSIONS: Naturally acquired immunoglobulin responses to S. aureus are heterogeneous in populations and their concentrations alter during adulthood. Elevated IgG1 or IgG3 titres against S. aureus enhance S. aureus recognition by phagocytosis and may be correlates of natural protection and/or vaccine efficacy in adult populations.

Allen ER, van Diemen P, Yamaguchi Y, Lindemann C, Soilleux E, Rollier C, Hill F, Schneider J, Wyllie DH. 2016. MRI Based Localisation and Quantification of Abscesses following Experimental S. aureus Intravenous Challenge: Application to Vaccine Evaluation. PLoS One, 11 (5), pp. e0154705. | Show Abstract | Read more

PURPOSE: To develop and validate a sensitive and specific method of abscess enumeration and quantification in a preclinical model of Staphylococcus aureus infection. METHODS: S. aureus infected murine kidneys were fixed in paraformaldehyde, impregnated with gadolinium, and embedded in agar blocks, which were subjected to 3D magnetic resonance microscopy on a 9.4T MRI scanner. Image analysis techniques were developed, which could identify and quantify abscesses. The result of this imaging was compared with histological examination. The impact of a S. aureus Sortase A vaccination regime was assessed using the technique. RESULTS: Up to 32 murine kidneys could be imaged in a single MRI run, yielding images with voxels of about 25 μm3. S. aureus abscesses could be readily identified in blinded analyses of the kidneys after 3 days of infection, with low inter-observer variability. Comparison with histological sections shows a striking correlation between the two techniques: all presumptive abscesses identified by MRI were confirmed histologically, and histology identified no abscesses not evident on MRI. In view of this, simulations were performed assuming that both MRI reconstruction, and histology examining all sections of the tissue, were fully sensitive and specific at abscess detection. This simulation showed that MRI provided more sensitive and precise estimates of abscess numbers and volume than histology, unless at least 5 histological sections are taken through the long axis of the kidney. We used the MRI technique described to investigate the impact of a S. aureus Sortase A vaccine. CONCLUSION: Post mortem MRI scanning of large batches of fixed organs has application in the preclinical assessment of S. aureus vaccines.

Flaxman A, Allen E, Lindemann C, Yamaguchi Y, O'Shea MK, Fallowfield JL, Lindsay M, Gunner F, Knox K, Wyllie DH. 2016. Risk factors for dermatitis in submariners during a submerged patrol: an observational cohort study. BMJ Open, 6 (6), pp. e010975. | Show Abstract | Read more

OBJECTIVE: The aim of this pilot study was to determine risk factors, including Staphylococcus aureus nasal carriage, for dermatitis in submariners during a submarine patrol. PARTICIPANTS AND METHODS: 36 submariners undertaking a submerged 6-week patrol participated in the study. Severity of dermatitis and its impact was assessed using visual analogue scales and questionnaires at baseline and weekly throughout the patrol. S. aureus carriage levels in submariners were determined by nasal swabbing at baseline and shortly before disembarking the submarine. Occurrence of any skin or soft tissue infections (SSTI) were reported to the medical officer and swabs of the area were taken for subsequent analysis. RESULTS: S. aureus carriers were significantly more likely than non-carriers to have previously received treatment for a cutaneous abscess (39% vs 5%, OR=13 (95% CI 1.3 to 130)) with a trend to being submariners longer (p=0.051). Skin scores at baseline and on patrol were not significantly associated with carriage status. Higher dermatitis scores were observed in those who had been submariners longer (p=0.045). Smoking and allergies were not found to be linked to carriage status or skin health score in this cohort. CONCLUSIONS: This small pilot study investigates S. aureus carriage status and skin health in submariners. Length of submarine service but not S. aureus carriage was identified as a risk factor for worsening skin health in this small cohort during a 6-week patrol. This does not support S. aureus decolonisation to improve skin health in this population. Further investigation into causes of dermatitis in submariners is required. This data supports a better understanding of the potential impact of exposure to environmental factors that could affect skin health in submariners.

Stoesser N, Sheppard AE, Pankhurst L, De Maio N, Moore CE, Sebra R, Turner P, Anson LW, Kasarskis A, Batty EM et al. 2016. Evolutionary History of the Global Emergence of the Escherichia coli Epidemic Clone ST131. MBio, 7 (2), pp. e02162. | Show Abstract | Read more

UNLABELLED: Escherichia colisequence type 131 (ST131) has emerged globally as the most predominant extraintestinal pathogenic lineage within this clinically important species, and its association with fluoroquinolone and extended-spectrum cephalosporin resistance impacts significantly on treatment. The evolutionary histories of this lineage, and of important antimicrobial resistance elements within it, remain unclearly defined. This study of the largest worldwide collection (n= 215) of sequenced ST131E. coliisolates to date demonstrates that the clonal expansion of two previously recognized antimicrobial-resistant clades, C1/H30R and C2/H30Rx, started around 25 years ago, consistent with the widespread introduction of fluoroquinolones and extended-spectrum cephalosporins in clinical medicine. These two clades appear to have emerged in the United States, with the expansion of the C2/H30Rx clade driven by the acquisition of ablaCTX-M-15-containing IncFII-like plasmid that has subsequently undergone extensive rearrangement. Several other evolutionary processes influencing the trajectory of this drug-resistant lineage are described, including sporadic acquisitions of CTX-M resistance plasmids and chromosomal integration ofblaCTX-Mwithin subclusters followed by vertical evolution. These processes are also occurring for another family of CTX-M gene variants more recently observed among ST131, theblaCTX-M-14/14-likegroup. The complexity of the evolutionary history of ST131 has important implications for antimicrobial resistance surveillance, epidemiological analysis, and control of emerging clinical lineages ofE. coli These data also highlight the global imperative to reduce specific antibiotic selection pressures and demonstrate the important and varied roles played by plasmids and other mobile genetic elements in the perpetuation of antimicrobial resistance within lineages. IMPORTANCE: Escherichia coli, perennially a major bacterial pathogen, is becoming increasingly difficult to manage due to emerging resistance to all preferred antimicrobials. Resistance is concentrated within specificE. colilineages, such as sequence type 131 (ST131). Clarification of the genetic basis for clonally associated resistance is key to devising intervention strategies. We used high-resolution genomic analysis of a large global collection of ST131 isolates to define the evolutionary history of extended-spectrum beta-lactamase production in ST131. We documented diverse contributory genetic processes, including stable chromosomal integrations of resistance genes, persistence and evolution of mobile resistance elements within sublineages, and sporadic acquisition of different resistance elements. Both global distribution and regional segregation were evident. The diversity of resistance element acquisition and propagation within ST131 indicates a need for control and surveillance strategies that target both bacterial strains and mobile genetic elements.

Daniels-Treffandier H, de Nie K, Marsay L, Dold C, Sadarangani M, Reyes-Sandoval A, Langford PR, Wyllie D, Hill F, Pollard AJ, Rollier CS. 2016. Impact of Reducing Complement Inhibitor Binding on the Immunogenicity of Native Neisseria meningitidis Outer Membrane Vesicles. PLoS One, 11 (2), pp. e0148840. | Show Abstract | Read more

Neisseria meningitidis recruits host human complement inhibitors to its surface to down-regulate complement activation and enhance survival in blood. We have investigated whether such complement inhibitor binding occurs after vaccination with native outer membrane vesicles (nOMVs), and limits immunogenicity of such vaccines. To this end, nOMVs reactogenic lipopolysaccharide was detoxified by deletion of the lpxl1 gene (nOMVlpxl1). nOMVs unable to bind human complement factor H (hfH) were generated by additional deletions of the genes encoding factor H binding protein (fHbp) and neisserial surface protein A (NspA) (nOMVdis). Antibody responses elicited in mice with nOMVdis were compared to those elicited with nOMVlpxl1 in the presence of hfH. Results demonstrate that the administration of human fH to mice immunized with fHbp containing OMVlpxl1 decreased immunogenicity against fHbp (but not against the OMV as a whole). The majority of the OMV-induced bactericidal immune response (OMVlpxl1 or OMVdis) was versus PorA. Despite a considerable reduction of hfH binding to nOMVdis, and the absence of the vaccine antigen fHbp, immunogenicity in mice was not different from nOMVlpxl1, in the absence or presence of hfH (serum bactericidal titers of 1:64 vs 1:128 after one dose in the nOMVdis and nOMVlpxl1-immunized groups respectively). Therefore, partial inhibition of fH binding did not enhance immunity in this model.

Pankhurst LJ, Del Ojo Elias C, Votintseva AA, Walker TM, Cole K, Davies J, Fermont JM, Gascoyne-Binzi DM, Kohl TA, Kong C et al. 2016. Rapid, comprehensive, and affordable mycobacterial diagnosis with whole-genome sequencing: a prospective study. Lancet Respir Med, 4 (1), pp. 49-58. | Show Abstract | Read more

BACKGROUND: Slow and cumbersome laboratory diagnostics for Mycobacterium tuberculosis complex (MTBC) risk delayed treatment and poor patient outcomes. Whole-genome sequencing (WGS) could potentially provide a rapid and comprehensive diagnostic solution. In this prospective study, we compare real-time WGS with routine MTBC diagnostic workflows. METHODS: We compared sequencing mycobacteria from all newly positive liquid cultures with routine laboratory diagnostic workflows across eight laboratories in Europe and North America for diagnostic accuracy, processing times, and cost between Sept 6, 2013, and April 14, 2014. We sequenced specimens once using local Illumina MiSeq platforms and processed data centrally using a semi-automated bioinformatics pipeline. We identified species or complex using gene presence or absence, predicted drug susceptibilities from resistance-conferring mutations identified from reference-mapped MTBC genomes, and calculated genetic distance to previously sequenced UK MTBC isolates to detect outbreaks. WGS data processing and analysis was done by staff masked to routine reference laboratory and clinical results. We also did a microcosting analysis to assess the financial viability of WGS-based diagnostics. FINDINGS: Compared with routine results, WGS predicted species with 93% (95% CI 90-96; 322 of 345 specimens; 356 mycobacteria specimens submitted) accuracy and drug susceptibility also with 93% (91-95; 628 of 672 specimens; 168 MTBC specimens identified) accuracy, with one sequencing attempt. WGS linked 15 (16% [95% CI 10-26]) of 91 UK patients to an outbreak. WGS diagnosed a case of multidrug-resistant tuberculosis before routine diagnosis was completed and discovered a new multidrug-resistant tuberculosis cluster. Full WGS diagnostics could be generated in a median of 9 days (IQR 6-10), a median of 21 days (IQR 14-32) faster than final reference laboratory reports were produced (median of 31 days [IQR 21-44]), at a cost of £481 per culture-positive specimen, whereas routine diagnosis costs £518, equating to a WGS-based diagnosis cost that is 7% cheaper annually than are present diagnostic workflows. INTERPRETATION: We have shown that WGS has a scalable, rapid turnaround, and is a financially feasible method for full MTBC diagnostics. Continued improvements to mycobacterial processing, bioinformatics, and analysis will improve the accuracy, speed, and scope of WGS-based diagnosis. FUNDING: National Institute for Health Research, Department of Health, Wellcome Trust, British Colombia Centre for Disease Control Foundation for Population and Public Health, Department of Clinical Microbiology, Trinity College Dublin.

Bradley P, Gordon NC, Walker TM, Dunn L, Heys S, Huang B, Earle S, Pankhurst LJ, Anson L, de Cesare M et al. 2015. Rapid antibiotic-resistance predictions from genome sequence data for Staphylococcus aureus and Mycobacterium tuberculosis. Nat Commun, 6 pp. 10063. | Show Abstract | Read more

The rise of antibiotic-resistant bacteria has led to an urgent need for rapid detection of drug resistance in clinical samples, and improvements in global surveillance. Here we show how de Bruijn graph representation of bacterial diversity can be used to identify species and resistance profiles of clinical isolates. We implement this method for Staphylococcus aureus and Mycobacterium tuberculosis in a software package ('Mykrobe predictor') that takes raw sequence data as input, and generates a clinician-friendly report within 3 minutes on a laptop. For S. aureus, the error rates of our method are comparable to gold-standard phenotypic methods, with sensitivity/specificity of 99.1%/99.6% across 12 antibiotics (using an independent validation set, n=470). For M. tuberculosis, our method predicts resistance with sensitivity/specificity of 82.6%/98.5% (independent validation set, n=1,609); sensitivity is lower here, probably because of limited understanding of the underlying genetic mechanisms. We give evidence that minor alleles improve detection of extremely drug-resistant strains, and demonstrate feasibility of the use of emerging single-molecule nanopore sequencing techniques for these purposes.

Votintseva AA, Pankhurst LJ, Anson LW, Morgan MR, Gascoyne-Binzi D, Walker TM, Quan TP, Wyllie DH, Del Ojo Elias C, Wilcox M et al. 2015. Mycobacterial DNA extraction for whole-genome sequencing from early positive liquid (MGIT) cultures. J Clin Microbiol, 53 (4), pp. 1137-1143. | Show Abstract | Read more

We developed a low-cost and reliable method of DNA extraction from as little as 1 ml of early positive mycobacterial growth indicator tube (MGIT) cultures that is suitable for whole-genome sequencing to identify mycobacterial species and predict antibiotic resistance in clinical samples. The DNA extraction method is based on ethanol precipitation supplemented by pretreatment steps with a MolYsis kit or saline wash for the removal of human DNA and a final DNA cleanup step with solid-phase reversible immobilization beads. The protocol yielded ≥0.2 ng/μl of DNA for 90% (MolYsis kit) and 83% (saline wash) of positive MGIT cultures. A total of 144 (94%) of the 154 samples sequenced on the MiSeq platform (Illumina) achieved the target of 1 million reads, with <5% of reads derived from human or nasopharyngeal flora for 88% and 91% of samples, respectively. A total of 59 (98%) of 60 samples that were identified by the national mycobacterial reference laboratory (NMRL) as Mycobacterium tuberculosis were successfully mapped to the H37Rv reference, with >90% coverage achieved. The DNA extraction protocol, therefore, will facilitate fast and accurate identification of mycobacterial species and resistance using a range of bioinformatics tools.

Walker TM, Kohl TA, Omar SV, Hedge J, Del Ojo Elias C, Bradley P, Iqbal Z, Feuerriegel S, Niehaus KE, Wilson DJ et al. 2015. Whole-genome sequencing for prediction of Mycobacterium tuberculosis drug susceptibility and resistance: a retrospective cohort study. Lancet Infect Dis, 15 (10), pp. 1193-1202. | Show Abstract | Read more

BACKGROUND: Diagnosing drug-resistance remains an obstacle to the elimination of tuberculosis. Phenotypic drug-susceptibility testing is slow and expensive, and commercial genotypic assays screen only common resistance-determining mutations. We used whole-genome sequencing to characterise common and rare mutations predicting drug resistance, or consistency with susceptibility, for all first-line and second-line drugs for tuberculosis. METHODS: Between Sept 1, 2010, and Dec 1, 2013, we sequenced a training set of 2099 Mycobacterium tuberculosis genomes. For 23 candidate genes identified from the drug-resistance scientific literature, we algorithmically characterised genetic mutations as not conferring resistance (benign), resistance determinants, or uncharacterised. We then assessed the ability of these characterisations to predict phenotypic drug-susceptibility testing for an independent validation set of 1552 genomes. We sought mutations under similar selection pressure to those characterised as resistance determinants outside candidate genes to account for residual phenotypic resistance. FINDINGS: We characterised 120 training-set mutations as resistance determining, and 772 as benign. With these mutations, we could predict 89·2% of the validation-set phenotypes with a mean 92·3% sensitivity (95% CI 90·7-93·7) and 98·4% specificity (98·1-98·7). 10·8% of validation-set phenotypes could not be predicted because uncharacterised mutations were present. With an in-silico comparison, characterised resistance determinants had higher sensitivity than the mutations from three line-probe assays (85·1% vs 81·6%). No additional resistance determinants were identified among mutations under selection pressure in non-candidate genes. INTERPRETATION: A broad catalogue of genetic mutations enable data from whole-genome sequencing to be used clinically to predict drug resistance, drug susceptibility, or to identify drug phenotypes that cannot yet be genetically predicted. This approach could be integrated into routine diagnostic workflows, phasing out phenotypic drug-susceptibility testing while reporting drug resistance early. FUNDING: Wellcome Trust, National Institute of Health Research, Medical Research Council, and the European Union.

Wyllie D, Davies J. 2015. Role of data warehousing in healthcare epidemiology Journal of Hospital Infection, 89 (4), pp. 267-270. | Show Abstract | Read more

© 2015 Published by Elsevier Ltd on behalf of the Healthcare Infection Society.Electronic storage of healthcare data, including individual-level risk factors for both infectious and other diseases, is increasing. These data can be integrated at hospital, regional and national levels. Data sources that contain risk factor and outcome information for a wide range of conditions offer the potential for efficient epidemiological analysis of multiple diseases. Opportunities may also arise for monitoring healthcare processes. Integrating diverse data sources presents epidemiological, practical, and ethical challenges. For example, diagnostic criteria, outcome definitions, and ascertainment methods may differ across the data sources. Data volumes may be very large, requiring sophisticated computing technology. Given the large populations involved, perhaps the most challenging aspect is how informed consent can be obtained for the development of integrated databases, particularly when it is not easy to demonstrate their potential. In this article, we discuss some of the ups and downs of recent projects as well as the potential of data warehousing for antimicrobial resistance monitoring.

Yamaguchi Y, Anson L, Crook D, Knox K, Wyllie D. 2014. Evidence for control of Staphylococcus aureus nasal carriage by innate-like T lymphocyte populations in humans: a cross sectional study IMMUNOLOGY, 143 pp. 73-73.

Spencer AJ, Furze J, Honeycutt JD, Calvert A, Saurya S, Colloca S, Wyllie DH, Gilbert SC, Bregu M, Cottingham MG, Hill AV. 2014. 4-1BBL enhances CD8+ T cell responses induced by vectored vaccines in mice but fails to improve immunogenicity in rhesus macaques. PLoS One, 9 (8), pp. e105520. | Show Abstract | Read more

T cells play a central role in the immune response to many of the world's major infectious diseases. In this study we investigated the tumour necrosis factor receptor superfamily costimulatory molecule, 4-1BBL (CD137L, TNFSF9), for its ability to increase T cell immunogenicity induced by a variety of recombinant vectored vaccines. To efficiently test this hypothesis, we assessed a number of promoters and developed a stable bi-cistronic vector expressing both the antigen and adjuvant. Co-expression of 4-1BBL, together with our model antigen TIP, was shown to increase the frequency of murine antigen-specific IFN-γ secreting CD8(+) T cells in three vector platforms examined. Enhancement of the response was not limited by co-expression with the antigen, as an increase in CD8(+) immunogenicity was also observed by co-administration of two vectors each expressing only the antigen or adjuvant. However, when this regimen was tested in non-human primates using a clinical malaria vaccine candidate, no adjuvant effect of 4-1BBL was observed limiting its potential use as a single adjuvant for translation into a clinical vaccine.

Votintseva AA, Fung R, Miller RR, Knox K, Godwin H, Wyllie DH, Bowden R, Crook DW, Walker AS. 2014. Prevalence of Staphylococcus aureus protein A (spa) mutants in the community and hospitals in Oxfordshire. BMC Microbiol, 14 (1), pp. 63. | Show Abstract | Read more

BACKGROUND: Staphylococcal protein A (spa) is an important virulence factor which enables Staphylococcus aureus to evade host immune responses. Genotypes known as "spa-types", based on highly variable Xr region sequences of the spa-gene, are frequently used to classify strains. A weakness of current spa-typing primers is that rearrangements in the IgG-binding region of the gene cause 1-2% of strains to be designated as "non-typeable". RESULTS: We developed an improved primer which enabled sequencing of all strains, containing any type of genetic rearrangement, in a large study among community carriers and hospital inpatients in Oxfordshire, UK (6110 isolates). We identified eight novel spa-gene variants, plus one previously described. Three of these rearrangements would be designated "non-typeable" using current spa-typing methods; they occurred in 1.8% (72/3905) asymptomatically carried and 0.6% (14/2205) inpatient S. aureus strains. Some individuals were simultaneously colonized by both formerly non-typeable and typeable strains; previously such patients would have been identified as carrying only currently typeable strains, underestimating mixed carriage prevalence and diversity. Formerly non-typeable strains were found in more spa-types associated with multilocus sequence type ST398 (35%), common among livestock, compared to other groups with any non-typeable strains (1-4%), suggesting particular spa-types may have been under-represented in previous human studies. CONCLUSIONS: This improved method allows us to spa-type previously non-typeable strains with rearrangements in the spa-gene and to resolve cases of mixed colonization with deletions in one or more strains, thus accounting for hidden diversity of S. aureus in both community and hospital environments.

Miller RM, Price JR, Batty EM, Didelot X, Wyllie D, Golubchik T, Crook DW, Paul J, Peto TE, Wilson DJ et al. 2014. Healthcare-associated outbreak of meticillin-resistant Staphylococcus aureus bacteraemia: role of a cryptic variant of an epidemic clone. J Hosp Infect, 86 (2), pp. 83-89. | Show Abstract | Read more

BACKGROUND: New strains of meticillin-resistant Staphylococcus aureus (MRSA) may be associated with changes in rates of disease or clinical presentation. Conventional typing techniques may not detect new clonal variants that underlie changes in epidemiology or clinical phenotype. AIM: To investigate the role of clonal variants of MRSA in an outbreak of MRSA bacteraemia at a hospital in England. METHODS: Bacteraemia isolates of the major UK lineages (EMRSA-15 and -16) from before and after the outbreak were analysed by whole-genome sequencing in the context of epidemiological and clinical data. For comparison, EMRSA-15 and -16 isolates from another hospital in England were sequenced. A clonal variant of EMRSA-16 was identified at the outbreak hospital and a molecular signature test designed to distinguish variant isolates among further EMRSA-16 strains. FINDINGS: By whole-genome sequencing, EMRSA-16 isolates during the outbreak showed strikingly low genetic diversity (P < 1 × 10(-6), Monte Carlo test), compared with EMRSA-15 and EMRSA-16 isolates from before the outbreak or the comparator hospital, demonstrating the emergence of a clonal variant. The variant was indistinguishable from the ancestral strain by conventional typing. This clonal variant accounted for 64/72 (89%) of EMRSA-16 bacteraemia isolates at the outbreak hospital from 2006. CONCLUSIONS: Evolutionary changes in epidemic MRSA strains not detected by conventional typing may be associated with changes in disease epidemiology. Rapid and affordable technologies for whole-genome sequencing are becoming available with the potential to identify and track the emergence of variants of highly clonal organisms.

Matthews PC, Chue AL, Wyllie D, Barnett A, Isinkaye T, Jefferies L, Lovering A, Scarborough M. 2014. Increased teicoplanin doses are associated with improved serum levels but not drug toxicity Journal of Infection, 68 (1), pp. 43-49. | Show Abstract | Read more

Objective: Teicoplanin is widely used for the treatment of severe gram-positive infection, aiming to achieve trough serum levels of 20-60mg/L for patients with severe infection. A standard 400mg daily dose is frequently associated with sub-therapeutic levels, and we have therefore changed our routine approach to 600mg daily (following loading doses in each case). We set out to investigate the impact of this dose increase on drug levels and potential side-effects. Methods: We undertook a retrospective study of 549 consecutive adult Out-Patient Antimicrobial Treatment (OPAT) episodes treated with intravenous teicoplanin. Results: Therapeutic teicoplanin levels were more frequently achieved in patients treated with 600mg compared to 400mg daily (68% vs. 37% respectively, p<0.0001), without an increased frequency of potentially toxic levels, defined as >60mg/L (6% vs. 8% respectively, p=0.4). There was no difference in the incidence of neutropaenia, eosinophilia, thrombocytopaenia, acute renal injury or treatment cessation in patients treated with the higher teicoplanin dose. Conclusions: In the majority of stable adult patients with normal renal function, we advocate a loading regimen (600mg b.d. for two doses) followed by a 600mg daily teicoplanin dose in order to achieve therapeutic trough levels. © 2013 The British Infection Association.

Wong TH, Dearlove BL, Hedge J, Giess AP, Piazza P, Trebes A, Paul J, Smit E, Smith EG, Sutton JK et al. 2013. Whole genome sequencing and de novo assembly identifies Sydney-like variant noroviruses and recombinants during the winter 2012/2013 outbreak in England. Virol J, 10 (1), pp. 335. | Show Abstract | Read more

BACKGROUND: Norovirus is the commonest cause of epidemic gastroenteritis among people of all ages. Outbreaks frequently occur in hospitals and the community, costing the UK an estimated £110 m per annum. An evolutionary explanation for periodic increases in norovirus cases, despite some host-specific post immunity is currently limited to the identification of obvious recombinants. Our understanding could be significantly enhanced by full length genome sequences for large numbers of intensively sampled viruses, which would also assist control and vaccine design. Our objective is to develop rapid, high-throughput, end-to-end methods yielding complete norovirus genome sequences. We apply these methods to recent English outbreaks, placing them in the wider context of the international norovirus epidemic of winter 2012. METHOD: Norovirus sequences were generated from 28 unique clinical samples by Illumina RNA sequencing (RNA-Seq) of total faecal RNA. A range of de novo sequence assemblers were attempted. The best assembler was identified by validation against three replicate samples and two norovirus qPCR negative samples, together with an additional 20 sequences determined by PCR and fractional capillary sequencing. Phylogenetic methods were used to reconstruct evolutionary relationships from the whole genome sequences. RESULTS: Full length norovirus genomes were generated from 23/28 samples. 5/28 partial norovirus genomes were associated with low viral copy numbers. The de novo assembled sequences differed from sequences determined by capillary sequencing by <0.003%. Intra-host nucleotide sequence diversity was rare, but detectable by mapping short sequence reads onto its de novo assembled consensus. Genomes similar to the Sydney 2012 strain caused 78% (18/23) of cases, consistent with its previously documented association with the winter 2012 global outbreak. Interestingly, phylogenetic analysis and recombination detection analysis of the consensus sequences identified two related viruses as recombinants, containing sequences in prior circulation to Sydney 2012 in open reading frame (ORF) 2. CONCLUSION: Our approach facilitates the rapid determination of complete norovirus genomes. This method provides high resolution of full norovirus genomes which, when coupled with detailed epidemiology, may improve the understanding of evolution and control of this important healthcare-associated pathogen.

Eyre DW, Cule ML, Wilson DJ, Griffiths D, Vaughan A, O'Connor L, Ip CL, Golubchik T, Batty EM, Finney JM et al. 2013. Diverse sources of C. difficile infection identified on whole-genome sequencing. N Engl J Med, 369 (13), pp. 1195-1205. | Show Abstract | Read more

BACKGROUND: It has been thought that Clostridium difficile infection is transmitted predominantly within health care settings. However, endemic spread has hampered identification of precise sources of infection and the assessment of the efficacy of interventions. METHODS: From September 2007 through March 2011, we performed whole-genome sequencing on isolates obtained from all symptomatic patients with C. difficile infection identified in health care settings or in the community in Oxfordshire, United Kingdom. We compared single-nucleotide variants (SNVs) between the isolates, using C. difficile evolution rates estimated on the basis of the first and last samples obtained from each of 145 patients, with 0 to 2 SNVs expected between transmitted isolates obtained less than 124 days apart, on the basis of a 95% prediction interval. We then identified plausible epidemiologic links among genetically related cases from data on hospital admissions and community location. RESULTS: Of 1250 C. difficile cases that were evaluated, 1223 (98%) were successfully sequenced. In a comparison of 957 samples obtained from April 2008 through March 2011 with those obtained from September 2007 onward, a total of 333 isolates (35%) had no more than 2 SNVs from at least 1 earlier case, and 428 isolates (45%) had more than 10 SNVs from all previous cases. Reductions in incidence over time were similar in the two groups, a finding that suggests an effect of interventions targeting the transition from exposure to disease. Of the 333 patients with no more than 2 SNVs (consistent with transmission), 126 patients (38%) had close hospital contact with another patient, and 120 patients (36%) had no hospital or community contact with another patient. Distinct subtypes of infection continued to be identified throughout the study, which suggests a considerable reservoir of C. difficile. CONCLUSIONS: Over a 3-year period, 45% of C. difficile cases in Oxfordshire were genetically distinct from all previous cases. Genetically diverse sources, in addition to symptomatic patients, play a major part in C. difficile transmission. (Funded by the U.K. Clinical Research Collaboration Translational Infection Research Initiative and others.).

Matthews PC, Chue AL, Wyllie D, Barnett A, Isinkaye T, Jefferies L, Lovering A, Scarborough M. 2014. Increased teicoplanin doses are associated with improved serum levels but not drug toxicity. J Infect, 68 (1), pp. 43-49. | Show Abstract | Read more

OBJECTIVE: Teicoplanin is widely used for the treatment of severe gram-positive infection, aiming to achieve trough serum levels of 20-60 mg/L for patients with severe infection. A standard 400 mg daily dose is frequently associated with sub-therapeutic levels, and we have therefore changed our routine approach to 600 mg daily (following loading doses in each case). We set out to investigate the impact of this dose increase on drug levels and potential side-effects. METHODS: We undertook a retrospective study of 549 consecutive adult Out-Patient Antimicrobial Treatment (OPAT) episodes treated with intravenous teicoplanin. RESULTS: Therapeutic teicoplanin levels were more frequently achieved in patients treated with 600 mg compared to 400 mg daily (68% vs. 37% respectively, p < 0.0001), without an increased frequency of potentially toxic levels, defined as >60 mg/L (6% vs. 8% respectively, p = 0.4). There was no difference in the incidence of neutropaenia, eosinophilia, thrombocytopaenia, acute renal injury or treatment cessation in patients treated with the higher teicoplanin dose. CONCLUSIONS: In the majority of stable adult patients with normal renal function, we advocate a loading regimen (600 mg b.d. for two doses) followed by a 600 mg daily teicoplanin dose in order to achieve therapeutic trough levels.

Batty EM, Wong TH, Trebes A, Argoud K, Attar M, Buck D, Ip CL, Golubchik T, Cule M, Bowden R et al. 2013. A modified RNA-Seq approach for whole genome sequencing of RNA viruses from faecal and blood samples. PLoS One, 8 (6), pp. e66129. | Show Abstract | Read more

To date, very large scale sequencing of many clinically important RNA viruses has been complicated by their high population molecular variation, which creates challenges for polymerase chain reaction and sequencing primer design. Many RNA viruses are also difficult or currently not possible to culture, severely limiting the amount and purity of available starting material. Here, we describe a simple, novel, high-throughput approach to Norovirus and Hepatitis C virus whole genome sequence determination based on RNA shotgun sequencing (also known as RNA-Seq). We demonstrate the effectiveness of this method by sequencing three Norovirus samples from faeces and two Hepatitis C virus samples from blood, on an Illumina MiSeq benchtop sequencer. More than 97% of reference genomes were recovered. Compared with Sanger sequencing, our method had no nucleotide differences in 14,019 nucleotides (nt) for Noroviruses (from a total of 2 Norovirus genomes obtained with Sanger sequencing), and 8 variants in 9,542 nt for Hepatitis C virus (1 variant per 1,193 nt). The three Norovirus samples had 2, 3, and 2 distinct positions called as heterozygous, while the two Hepatitis C virus samples had 117 and 131 positions called as heterozygous. To confirm that our sample and library preparation could be scaled to true high-throughput, we prepared and sequenced an additional 77 Norovirus samples in a single batch on an Illumina HiSeq 2000 sequencer, recovering >90% of the reference genome in all but one sample. No discrepancies were observed across 118,757 nt compared between Sanger and our custom RNA-Seq method in 16 samples. By generating viral genomic sequences that are not biased by primer-specific amplification or enrichment, this method offers the prospect of large-scale, affordable studies of RNA viruses which could be adapted to routine diagnostic laboratory workflows in the near future, with the potential to directly characterize within-host viral diversity.

van Diemen PM, Yamaguchi Y, Paterson GK, Rollier CS, Hill AV, Wyllie DH. 2013. Irradiated wild-type and Spa mutant Staphylococcus aureus induce anti-S. aureus immune responses in mice which do not protect against subsequent intravenous challenge. Pathog Dis, 68 (1), pp. 20-26. | Show Abstract | Read more

Staphylococcus aureus remains an important human and animal pathogen. Its pathogenicity is determined in part by expression of the Spa-immune subversion protein, neutralising the activity of which provides partial protection in murine models, as does experimental infection with live S. aureus with Spa gene deletions followed by antibiotic-mediated cure in mice. Together, these data raise the question of whether Spa mutant S. aureus might represent a viable vaccine. Here, we find that gamma-irradiated S. aureus strains, both wild-type and null mutant of spa, are immunogenic in mice when administered intramuscularly, eliciting large amounts of anti-S. aureus antibodies, as judged by whole-cell immunoassay on fixed microorganisms. We used an intravenous challenge system to assess vaccine efficacy, the sensitivity of which was increased by studying renal bacterial concentrations in both kidneys. Despite this, protection from intravenous challenge was not observed (mean difference between vaccinated and unvaccinated mice 0.27 log(10) with 95% confidence interval -0.922 to 1.467). Surprisingly, antibody responses elicited against a panel of protective cell surface proteins were very low, indicating that most antibody induced is not protective. Additionally, these data suggest a limited role for irradiated wild-type or spa mutant S. aureus as vaccines.

Stoesser N, Batty EM, Eyre DW, Morgan M, Wyllie DH, Del Ojo Elias C, Johnson JR, Walker AS, Peto TE, Crook DW. 2013. Predicting antimicrobial susceptibilities for Escherichia coli and Klebsiella pneumoniae isolates using whole genomic sequence data. J Antimicrob Chemother, 68 (10), pp. 2234-2244. | Show Abstract | Read more

OBJECTIVES: Whole-genome sequencing potentially represents a single, rapid and cost-effective approach to defining resistance mechanisms and predicting phenotype, and strain type, for both clinical and epidemiological purposes. This retrospective study aimed to determine the efficacy of whole genome-based antimicrobial resistance prediction in clinical isolates of Escherichia coli and Klebsiella pneumoniae. METHODS: Seventy-four E. coli and 69 K. pneumoniae bacteraemia isolates from Oxfordshire, UK, were sequenced (Illumina HiSeq 2000). Resistance phenotypes were predicted from genomic sequences using BLASTn-based comparisons of de novo-assembled contigs with a study database of >100 known resistance-associated loci, including plasmid-associated and chromosomal genes. Predictions were made for seven commonly used antimicrobials: amoxicillin, co-amoxiclav, ceftriaxone, ceftazidime, ciprofloxacin, gentamicin and meropenem. Comparisons were made with phenotypic results obtained in duplicate by broth dilution (BD Phoenix). Discrepancies, either between duplicate BD Phoenix results or between genotype and phenotype, were resolved with gradient diffusion analyses. RESULTS: A wide variety of antimicrobial resistance genes were identified, including blaCTX-M, blaLEN, blaOKP, blaOXA, blaSHV, blaTEM, aac(3')-Ia, aac-(3')-IId, aac-(3')-IIe, aac(6')-Ib-cr, aadA1a, aadA4, aadA5, aadA16, aph(6')-Id, aph(3')-Ia, qnrB and qnrS, as well as resistance-associated mutations in chromosomal gyrA and parC genes. The sensitivity of genome-based resistance prediction across all antibiotics for both species was 0.96 (95% CI: 0.94-0.98) and the specificity was 0.97 (95% CI: 0.95-0.98). Very major and major error rates were 1.2% and 2.1%, respectively. CONCLUSIONS: Our method was as sensitive and specific as routinely deployed phenotypic methods. Validation against larger datasets and formal assessments of cost and turnaround time in a routine laboratory setting are warranted.

Walker AS, Eyre DW, Wyllie DH, Dingle KE, Griffiths D, Shine B, Oakley S, O'Connor L, Finney J, Vaughan A et al. 2013. Relationship between bacterial strain type, host biomarkers, and mortality in Clostridium difficile infection. Clin Infect Dis, 56 (11), pp. 1589-1600. | Show Abstract | Read more

BACKGROUND: Despite substantial interest in biomarkers, their impact on clinical outcomes and variation with bacterial strain has rarely been explored using integrated databases. METHODS: From September 2006 to May 2011, strains isolated from Clostridium difficile toxin enzyme immunoassay (EIA)-positive fecal samples from Oxfordshire, United Kingdom (approximately 600,000 people) underwent multilocus sequence typing. Fourteen-day mortality and levels of 15 baseline biomarkers were compared between consecutive C. difficile infections (CDIs) from different clades/sequence types (STs) and EIA-negative controls using Cox and normal regression adjusted for demographic/clinical factors. RESULTS: Fourteen-day mortality was 13% in 2222 adults with 2745 EIA-positive samples (median, 78 years) vs 5% in 20,722 adults with 27,550 EIA-negative samples (median, 74 years) (absolute attributable mortality, 7.7%; 95% CI, 6.4%-9.0%). Mortality was highest in clade 5 CDIs (25% [16 of 63]; polymerase chain reaction (PCR) ribotype 078/ST 11), then clade 2 (20% [111 of 560]; 99% PCR ribotype 027/ST 1) versus clade 1 (12% [137 of 1168]; adjusted P < .0001). Within clade 1, 14-day mortality was only 4% (3 of 84) in ST 44 (PCR ribotype 015) (adjusted P = .05 vs other clade 1). Mean baseline neutrophil counts also varied significantly by genotype: 12.4, 11.6, and 9.5 × 10(9) neutrophils/L for clades 5, 2 and 1, respectively, vs 7.0 × 10(9) neutrophils/L in EIA-negative controls (P < .0001) and 7.9 × 10(9) neutrophils/L in ST 44 (P = .08). There were strong associations between C. difficile-type-specific effects on mortality and neutrophil/white cell counts (rho = 0.48), C-reactive-protein (rho = 0.43), eosinophil counts (rho = -0.45), and serum albumin (rho = -0.47). Biomarkers predicted 30%-40% of clade-specific mortality differences. CONCLUSIONS: C. difficile genotype predicts mortality, and excess mortality correlates with genotype-specific changes in biomarkers, strongly implicating inflammatory pathways as a major influence on poor outcome after CDI. PCR ribotype 078/ST 11 (clade 5) leads to severe CDI; thus ongoing surveillance remains essential.

Schlackow I, Walker AS, Dingle K, Griffiths D, Oakley S, Finney J, Vaughan A, Gill MJ, Crook DW, Peto TE, Wyllie DH. 2012. Surveillance of infection severity: a registry study of laboratory diagnosed Clostridium difficile. PLoS Med, 9 (7), pp. e1001279. | Show Abstract | Read more

BACKGROUND: Changing clinical impact, as virulent clones replace less virulent ones, is a feature of many pathogenic bacterial species and can be difficult to detect. Consequently, innovative techniques monitoring infection severity are of potential clinical value. METHODS AND FINDINGS: We studied 5,551 toxin-positive and 20,098 persistently toxin-negative patients tested for Clostridium difficile infection between February 1998 and July 2009 in a group of hospitals based in Oxford, UK, and investigated 28-day mortality and biomarkers of inflammation (blood neutrophil count, urea, and creatinine concentrations) collected at diagnosis using iterative sequential regression (ISR), a novel joinpoint-based regression technique suitable for serial monitoring of continuous or dichotomous outcomes. Among C. difficile toxin-positive patients in the Oxford hospitals, mean neutrophil counts on diagnosis increased from 2003, peaked in 2006-2007, and then declined; 28-day mortality increased from early 2006, peaked in late 2006-2007, and then declined. Molecular typing confirmed these changes were likely due to the ingress of the globally distributed severe C. difficile strain, ST1. We assessed the generalizability of ISR-based severity monitoring in three ways. First, we assessed and found strong (p<0.0001) associations between isolation of the ST1 severe strain and higher neutrophil counts at diagnosis in two unrelated large multi-centre studies, suggesting the technique described might be useful elsewhere. Second, we assessed and found similar trends in a second group of hospitals in Birmingham, UK, from which 5,399 cases were analysed. Third, we used simulation to assess the performance of this surveillance system given the ingress of future severe strains under a variety of assumptions. ISR-based severity monitoring allowed the detection of the severity change years earlier than mortality monitoring. CONCLUSIONS: Automated electronic systems providing early warning of the changing severity of infectious conditions can be established using routinely collected laboratory hospital data. In the settings studied here these systems have higher performance than those monitoring mortality, at least in C. difficile infection. Such systems could have wider applicability for monitoring infections presenting in hospital.

Schlackow I, Stoesser N, Walker AS, Crook DW, Peto TE, Wyllie DH, Infections in Oxfordshire Research Database Team. 2012. Increasing incidence of Escherichia coli bacteraemia is driven by an increase in antibiotic-resistant isolates: electronic database study in Oxfordshire 1999-2011. J Antimicrob Chemother, 67 (6), pp. 1514-1524. | Show Abstract | Read more

OBJECTIVES: To investigate trends in Escherichia coli resistance, bacteraemia rates and post-bacteraemia outcomes over time. METHODS: Trends in E. coli bacteraemia incidence were monitored from January 1999 to June 2011 using an infection surveillance database including microbiological, clinical risk factor, infection severity and outcome data in Oxfordshire, UK, with imported temperature/rainfall data. RESULTS: A total of 2240 E. coli (from 2080 patients) were studied, of which 1728 (77%) were susceptible to co-amoxiclav, cefotaxime, ciprofloxacin and gentamicin. E. coli bacteraemia incidence increased from 3.4/10,000 bedstays in 1999 to 5.7/10,000 bedstays in 2011. The increase was fastest around 2006, and was essentially confined to organisms resistant to ciprofloxacin, co-amoxiclav, cefotaxime and/or aminoglycosides. Resistant E. coli isolation rates increased similarly in those with and without recent hospital contact. The sharp increase also occurred in urinary isolates, with similar timing. In addition to these long-term trends, increases in ambient temperature, but not rainfall, were associated with increased E. coli bacteraemia rates. It is unclear whether resistant E. coli bacteraemia rates are currently still increasing [incidence rate ratio = 1.07 per annum (95% CI = 0.99-1.16), P = 0.07], whereas current susceptible E. coli bacteraemia rates are not changing significantly [incidence rate ratio = 1.01 (95% CI = 0.99-1.02)]. However, neither mortality nor biomarkers associated with mortality (blood creatinine, urea/albumin concentrations, neutrophil counts) changed during the study. CONCLUSIONS: E. coli bacteraemia rates have risen due to rising rates of resistant organisms; little change occurred in susceptible E. coli. Although the severity of resistant infections, and their outcome, appear similar to susceptible E. coli in the setting studied, the increasing burden of highly resistant organisms is alarming and merits on-going surveillance.

Reyes-Sandoval A, Rollier CS, Milicic A, Bauza K, Cottingham MG, Tang CK, Dicks MD, Wang D, Longley RJ, Wyllie DH, Hill AV. 2012. Mixed vector immunization with recombinant adenovirus and MVA can improve vaccine efficacy while decreasing antivector immunity. Mol Ther, 20 (8), pp. 1633-1647. | Show Abstract | Read more

Substantial protection can be provided against the pre-erythrocytic stages of malaria by vaccination first with an adenoviral and then with an modified vaccinia virus Ankara (MVA) poxviral vector encoding the same ME.TRAP transgene. We investigated whether the two vaccine components adenovirus (Ad) and MVA could be coinjected as a mixture to enhance protection against malaria. A single-shot mixture at specific ratios of Ad and MVA (Ad+MVA) enhanced CD8(+) T cell-dependant protection of mice against challenge with Plasmodium berghei. Moreover, the degree of protection could be enhanced after homologous boosting with the same Ad+MVA mixture to levels comparable with classic heterologous Ad prime-MVA boost regimes. The mixture increased transgene-specific responses while decreasing the CD8(+) T cell antivector immunity compared to each vector used alone, particularly against the MVA backbone. Mixed vector immunization led to increased early circulating interferon-γ (IFN-γ) response levels and altered transcriptional microarray profiles. Furthermore, we found that sequential immunizations with the Ad+MVA mixture led to consistent boosting of the transgene-specific CD8(+) response for up to three mixture immunizations, whereas each vector used alone elicited progressively lower responses. Our findings offer the possibility of simplifying the deployment of viral vectors as a single mixture product rather than in heterologous prime-boost regimens.

Walker AS, Eyre DW, Wyllie DH, Dingle KE, Harding RM, O'Connor L, Griffiths D, Vaughan A, Finney J, Wilcox MH et al. 2012. Characterisation of Clostridium difficile hospital ward-based transmission using extensive epidemiological data and molecular typing. PLoS Med, 9 (2), pp. e1001172. | Show Abstract | Read more

BACKGROUND: Clostridium difficile infection (CDI) is a leading cause of antibiotic-associated diarrhoea and is endemic in hospitals, hindering the identification of sources and routes of transmission based on shared time and space alone. This may compromise rational control despite costly prevention strategies. This study aimed to investigate ward-based transmission of C. difficile, by subdividing outbreaks into distinct lineages defined by multi-locus sequence typing (MLST). METHODS AND FINDINGS: All C. difficile toxin enzyme-immunoassay-positive and culture-positive samples over 2.5 y from a geographically defined population of ~600,000 persons underwent MLST. Sequence types (STs) were combined with admission and ward movement data from an integrated comprehensive healthcare system incorporating three hospitals (1,700 beds) providing all acute care for the defined geographical population. Networks of cases and potential transmission events were constructed for each ST. Potential infection sources for each case and transmission timescales were defined by prior ward-based contact with other cases sharing the same ST. From 1 September 2007 to 31 March 2010, there were means of 102 tests and 9.4 CDIs per 10,000 overnight stays in inpatients, and 238 tests and 15.7 CDIs per month in outpatients/primary care. In total, 1,276 C. difficile isolates of 69 STs were studied. From MLST, no more than 25% of cases could be linked to a potential ward-based inpatient source, ranging from 37% in renal/transplant, 29% in haematology/oncology, and 28% in acute/elderly medicine to 6% in specialist surgery. Most of the putative transmissions identified occurred shortly (≤ 1 wk) after the onset of symptoms (141/218, 65%), with few >8 wk (21/218, 10%). Most incubation periods were ≤ 4 wk (132/218, 61%), with few >12 wk (28/218, 13%). Allowing for persistent ward contamination following ward discharge of a CDI case did not increase the proportion of linked cases after allowing for random meeting of matched controls. CONCLUSIONS: In an endemic setting with well-implemented infection control measures, ward-based contact with symptomatic enzyme-immunoassay-positive patients cannot account for most new CDI cases.

Cited:

28

Scopus

Reyes-Sandoval A, Rollier CS, Milicic A, Bauza K, Cottingham MG, Tang CK, Dicks MD, Wang D, Longley RJ, Wyllie DH, Hill AVS. 2012. Mixed vector immunization with recombinant adenovirus and MVA can improve vaccine efficacy while decreasing antivector immunity Molecular Therapy, 20 (8), pp. 1633-1647. | Show Abstract | Read more

Substantial protection can be provided against the pre-erythrocytic stages of malaria by vaccination first with an adenoviral and then with an modified vaccinia virus Ankara (MVA) poxviral vector encoding the same ME.TRAP transgene. We investigated whether the two vaccine components adenovirus (Ad) and MVA could be coinjected as a mixture to enhance protection against malaria. A single-shot mixture at specific ratios of Ad and MVA (AdMVA) enhanced CD8 ++ T cell-dependant protection of mice against challenge with Plasmodium berghei. Moreover, the degree of protection could be enhanced after homologous boosting with the same AdMVA mixture to levels comparable with classic heterologous Ad prime-MVA boost regimes. The mixture increased transgene-specific responses while decreasing the CD8++ T cell antivector immunity compared to each vector used alone, particularly against the MVA backbone. Mixed vector immunization led to increased early circulating interferon-γ (IFN-γ) response levels and altered transcriptional microarray profiles. Furthermore, we found that sequential immunizations with the AdMVA mixture led to consistent boosting of the transgene-specific CD8++ response for up to three mixture immunizations, whereas each vector used alone elicited progressively lower responses. Our findings offer the possibility of simplifying the deployment of viral vectors as a single mixture product rather than in heterologous prime-boost regimens. © The American Society of Gene & Cell Therapy.

Wyllie DH, Søgaard KC, Holland K, Yaobo X, Bregu M, Hill AV, Kiss-Toth E. 2012. Identification of 34 novel proinflammatory proteins in a genome-wide macrophage functional screen. PLoS One, 7 (7), pp. e42388. | Show Abstract | Read more

Signal transduction pathways activated by Toll-like Receptors and the IL-1 family of cytokines are fundamental to mounting an innate immune response and thus to clearing pathogens and promoting wound healing. Whilst mechanistic understanding of the regulation of innate signalling pathways has advanced considerably in recent years, there are still a number of critical controllers to be discovered. In order to characterise novel regulators of macrophage inflammation, we have carried out an extensive, cDNA-based forward genetic screen and identified 34 novel activators, based on their ability to induce the expression of cxcl2. Many are physiologically expressed in macrophages, although the majority of genes uncovered in our screen have not previously been linked to innate immunity. We show that expression of particular activators has profound but distinct impacts on LPS-induced inflammatory gene expression, including switch-type, amplifier and sensitiser behaviours. Furthermore, the novel genes identified here interact with the canonical inflammatory signalling network via specific mechanisms, as demonstrated by the use of dominant negative forms of IL1/TLR signalling mediators.

Eyre DW, Walker AS, Wyllie D, Dingle KE, Griffiths D, Finney J, O'Connor L, Vaughan A, Crook DW, Wilcox MH et al. 2012. Predictors of first recurrence of Clostridium difficile infection: implications for initial management. Clin Infect Dis, 55 Suppl 2 (suppl 2), pp. S77-S87. | Show Abstract | Read more

Symptomatic recurrence of Clostridium difficile infection (CDI) occurs in approximately 20% of patients and is challenging to treat. Identifying those at high risk could allow targeted initial management and improve outcomes. Adult toxin enzyme immunoassay-positive CDI cases in a population of approximately 600,000 persons from September 2006 through December 2010 were combined with epidemiological/clinical data. The cumulative incidence of recurrence ≥ 14 days after the diagnosis and/or onset of first-ever CDI was estimated, treating death without recurrence as a competing risk, and predictors were identified from cause-specific proportional hazards regression models. A total of 1678 adults alive 14 days after their first CDI were included; median age was 77 years, and 1191 (78%) were inpatients. Of these, 363 (22%) experienced a recurrence ≥ 14 days after their first CDI, and 594 (35%) died without recurrence through March 2011. Recurrence risk was independently and significantly higher among patients admitted as emergencies, with previous gastrointestinal ward admission(s), last discharged 4-12 weeks before first diagnosis, and with CDI diagnosed at admission. Recurrence risk also increased with increasing age, previous total hours admitted, and C-reactive protein level at first CDI (all P < .05). The 4-month recurrence risk increased by approximately 5% (absolute) for every 1-point increase in a risk score based on these factors. Risk factors, including increasing age, initial disease severity, and hospital exposure, predict CDI recurrence and identify patients likely to benefit from enhanced initial CDI treatment.

Spencer AJ, Hill F, Honeycutt JD, Cottingham MG, Bregu M, Rollier CS, Furze J, Draper SJ, Søgaard KC, Gilbert SC et al. 2012. Fusion of the Mycobacterium tuberculosis antigen 85A to an oligomerization domain enhances its immunogenicity in both mice and non-human primates. PLoS One, 7 (3), pp. e33555. | Show Abstract | Read more

To prevent important infectious diseases such as tuberculosis, malaria and HIV, vaccines inducing greater T cell responses are required. In this study, we investigated whether fusion of the M. tuberculosis antigen 85A to recently described adjuvant IMX313, a hybrid avian C4bp oligomerization domain, could increase T cell responses in pre-clinical vaccine model species. In mice, the fused antigen 85A showed consistent increases in CD4(+) and CD8(+) T cell responses after DNA and MVA vaccination. In rhesus macaques, higher IFN-γ responses were observed in animals vaccinated with MVA-Ag85A IMX313 after both primary and secondary immunizations. In both animal models, fusion to IMX313 induced a quantitative enhancement in the response without altering its quality: multifunctional cytokines were uniformly increased and differentiation into effector and memory T cell subsets was augmented rather than skewed. An extensive in vivo characterization suggests that IMX313 improves the initiation of immune responses as an increase in antigen 85A specific cells was observed as early as day 3 after vaccination. This report demonstrates that antigen multimerization using IMX313 is a simple and effective cross-species method to improve vaccine immunogenicity with potentially broad applicability.

Eyre DW, Walker AS, Griffiths D, Wilcox MH, Wyllie DH, Dingle KE, Crook DW, Peto TE. 2012. Clostridium difficile mixed infection and reinfection. J Clin Microbiol, 50 (1), pp. 142-144. | Show Abstract | Read more

Isolates from consecutive Clostridium difficile infection (CDI) fecal samples underwent multilocus sequence typing. Potential reinfections with different genotypes were identified in 88/560 (16%) sample pairs taken 1 to 1,414 days (median, 24; interquartile range [IQR], 1 to 52 days) apart; odds of reinfection increased by 58% for every doubling of time between samples. Of 109 sample pairs taken on the same day, 3 (3%) had different genotypes. Considering samples 0 to 7 days apart as the same CDI, 7% of cases had mixed infections with >1 genotype.

Wyllie D, Paul J, Crook D. 2011. Waves of trouble: MRSA strain dynamics and assessment of the impact of infection control. J Antimicrob Chemother, 66 (12), pp. 2685-2688. | Show Abstract | Read more

There has been a sustained decline in bloodstream infections due to methicillin-resistant Staphylococcus aureus (MRSA) throughout the UK. The UK MRSA epidemic, which began in the 1990s, has been dominated by two epidemic MRSA (EMRSA) clones {EMRSA-15, of clonal complex (CC) 22 [sequence type (ST) 22], and EMRSA-16, of CC30 (ST36)}. It appears that both these clones followed a wave trajectory (initial expansion, relative stasis, then decline). Three recent studies have shown that ST36 has declined faster than ST22, a change that appears to have begun before the recent intensification of intensive control measures in the UK. The biological basis of infectious disease waves, including those of MRSA, is discussed, as are the implications of such waves for the assessment of the impact of infection control measures.

Reyes-Sandoval A, Wyllie DH, Bauza K, Milicic A, Forbes EK, Rollier CS, Hill AV. 2011. CD8+ T effector memory cells protect against liver-stage malaria. J Immunol, 187 (3), pp. 1347-1357. | Show Abstract | Read more

Identification of correlates of protection for infectious diseases including malaria is a major challenge and has become one of the main obstacles in developing effective vaccines. We investigated protection against liver-stage malaria conferred by vaccination with adenoviral (Ad) and modified vaccinia Ankara (MVA) vectors expressing pre-erythrocytic malaria Ags. By classifying CD8(+) T cells into effector, effector memory (T(EM)), and central memory subsets using CD62L and CD127 markers, we found striking differences in T cell memory generation. Although MVA induced accelerated central memory T cell generation, which could be efficiently boosted by subsequent Ad administration, it failed to protect against malaria. In contrast, Ad vectors, which permit persistent Ag delivery, elicit a prolonged effector T cell and T(EM) response that requires long intervals for an efficient boost. A preferential T(EM) phenotype was maintained in liver, blood, and spleen after Ad/MVA prime-boost regimens, and animals were protected against malaria sporozoite challenge. Blood CD8(+) T(EM) cells correlated with protection against malaria liver-stage infection, assessed by estimation of number of parasites emerging from the liver into the blood. The protective ability of Ag-specific T(EM) cells was confirmed by transfer experiments into naive recipient mice. Thus, we identify persistent CD8 T(EM) populations as essential for vaccine-induced pre-erythrocytic protection against malaria, a finding that has important implications for vaccine design.

Douglas AD, Williams AR, Illingworth JJ, Kamuyu G, Biswas S, Goodman AL, Wyllie DH, Crosnier C, Miura K, Wright GJ et al. 2011. The blood-stage malaria antigen PfRH5 is susceptible to vaccine-inducible cross-strain neutralizing antibody. Nat Commun, 2 (1), pp. 601. | Show Abstract | Read more

Current vaccine strategies against the asexual blood stage of Plasmodium falciparum are mostly focused on well-studied merozoite antigens that induce immune responses after natural exposure, but have yet to induce robust protection in any clinical trial. Here we compare human-compatible viral-vectored vaccines targeting ten different blood-stage antigens. We show that the full-length P. falciparum reticulocyte-binding protein homologue 5 (PfRH5) is highly susceptible to cross-strain neutralizing vaccine-induced antibodies, out-performing all other antigens delivered by the same vaccine platform. We find that, despite being susceptible to antibody, PfRH5 is unlikely to be under substantial immune selection pressure; there is minimal acquisition of anti-PfRH5 IgG antibodies in malaria-exposed Kenyans. These data challenge the widespread beliefs that any merozoite antigen that is highly susceptible to immune attack would be subject to significant levels of antigenic polymorphism, and that erythrocyte invasion by P. falciparum is a degenerate process involving a series of parallel redundant pathways.

Finney JM, Walker AS, Peto TE, Wyllie DH. 2011. An efficient record linkage scheme using graphical analysis for identifier error detection. BMC Med Inform Decis Mak, 11 (1), pp. 7. | Show Abstract | Read more

BACKGROUND: Integration of information on individuals (record linkage) is a key problem in healthcare delivery, epidemiology, and "business intelligence" applications. It is now common to be required to link very large numbers of records, often containing various combinations of theoretically unique identifiers, such as NHS numbers, which are both incomplete and error-prone. METHODS: We describe a two-step record linkage algorithm in which identifiers with high cardinality are identified or generated, and used to perform an initial exact match based linkage. Subsequently, the resulting clusters are studied and, if appropriate, partitioned using a graph based algorithm detecting erroneous identifiers. RESULTS: The system was used to cluster over 250 million health records from five data sources within a large UK hospital group. Linkage, which was completed in about 30 minutes, yielded 3.6 million clusters of which about 99.8% contain, with high likelihood, records from one patient. Although computationally efficient, the algorithm's requirement for exact matching of at least one identifier of each record to another for cluster formation may be a limitation in some databases containing records of low identifier quality. CONCLUSIONS: The technique described offers a simple, fast and highly efficient two-step method for large scale initial linkage for records commonly found in the UK's National Health Service.

Wyllie DH, Walker AS, Miller R, Moore C, Williamson SR, Schlackow I, Finney JM, O'Connor L, Peto TE, Crook DW. 2011. Decline of meticillin-resistant Staphylococcus aureus in Oxfordshire hospitals is strain-specific and preceded infection-control intensification. BMJ Open, 1 (1), pp. e000160. | Show Abstract | Read more

Background In the past, strains of Staphylococcus aureus have evolved, expanded, made a marked clinical impact and then disappeared over several years. Faced with rising meticillin-resistant S aureus (MRSA) rates, UK government-supported infection control interventions were rolled out in Oxford Radcliffe Hospitals NHS Trust from 2006 onwards. Methods Using an electronic Database, the authors identified isolation of MRS among 611 434 hospital inpatients admitted to acute hospitals in Oxford, UK, 1 April 1998 to 30 June 2010. Isolation rates were modelled using segmented negative binomial regression for three groups of isolates: from blood cultures, from samples suggesting invasion (eg, cerebrospinal fluid, joint fluid, pus samples) and from surface swabs (eg, from wounds). Findings MRSA isolation rates rose rapidly from 1998 to the end of 2003 (annual increase from blood cultures 23%, 95% CI 16% to 30%), and then declined. The decline accelerated from mid-2006 onwards (annual decrease post-2006 38% from blood cultures, 95% CI 29% to 45%, p=0.003 vs previous decline). Rates of meticillin-sensitive S aureus changed little by comparison, with no evidence for declines 2006 onward (p=0.40); by 2010, sensitive S aureus was far more common than MRSA (blood cultures: 2.9 vs 0.25; invasive samples 14.7 vs 2.0 per 10 000 bedstays). Interestingly, trends in isolation of erythromycin-sensitive and resistant MRSA differed. Erythromycin-sensitive strains rose significantly faster (eg, from blood cultures p=0.002), and declined significantly more slowly (p=0.002), than erythromycin-resistant strains (global p<0.0001). Bacterial typing suggests this reflects differential spread of two major UK MRSA strains (ST22/36), ST36 having declined markedly 2006-2010, with ST22 becoming the dominant MRSA strain. Conclusions MRSA isolation rates were falling before recent intensification of infection-control measures. This, together with strain-specific changes in MRSA isolation, strongly suggests that incompletely understood biological factors are responsible for the much recent variation in MRSA isolation. A major, mainly meticillin-sensitive, S aureus burden remains.

Cited:

67

Scopus

Reyes-Sandoval A, Wyllie DH, Bauza K, Milicic A, Forbes EK, Rollier CS, Hill AVS. 2011. CD8+ T effector memory cells protect against liver-stage malaria Journal of Immunology, 187 (3), pp. 1347-1357. | Show Abstract | Read more

Identification of correlates of protection for infectious diseases including malaria is a major challenge and has become one of the main obstacles in developing effective vaccines.We investigated protection against liver-stage malaria conferred by vaccination with adenoviral (Ad) and modified vaccinia Ankara (MVA) vectors expressing pre-erythrocytic malaria Ags. By classifying CD8+ T cells into effector, effector memory (TEM), and central memory subsets using CD62L and CD127 markers, we found striking differences in T cell memory generation. Although MVA induced accelerated central memory T cell generation, which could be efficiently boosted by subsequent Ad administration, it failed to protect against malaria. In contrast, Ad vectors, which permit persistent Ag delivery, elicit a prolonged effector T cell and TEM response that requires long intervals for an efficient boost. A preferential TEM phenotype was maintained in liver, blood, and spleen after Ad/MVA prime-boost regimens, and animals were protected against malaria sporozoite challenge. Blood CD8+ TEM cells correlated with protection against malaria liver-stage infection, assessed by estimation of number of parasites emerging from the liver into the blood. The protective ability of Agspecific TEM cells was confirmed by transfer experiments into naive recipient mice. Thus, we identify persistent CD8 TEMpopulations as essential for vaccine-induced pre-erythrocytic protection against malaria, a finding that has important implications for vaccine design. Copyright © 2011 by The American Association of Immunologists, Inc.

Checkley AM, Wyllie DH, Scriba TJ, Golubchik T, Hill AV, Hanekom WA, McShane H. 2011. Identification of antigens specific to non-tuberculous mycobacteria: the Mce family of proteins as a target of T cell immune responses. PLoS One, 6 (10), pp. e26434. | Show Abstract | Read more

The lack of an effective TB vaccine hinders current efforts in combating the TB pandemic. One theory as to why BCG is less protective in tropical countries is that exposure to non-tuberculous mycobacteria (NTM) reduces BCG efficacy. There are currently several new TB vaccines in clinical trials, and NTM exposure may also be relevant in this context. NTM exposure cannot be accurately evaluated in the absence of specific antigens; those which are known to be present in NTM and absent from M. tuberculosis and BCG. We therefore used a bioinformatic pipeline to define proteins which are present in common NTM and absent from the M. tuberculosis complex, using protein BLAST, TBLASTN and a short sequence protein BLAST to ensure the specificity of this process. We then assessed immune responses to these proteins, in healthy South Africans and in patients from the United Kingdom and United States with documented exposure to NTM. Low level responses were detected to a cluster of proteins from the mammalian cell entry family, and to a cluster of hypothetical proteins, using ex vivo ELISpot and a 6 day proliferation assay. These early findings may provide a basis for characterising exposure to NTM at a population level, which has applications in the field of TB vaccine design as well as in the development of diagnostic tests.

Miller R, Walker AS, Knox K, Wyllie D, Paul J, Haworth E, Mant D, Peto T, Crook DW. 2010. 'Feral' and 'wild'-type methicillin-resistant Staphylococcus aureus in the United Kingdom. Epidemiol Infect, 138 (5), pp. 655-665. | Show Abstract | Read more

Circulation of methicillin-resistant Staphylococcus aureus (MRSA) outside hospitals could alter the impact of hospital-based control strategies. We investigated two groups of cases (each matched to controls with MRSA): 61 'community cases' not in acute hospital in the year before MRSA isolation; and 21 cases with ciprofloxacin-sensitive (CipS) MRSA. Multi-locus sequence typing, spa-typing and Panton-Valentine leukocidin gene testing were performed and demographics obtained. Additional questionnaires were completed by community case GPs. Community cases comprised 6% of Oxfordshire MRSA. Three community cases had received no regular healthcare or antibiotics: one was infected with CipS. Ninety-one percent of community cases had healthcare-associated sequence type (ST)22/36; CipS MRSA cases had heterogeneous STs but many had recent healthcare exposure. A substantial minority of UK MRSA transmission may occur outside hospitals. Hospital strains are becoming 'feral' or persisting in long-term carriers in the community with regular healthcare contacts; those with recent healthcare exposure may nevertheless acquire non-hospital epidemic MRSA strains in the community.

Sheehy SH, Atkins BA, Bejon P, Byren I, Wyllie D, Athanasou NA, Berendt AR, McNally MA. 2010. The microbiology of chronic osteomyelitis: prevalence of resistance to common empirical anti-microbial regimens. J Infect, 60 (5), pp. 338-343. | Show Abstract | Read more

OBJECTIVES: This study describes the microbiological spectrum of chronic osteomyelitis and so guides the choice of empirical antibiotics for this condition. METHODS: We performed a prospective review of a 166 prospective patient series of chronic osteomyelitis from Oxford, UK in which a standardised surgical sampling protocol was used. RESULTS: Staphylococcus aureus was most commonly isolated (32%) amongst a wide range of organisms including gram negative bacilli, anaerobes and coagulase negative staphylococci. Low grade pathogens were not confined to patients with a history of metalwork, a high proportion of cases were polymicrobial (29%) and culture negative cases were common (28%). No clear predictors of causative organism could be established. Many isolates were found to be resistant to commonly used empirical anti-microbial regimens. CONCLUSIONS: The wide range of causative organisms and degree of resistance to commonly used anti-microbials supports the importance of extensive intra-operative sampling and provides important information to guide clinicians' choice of empirical antibiotics.

Wong SH, Vannberg FO, Spencer AJ, van der Weyden L, Hill AV, Wyllie DH. 2010. Comment on "CRTAM confers late-stage activation of CD8+ T cells to regulate retention within lymph node". J Immunol, 184 (8), pp. 4052-4053. | Read more

Alcock R, Cottingham MG, Rollier CS, Furze J, De Costa SD, Hanlon M, Spencer AJ, Honeycutt JD, Wyllie DH, Gilbert SC et al. 2010. Long-term thermostabilization of live poxviral and adenoviral vaccine vectors at supraphysiological temperatures in carbohydrate glass. Sci Transl Med, 2 (19), pp. 19ra12. | Show Abstract | Read more

Live recombinant viral vectors based on adenoviruses and poxviruses are among the most promising platforms for development of new vaccines against diseases such as malaria, tuberculosis, and HIV-AIDS. Vaccines based on live viruses must remain infectious to be effective, so therefore need continuous refrigeration to maintain stability and viability, a requirement that can be costly and difficult, especially in developing countries. The sugars sucrose and trehalose are commonly used as stabilizing agents and cryoprotectants for biological products. Here, we have exploited the ability of these sugars to vitrify on desiccation to develop a thermostabilization technique for live viral vaccine vectors. By slowly drying vaccines suspended in solutions of these disaccharide stabilizers onto a filter-like support membrane at ambient temperature, an ultrathin glass is deposited on the fibers of the inert matrix. Immobilization of two recombinant vaccine vectors-E1/E3-deleted human adenovirus type 5 and modified vaccinia virus Ankara-in this glass on the membranes enabled complete recovery of viral titer and immunogenicity after storage at up to 45 degrees C for 6 months and even longer with minimal losses. Furthermore, the membrane carrying the stabilized vaccine can be incorporated into a holder attached to a syringe for almost simultaneous reconstitution and injection at point of use. The technology may potentially be developed for the deployment of viral vector-based biopharmaceuticals in resource-poor settings.

Larsen KC, Spencer AJ, Goodman AL, Gilchrist A, Furze J, Rollier CS, Kiss-Toth E, Gilbert SC, Bregu M, Soilleux EJ et al. 2009. Expression of tak1 and tram induces synergistic pro-inflammatory signalling and adjuvants DNA vaccines. Vaccine, 27 (41), pp. 5589-5598. | Show Abstract | Read more

Improving vaccine immunogenicity remains a major challenge in the fight against developing country diseases like malaria and AIDS. We describe a novel strategy to identify new DNA vaccine adjuvants. We have screened components of the Toll-like receptor signalling pathways for their ability to activate pro-inflammatory target genes in transient transfection assays and assessed in vivo adjuvant activity by expressing the activators from the DNA backbone of vaccines. We find that a robust increase in the immune response necessitates co-expression of two activators. Accordingly, the combination of tak1 and tram elicits synergistic reporter activation in transient transfection assays. In a mouse model this combination, but not the individual molecules, induced approximately twofold increases in CD8+ T-cell immune responses. These results indicate that optimal immunogenicity may require activation of distinct innate immune signalling pathways. Thus this strategy offers a novel route to the discovery of a new generation of adjuvants.

Miller R, Esmail H, Peto T, Walker S, Crook D, Wyllie D. 2008. Is MRSA admission bacteraemia community-acquired? A case control study. J Infect, 56 (3), pp. 163-170. | Show Abstract | Read more

OBJECTIVES: To compare characteristics of methicillin resistant Staphylococcus aureus (MRSA) and methicillin susceptible S. aureus (MSSA) bacteraemia detected on admission to a UK hospital and to determine whether these organisms are community-acquired. METHODS: Consecutive cases of MRSA bacteraemia admitted to general medicine between 2003 and 2006 were identified and compared to MSSA age-matched and unmatched controls (35, 35 and 34 patients, respectively). Demographics, MRSA risk factors, previous health-care contact and clinical presentation were compared using patient notes. Multi-locus sequence typing was performed. RESULTS: 34/35 strains of admission MRSA bacteraemia were the health-care associated Sequence Types (ST)-22 (77%) or ST-36 (21%), whereas 20 different MSSA strains were identified. No MRSA cases fitted the CDC definition of community-acquired MRSA. Compatible with health-care associated acquisition, after matching for age MRSA cases had significantly higher levels of previous hospital exposure than MSSA controls, and more co-morbidities. Notably, 63% of MRSA cases were admitted from their own home, as opposed to secondary care facilities. Clinical presentation of MRSA and MSSA bacteraemias was similar. CONCLUSIONS: MRSA strains associated with health-care were responsible for almost all cases of MRSA bacteraemia on admission to hospital during the period studied. Despite this the majority of cases with MRSA admission bacteraemia were admitted from their own homes. Further research is needed into the determinants of MRSA bacteraemia among patients outside hospital.

Walker S, Peto TE, O'Connor L, Crook DW, Wyllie D. 2008. Are there better methods of monitoring MRSA control than bacteraemia surveillance? An observational database study. PLoS One, 3 (6), pp. e2378. | Show Abstract | Read more

BACKGROUND: Despite a substantial burden of non-bacteraemic methicillin resistant Staphylococcus aureus (MRSA) disease, most MRSA surveillance schemes are based on bacteraemias. Using bacteraemia as an outcome, trends at hospital level are difficult to discern, due to random variation. We investigated rates of nosocomial bacteraemic and non-bacteraemic MRSA infection as surveillance outcomes. METHODS AND FINDINGS: We used microbiology and patient administration system data from an Oxford hospital to estimate monthly rates of first nosocomial MRSA bacteraemia, and nosocomial MRSA isolation from blood/respiratory/sterile site specimens ("sterile sites") or all clinical samples (screens excluded) in all patients admitted from the community for at least 2 days between April 1998 and June 2006. During this period there were 441 nosocomial MRSA bacteraemias, 1464 MRSA isolations from sterile sites, and 3450 isolations from clinical specimens (8% blood, 15% sterile site, 10% respiratory, 59% surface swabs, 8% urine) in over 2.6 million patient-days. The ratio of bacteraemias to sterile site and all clinical isolations was similar over this period (around 3 and 8-fold lower respectively), during which rates of nosocomial MRSA bacteraemia increased by 27% per year to July 2003 before decreasing by 18% per year thereafter (heterogeneity p<0.001). Trends in sterile site and all clinical isolations were similar. Notably, a change in rate of all clinical MRSA isolations in December 2002 could first be detected with conventional statistical significance by August 2003 (p = 0.03). In contrast, when monitoring MRSA bacteraemia, identification of probable changes in trend took longer, first achieving p<0.05 in July 2004. CONCLUSIONS: MRSA isolation from all sites of suspected infection, including bacteraemic and non-bacteraemic isolation, is a potential new surveillance method for MRSA control. It occurs about 8 times more frequently than bacteraemia, allowing robust statistical determination of changing rates over substantially shorter times or smaller areas than using bacteraemia as an outcome.

Walker AS, Spiegelhalter D, Crook DW, Wyllie D, Morris J, Peto TE. 2008. Fairness of financial penalties to improve control of Clostridium difficile. BMJ, 337 (nov20 2), pp. a2097. | Read more

Wyllie DH, Walker AS, Peto TE, Crook DW. 2007. Hospital exposure in a UK population, and its association with bacteraemia. J Hosp Infect, 67 (4), pp. 301-307. | Show Abstract | Read more

Despite the importance of healthcare-associated infection, few studies have quantified the association between severe infection and hospital exposure in UK populations. Our aim was to estimate the proportion of the population with recent hospital admission, together with rates of infection in hospital-exposed and hospital-naïve populations. We studied bacteraemia as a marker of severe infection in a population of 550,000, served by two hospitals, between 1 April 2000 and 31 March 2005. Hospital-exposed persons accounted for 8.3% of the population, defined as having been resident in a hospital in the last year. The hospital-exposed population accounted for 55% of all admissions, and 42% of emergency admissions to medical, paediatric or surgery departments. After adjustment for age, the hospital-exposed group had much higher rates of admission bacteraemia. Age-standardised incidence rate ratios relative to hospital-naïve patients were 43 [95% confidence interval (CI): 22-85] for meticillin-resistant Staphylococcus aureus (MRSA), 20 (15-27) for S. aureus other than MRSA, 7.3 (5.2-10) for Streptococcus pneumoniae, and 14 (11-18) for E. coli. MRSA was common among hospital-exposed admissions, including emergencies in hospital-exposed men, rates of admission MRSA bacteraemia (31 per 100,000 per annum) and S. pneumoniae bacteraemia (33 per 100,000 per annum) were similar. This quantitative analysis confirms that prior hospital admission is a major risk factor for bacteraemia on hospital admission; it is unclear whether acquisition of pathogens in hospital, co-morbidity or other factors explain this.

Wyllie DH, Crook DW, Peto TE. 2006. Mortality after Staphylococcus aureus bacteraemia in two hospitals in Oxfordshire, 1997-2003: cohort study. BMJ, 333 (7562), pp. 281. | Show Abstract | Read more

OBJECTIVE: To determine the incidence of methicillin resistant and methicillin sensitive Staphylococcus aureus (MRSA and MSSA) bacteraemia in inpatients and associated mortality within 30 days after diagnosis. DESIGN: Anonymised record linkage study of data from hospital information systems and microbiology databases. SETTING: Teaching hospital and district general hospital in Oxfordshire. PARTICIPANTS: Inpatients aged 18 or over admitted to a teaching hospital between 1 April 1997 and 31 March 2004 and to a district general hospital between 1 April 1999 and 31 March 2004. The main part of the study comprised 216 644 inpatients; patients admitted to haematology, nephrology, or oncology services were not included because most were managed as outpatients. OUTCOME MEASURES: Nosocomial MSSA and MRSA bacteraemia; death in hospital within 30 days after bacteraemia. RESULTS: Rates of S aureus bacteraemia rose between 1997 and 2003, and MRSA was responsible for this increase. Overall mortality 30 days after bacteraemia was 29%. The crude odds ratio for death after MRSA bacteraemia compared with MSSA bacteraemia was 1.49 (95% confidence interval 0.99 to 2.26). CONCLUSION: The spread of MRSA has greatly increased the overall number of cases of S aureus bacteraemia and has contributed to short term mortality after S aureus bacteraemia.

Wyllie DH, Peto TE, Crook D. 2005. MRSA bacteraemia in patients on arrival in hospital: a cohort study in Oxfordshire 1997-2003. BMJ, 331 (7523), pp. 992. | Show Abstract | Read more

OBJECTIVE: To describe the incidence and determinants of methicillin resistant and methicillin sensitive Staphylococcus aureus (MRSA and MSSA) bacteraemia in patients presenting to acute hospitals. DESIGN: Anonymised record linkage study with information from hospital information systems and microbiology databases. SETTING: One teaching hospital and one district general hospital in Oxfordshire. PARTICIPANTS: All patients admitted to a teaching hospital 1 April 1997 to 31 March 2003 and to a district general hospital 1 April 1999 to 31 March 2003. MAIN OUTCOME MEASURES: Detection of MRSA and MSSA from blood cultures taken during the first two days of admission to hospital. RESULTS: In the teaching hospital, there were 479 patients with MSSA and 116 with MRSA bacteraemia admitted from the community. Among this group, which comprised 24% of all hospital MRSA cases, 31% (36 cases) of patients had been admitted to renal, oncology, or haematology services for intensive day case therapy. The 69% remaining were most commonly patients admitted as medical or surgical emergencies. At least 91% had been in hospital previously; the median time since discharge was 46 days. About half of cases were in patients in whom MRSA had not been isolated before. Similar epidemiology was observed in the district general hospital. CONCLUSION: Diagnostic algorithms and policies on use of antibiotics need to reflect the fact that a quarter of hospital MRSA cases occur in patients who have previously been in hospital and are subsequently readmitted.

Wyllie DH, Bowler IC, Peto TE. 2005. Bacteraemia prediction in emergency medical admissions: role of C reactive protein. J Clin Pathol, 58 (4), pp. 352-356. | Show Abstract | Read more

AIM: To define the contribution made by C reactive protein (CRP) measurement to bacteraemia prediction in adults with medical emergencies in the UK. METHODS: This two year cohort study involved 6234 patients admitted as emergency cases to the acute medical or infectious diseases services of the Oxford Radcliffe Hospitals, in whom blood cultures were taken on arrival. The main outcome measures were bacteraemia risk associated with admission CRP concentrations, lymphocyte counts, and neutrophil counts. RESULTS: The quantitative associations between CRP concentration, admission lymphocyte count, and neutrophil count were defined. Risk of bacteraemia rose continuously as the CRP increased: no "cutoff" value was evident. Models examining combinations of CRP, neutrophil count, and lymphocyte count were developed and validated using a split sample technique. CRP contributed to a model including lymphocyte and neutrophil counts, but its effect was small. CRP alone performed no better than either a model combining lymphopenia and neutrophilia, or than lymphopenia alone. CONCLUSIONS: In patients with acute medical emergencies who are suspected of bacteraemia clinically, CRP concentrations, although associated with bacteraemia, have a limited role in bacteraemia prediction.

Wyllie DH, Baxter E, Morgan M, Bowler IC. 2005. Spread of multiresistance and extended-spectrum beta-lactamases amongst urinary Escherichia coli in Oxford, UK. J Antimicrob Chemother, 56 (5), pp. 986-988. | Read more

Wyllie DH, Bowler IC, Peto TE. 2004. Relation between lymphopenia and bacteraemia in UK adults with medical emergencies. J Clin Pathol, 57 (9), pp. 950-955. | Show Abstract | Read more

AIMS: To determine the relevance of lymphopenia to the diagnosis of bacteraemia in patients admitted with medical emergencies, relative to peripheral blood white cell count and neutrophilia. PATIENTS/METHODS: A two year cohort study carried out in a teaching hospital in Oxford, UK of 21,495 consecutive adult emergency admissions to general medical or infectious disease wards. Full blood data were available in 21,372 cases; 41 cases with extreme full blood count results (neutrophil count, > 75 x 10(9)/litre; lymphocyte count, > 10 x 10(9)/litre) were excluded, leaving 21,331 cases for analysis. The association between the admission lymphocyte and neutrophil counts and the risk of bacteraemia was assessed. RESULTS: Neutrophilia and lymphopenia were both associated with bacteraemia. Lymphopenia was the better predictor in this cohort. Both neutrophilia and lymphopenia were more predictive of bacteraemia than the total white blood cell count. CONCLUSIONS: Both lymphocyte and neutrophil counts, rather than total white blood cell count, should be considered in adult medical admissions with suspected bacteraemia.

Read RC, Wyllie DH. 2001. Toll receptors and sepsis. Curr Opin Crit Care, 7 (5), pp. 371-375. | Show Abstract | Read more

Toll-like receptors are a family of receptors that recognize components of bacteria and induce a proinflammatory response by cells, including macrophages and endothelial cells. Ten human Toll receptors differing in their specificity for microbial components have been cloned. They respond to various components, including lipopolysaccharide of Gram-negative bacteria, lipopeptides of Gram-positive cell walls, bacterial DNA, and flagella. Some Toll-like receptors require the cooperation of an adapter protein. Toll-like receptor 4 function requires the presence of the protein MD2. Recently, it has been shown that Toll-like receptors function cooperatively to increase the specificity of response to a given microbe. Human polymorphisms of Toll-like receptor genes have been discovered and are associated with hyporesponsiveness to bacterial components.

Walker AS, Mason A, Quan TP, Fawcett NJ, Watkinson P, Llewelyn M, Stoesser N, Finney J, Davies J, Wyllie DH et al. 2017. Mortality risks associated with emergency admissions during weekends and public holidays: an analysis of electronic health records. Lancet, | Show Abstract | Read more

BACKGROUND: Weekend hospital admission is associated with increased mortality, but the contributions of varying illness severity and admission time to this weekend effect remain unexplored. METHODS: We analysed unselected emergency admissions to four Oxford University National Health Service hospitals in the UK from Jan 1, 2006, to Dec 31, 2014. The primary outcome was death within 30 days of admission (in or out of hospital), analysed using Cox models measuring time from admission. The primary exposure was day of the week of admission. We adjusted for multiple confounders including demographics, comorbidities, and admission characteristics, incorporating non-linearity and interactions. Models then considered the effect of adjusting for 15 common haematology and biochemistry test results or proxies for hospital workload. FINDINGS: 257 596 individuals underwent 503 938 emergency admissions. 18 313 (4·7%) patients admitted as weekday energency admissions and 6070 (5·1%) patients admitted as weekend emergency admissions died within 30 days (p<0·0001). 9347 individuals underwent 9707 emergency admissions on public holidays. 559 (5·8%) died within 30 days (p<0·0001 vs weekday). 15 routine haematology and biochemistry test results were highly prognostic for mortality. In 271 465 (53·9%) admissions with complete data, adjustment for test results explained 33% (95% CI 21 to 70) of the excess mortality associated with emergency admission on Saturdays compared with Wednesdays, 52% (lower 95% CI 34) on Sundays, and 87% (lower 95% CI 45) on public holidays after adjustment for standard patient characteristics. Excess mortality was predominantly restricted to admissions between 1100 h and 1500 h (pinteraction=0·04). No hospital workload measure was independently associated with mortality (all p values >0·06). INTERPRETATION: Adjustment for routine test results substantially reduced excess mortality associated with emergency admission at weekends and public holidays. Adjustment for patient-level factors not available in our study might further reduce the residual excess mortality, particularly as this clustered around midday at weekends. Hospital workload was not associated with mortality. Together, these findings suggest that the weekend effect arises from patient-level differences at admission rather than reduced hospital staffing or services. FUNDING: NIHR Oxford Biomedical Research Centre.

Wyllie D, Davies J. 2015. Role of data warehousing in healthcare epidemiology Journal of Hospital Infection, 89 (4), pp. 267-270. | Show Abstract | Read more

© 2015 Published by Elsevier Ltd on behalf of the Healthcare Infection Society.Electronic storage of healthcare data, including individual-level risk factors for both infectious and other diseases, is increasing. These data can be integrated at hospital, regional and national levels. Data sources that contain risk factor and outcome information for a wide range of conditions offer the potential for efficient epidemiological analysis of multiple diseases. Opportunities may also arise for monitoring healthcare processes. Integrating diverse data sources presents epidemiological, practical, and ethical challenges. For example, diagnostic criteria, outcome definitions, and ascertainment methods may differ across the data sources. Data volumes may be very large, requiring sophisticated computing technology. Given the large populations involved, perhaps the most challenging aspect is how informed consent can be obtained for the development of integrated databases, particularly when it is not easy to demonstrate their potential. In this article, we discuss some of the ups and downs of recent projects as well as the potential of data warehousing for antimicrobial resistance monitoring.

Spencer AJ, Furze J, Honeycutt JD, Calvert A, Saurya S, Colloca S, Wyllie DH, Gilbert SC, Bregu M, Cottingham MG, Hill AV. 2014. 4-1BBL enhances CD8+ T cell responses induced by vectored vaccines in mice but fails to improve immunogenicity in rhesus macaques. PLoS One, 9 (8), pp. e105520. | Show Abstract | Read more

T cells play a central role in the immune response to many of the world's major infectious diseases. In this study we investigated the tumour necrosis factor receptor superfamily costimulatory molecule, 4-1BBL (CD137L, TNFSF9), for its ability to increase T cell immunogenicity induced by a variety of recombinant vectored vaccines. To efficiently test this hypothesis, we assessed a number of promoters and developed a stable bi-cistronic vector expressing both the antigen and adjuvant. Co-expression of 4-1BBL, together with our model antigen TIP, was shown to increase the frequency of murine antigen-specific IFN-γ secreting CD8(+) T cells in three vector platforms examined. Enhancement of the response was not limited by co-expression with the antigen, as an increase in CD8(+) immunogenicity was also observed by co-administration of two vectors each expressing only the antigen or adjuvant. However, when this regimen was tested in non-human primates using a clinical malaria vaccine candidate, no adjuvant effect of 4-1BBL was observed limiting its potential use as a single adjuvant for translation into a clinical vaccine.

Votintseva AA, Fung R, Miller RR, Knox K, Godwin H, Wyllie DH, Bowden R, Crook DW, Walker AS. 2014. Prevalence of Staphylococcus aureus protein A (spa) mutants in the community and hospitals in Oxfordshire. BMC Microbiol, 14 (1), pp. 63. | Show Abstract | Read more

BACKGROUND: Staphylococcal protein A (spa) is an important virulence factor which enables Staphylococcus aureus to evade host immune responses. Genotypes known as "spa-types", based on highly variable Xr region sequences of the spa-gene, are frequently used to classify strains. A weakness of current spa-typing primers is that rearrangements in the IgG-binding region of the gene cause 1-2% of strains to be designated as "non-typeable". RESULTS: We developed an improved primer which enabled sequencing of all strains, containing any type of genetic rearrangement, in a large study among community carriers and hospital inpatients in Oxfordshire, UK (6110 isolates). We identified eight novel spa-gene variants, plus one previously described. Three of these rearrangements would be designated "non-typeable" using current spa-typing methods; they occurred in 1.8% (72/3905) asymptomatically carried and 0.6% (14/2205) inpatient S. aureus strains. Some individuals were simultaneously colonized by both formerly non-typeable and typeable strains; previously such patients would have been identified as carrying only currently typeable strains, underestimating mixed carriage prevalence and diversity. Formerly non-typeable strains were found in more spa-types associated with multilocus sequence type ST398 (35%), common among livestock, compared to other groups with any non-typeable strains (1-4%), suggesting particular spa-types may have been under-represented in previous human studies. CONCLUSIONS: This improved method allows us to spa-type previously non-typeable strains with rearrangements in the spa-gene and to resolve cases of mixed colonization with deletions in one or more strains, thus accounting for hidden diversity of S. aureus in both community and hospital environments.

Wong TH, Dearlove BL, Hedge J, Giess AP, Piazza P, Trebes A, Paul J, Smit E, Smith EG, Sutton JK et al. 2013. Whole genome sequencing and de novo assembly identifies Sydney-like variant noroviruses and recombinants during the winter 2012/2013 outbreak in England. Virol J, 10 (1), pp. 335. | Show Abstract | Read more

BACKGROUND: Norovirus is the commonest cause of epidemic gastroenteritis among people of all ages. Outbreaks frequently occur in hospitals and the community, costing the UK an estimated £110 m per annum. An evolutionary explanation for periodic increases in norovirus cases, despite some host-specific post immunity is currently limited to the identification of obvious recombinants. Our understanding could be significantly enhanced by full length genome sequences for large numbers of intensively sampled viruses, which would also assist control and vaccine design. Our objective is to develop rapid, high-throughput, end-to-end methods yielding complete norovirus genome sequences. We apply these methods to recent English outbreaks, placing them in the wider context of the international norovirus epidemic of winter 2012. METHOD: Norovirus sequences were generated from 28 unique clinical samples by Illumina RNA sequencing (RNA-Seq) of total faecal RNA. A range of de novo sequence assemblers were attempted. The best assembler was identified by validation against three replicate samples and two norovirus qPCR negative samples, together with an additional 20 sequences determined by PCR and fractional capillary sequencing. Phylogenetic methods were used to reconstruct evolutionary relationships from the whole genome sequences. RESULTS: Full length norovirus genomes were generated from 23/28 samples. 5/28 partial norovirus genomes were associated with low viral copy numbers. The de novo assembled sequences differed from sequences determined by capillary sequencing by <0.003%. Intra-host nucleotide sequence diversity was rare, but detectable by mapping short sequence reads onto its de novo assembled consensus. Genomes similar to the Sydney 2012 strain caused 78% (18/23) of cases, consistent with its previously documented association with the winter 2012 global outbreak. Interestingly, phylogenetic analysis and recombination detection analysis of the consensus sequences identified two related viruses as recombinants, containing sequences in prior circulation to Sydney 2012 in open reading frame (ORF) 2. CONCLUSION: Our approach facilitates the rapid determination of complete norovirus genomes. This method provides high resolution of full norovirus genomes which, when coupled with detailed epidemiology, may improve the understanding of evolution and control of this important healthcare-associated pathogen.

van Diemen PM, Yamaguchi Y, Paterson GK, Rollier CS, Hill AV, Wyllie DH. 2013. Irradiated wild-type and Spa mutant Staphylococcus aureus induce anti-S. aureus immune responses in mice which do not protect against subsequent intravenous challenge. Pathog Dis, 68 (1), pp. 20-26. | Show Abstract | Read more

Staphylococcus aureus remains an important human and animal pathogen. Its pathogenicity is determined in part by expression of the Spa-immune subversion protein, neutralising the activity of which provides partial protection in murine models, as does experimental infection with live S. aureus with Spa gene deletions followed by antibiotic-mediated cure in mice. Together, these data raise the question of whether Spa mutant S. aureus might represent a viable vaccine. Here, we find that gamma-irradiated S. aureus strains, both wild-type and null mutant of spa, are immunogenic in mice when administered intramuscularly, eliciting large amounts of anti-S. aureus antibodies, as judged by whole-cell immunoassay on fixed microorganisms. We used an intravenous challenge system to assess vaccine efficacy, the sensitivity of which was increased by studying renal bacterial concentrations in both kidneys. Despite this, protection from intravenous challenge was not observed (mean difference between vaccinated and unvaccinated mice 0.27 log(10) with 95% confidence interval -0.922 to 1.467). Surprisingly, antibody responses elicited against a panel of protective cell surface proteins were very low, indicating that most antibody induced is not protective. Additionally, these data suggest a limited role for irradiated wild-type or spa mutant S. aureus as vaccines.

Walker AS, Eyre DW, Wyllie DH, Dingle KE, Griffiths D, Shine B, Oakley S, O'Connor L, Finney J, Vaughan A et al. 2013. Relationship between bacterial strain type, host biomarkers, and mortality in Clostridium difficile infection. Clin Infect Dis, 56 (11), pp. 1589-1600. | Show Abstract | Read more

BACKGROUND: Despite substantial interest in biomarkers, their impact on clinical outcomes and variation with bacterial strain has rarely been explored using integrated databases. METHODS: From September 2006 to May 2011, strains isolated from Clostridium difficile toxin enzyme immunoassay (EIA)-positive fecal samples from Oxfordshire, United Kingdom (approximately 600,000 people) underwent multilocus sequence typing. Fourteen-day mortality and levels of 15 baseline biomarkers were compared between consecutive C. difficile infections (CDIs) from different clades/sequence types (STs) and EIA-negative controls using Cox and normal regression adjusted for demographic/clinical factors. RESULTS: Fourteen-day mortality was 13% in 2222 adults with 2745 EIA-positive samples (median, 78 years) vs 5% in 20,722 adults with 27,550 EIA-negative samples (median, 74 years) (absolute attributable mortality, 7.7%; 95% CI, 6.4%-9.0%). Mortality was highest in clade 5 CDIs (25% [16 of 63]; polymerase chain reaction (PCR) ribotype 078/ST 11), then clade 2 (20% [111 of 560]; 99% PCR ribotype 027/ST 1) versus clade 1 (12% [137 of 1168]; adjusted P < .0001). Within clade 1, 14-day mortality was only 4% (3 of 84) in ST 44 (PCR ribotype 015) (adjusted P = .05 vs other clade 1). Mean baseline neutrophil counts also varied significantly by genotype: 12.4, 11.6, and 9.5 × 10(9) neutrophils/L for clades 5, 2 and 1, respectively, vs 7.0 × 10(9) neutrophils/L in EIA-negative controls (P < .0001) and 7.9 × 10(9) neutrophils/L in ST 44 (P = .08). There were strong associations between C. difficile-type-specific effects on mortality and neutrophil/white cell counts (rho = 0.48), C-reactive-protein (rho = 0.43), eosinophil counts (rho = -0.45), and serum albumin (rho = -0.47). Biomarkers predicted 30%-40% of clade-specific mortality differences. CONCLUSIONS: C. difficile genotype predicts mortality, and excess mortality correlates with genotype-specific changes in biomarkers, strongly implicating inflammatory pathways as a major influence on poor outcome after CDI. PCR ribotype 078/ST 11 (clade 5) leads to severe CDI; thus ongoing surveillance remains essential.

Reyes-Sandoval A, Rollier CS, Milicic A, Bauza K, Cottingham MG, Tang CK, Dicks MD, Wang D, Longley RJ, Wyllie DH, Hill AV. 2012. Mixed vector immunization with recombinant adenovirus and MVA can improve vaccine efficacy while decreasing antivector immunity. Mol Ther, 20 (8), pp. 1633-1647. | Show Abstract | Read more

Substantial protection can be provided against the pre-erythrocytic stages of malaria by vaccination first with an adenoviral and then with an modified vaccinia virus Ankara (MVA) poxviral vector encoding the same ME.TRAP transgene. We investigated whether the two vaccine components adenovirus (Ad) and MVA could be coinjected as a mixture to enhance protection against malaria. A single-shot mixture at specific ratios of Ad and MVA (Ad+MVA) enhanced CD8(+) T cell-dependant protection of mice against challenge with Plasmodium berghei. Moreover, the degree of protection could be enhanced after homologous boosting with the same Ad+MVA mixture to levels comparable with classic heterologous Ad prime-MVA boost regimes. The mixture increased transgene-specific responses while decreasing the CD8(+) T cell antivector immunity compared to each vector used alone, particularly against the MVA backbone. Mixed vector immunization led to increased early circulating interferon-γ (IFN-γ) response levels and altered transcriptional microarray profiles. Furthermore, we found that sequential immunizations with the Ad+MVA mixture led to consistent boosting of the transgene-specific CD8(+) response for up to three mixture immunizations, whereas each vector used alone elicited progressively lower responses. Our findings offer the possibility of simplifying the deployment of viral vectors as a single mixture product rather than in heterologous prime-boost regimens.

Spencer AJ, Hill F, Honeycutt JD, Cottingham MG, Bregu M, Rollier CS, Furze J, Draper SJ, Søgaard KC, Gilbert SC et al. 2012. Fusion of the Mycobacterium tuberculosis antigen 85A to an oligomerization domain enhances its immunogenicity in both mice and non-human primates. PLoS One, 7 (3), pp. e33555. | Show Abstract | Read more

To prevent important infectious diseases such as tuberculosis, malaria and HIV, vaccines inducing greater T cell responses are required. In this study, we investigated whether fusion of the M. tuberculosis antigen 85A to recently described adjuvant IMX313, a hybrid avian C4bp oligomerization domain, could increase T cell responses in pre-clinical vaccine model species. In mice, the fused antigen 85A showed consistent increases in CD4(+) and CD8(+) T cell responses after DNA and MVA vaccination. In rhesus macaques, higher IFN-γ responses were observed in animals vaccinated with MVA-Ag85A IMX313 after both primary and secondary immunizations. In both animal models, fusion to IMX313 induced a quantitative enhancement in the response without altering its quality: multifunctional cytokines were uniformly increased and differentiation into effector and memory T cell subsets was augmented rather than skewed. An extensive in vivo characterization suggests that IMX313 improves the initiation of immune responses as an increase in antigen 85A specific cells was observed as early as day 3 after vaccination. This report demonstrates that antigen multimerization using IMX313 is a simple and effective cross-species method to improve vaccine immunogenicity with potentially broad applicability.

Wyllie D, Paul J, Crook D. 2011. Waves of trouble: MRSA strain dynamics and assessment of the impact of infection control. J Antimicrob Chemother, 66 (12), pp. 2685-2688. | Show Abstract | Read more

There has been a sustained decline in bloodstream infections due to methicillin-resistant Staphylococcus aureus (MRSA) throughout the UK. The UK MRSA epidemic, which began in the 1990s, has been dominated by two epidemic MRSA (EMRSA) clones {EMRSA-15, of clonal complex (CC) 22 [sequence type (ST) 22], and EMRSA-16, of CC30 (ST36)}. It appears that both these clones followed a wave trajectory (initial expansion, relative stasis, then decline). Three recent studies have shown that ST36 has declined faster than ST22, a change that appears to have begun before the recent intensification of intensive control measures in the UK. The biological basis of infectious disease waves, including those of MRSA, is discussed, as are the implications of such waves for the assessment of the impact of infection control measures.

Wyllie DH, Walker AS, Miller R, Moore C, Williamson SR, Schlackow I, Finney JM, O'Connor L, Peto TE, Crook DW. 2011. Decline of meticillin-resistant Staphylococcus aureus in Oxfordshire hospitals is strain-specific and preceded infection-control intensification. BMJ Open, 1 (1), pp. e000160. | Show Abstract | Read more

Background In the past, strains of Staphylococcus aureus have evolved, expanded, made a marked clinical impact and then disappeared over several years. Faced with rising meticillin-resistant S aureus (MRSA) rates, UK government-supported infection control interventions were rolled out in Oxford Radcliffe Hospitals NHS Trust from 2006 onwards. Methods Using an electronic Database, the authors identified isolation of MRS among 611 434 hospital inpatients admitted to acute hospitals in Oxford, UK, 1 April 1998 to 30 June 2010. Isolation rates were modelled using segmented negative binomial regression for three groups of isolates: from blood cultures, from samples suggesting invasion (eg, cerebrospinal fluid, joint fluid, pus samples) and from surface swabs (eg, from wounds). Findings MRSA isolation rates rose rapidly from 1998 to the end of 2003 (annual increase from blood cultures 23%, 95% CI 16% to 30%), and then declined. The decline accelerated from mid-2006 onwards (annual decrease post-2006 38% from blood cultures, 95% CI 29% to 45%, p=0.003 vs previous decline). Rates of meticillin-sensitive S aureus changed little by comparison, with no evidence for declines 2006 onward (p=0.40); by 2010, sensitive S aureus was far more common than MRSA (blood cultures: 2.9 vs 0.25; invasive samples 14.7 vs 2.0 per 10 000 bedstays). Interestingly, trends in isolation of erythromycin-sensitive and resistant MRSA differed. Erythromycin-sensitive strains rose significantly faster (eg, from blood cultures p=0.002), and declined significantly more slowly (p=0.002), than erythromycin-resistant strains (global p<0.0001). Bacterial typing suggests this reflects differential spread of two major UK MRSA strains (ST22/36), ST36 having declined markedly 2006-2010, with ST22 becoming the dominant MRSA strain. Conclusions MRSA isolation rates were falling before recent intensification of infection-control measures. This, together with strain-specific changes in MRSA isolation, strongly suggests that incompletely understood biological factors are responsible for the much recent variation in MRSA isolation. A major, mainly meticillin-sensitive, S aureus burden remains.

Cited:

67

Scopus

Reyes-Sandoval A, Wyllie DH, Bauza K, Milicic A, Forbes EK, Rollier CS, Hill AVS. 2011. CD8+ T effector memory cells protect against liver-stage malaria Journal of Immunology, 187 (3), pp. 1347-1357. | Show Abstract | Read more

Identification of correlates of protection for infectious diseases including malaria is a major challenge and has become one of the main obstacles in developing effective vaccines.We investigated protection against liver-stage malaria conferred by vaccination with adenoviral (Ad) and modified vaccinia Ankara (MVA) vectors expressing pre-erythrocytic malaria Ags. By classifying CD8+ T cells into effector, effector memory (TEM), and central memory subsets using CD62L and CD127 markers, we found striking differences in T cell memory generation. Although MVA induced accelerated central memory T cell generation, which could be efficiently boosted by subsequent Ad administration, it failed to protect against malaria. In contrast, Ad vectors, which permit persistent Ag delivery, elicit a prolonged effector T cell and TEM response that requires long intervals for an efficient boost. A preferential TEM phenotype was maintained in liver, blood, and spleen after Ad/MVA prime-boost regimens, and animals were protected against malaria sporozoite challenge. Blood CD8+ TEM cells correlated with protection against malaria liver-stage infection, assessed by estimation of number of parasites emerging from the liver into the blood. The protective ability of Agspecific TEM cells was confirmed by transfer experiments into naive recipient mice. Thus, we identify persistent CD8 TEMpopulations as essential for vaccine-induced pre-erythrocytic protection against malaria, a finding that has important implications for vaccine design. Copyright © 2011 by The American Association of Immunologists, Inc.

Larsen KC, Spencer AJ, Goodman AL, Gilchrist A, Furze J, Rollier CS, Kiss-Toth E, Gilbert SC, Bregu M, Soilleux EJ et al. 2009. Expression of tak1 and tram induces synergistic pro-inflammatory signalling and adjuvants DNA vaccines. Vaccine, 27 (41), pp. 5589-5598. | Show Abstract | Read more

Improving vaccine immunogenicity remains a major challenge in the fight against developing country diseases like malaria and AIDS. We describe a novel strategy to identify new DNA vaccine adjuvants. We have screened components of the Toll-like receptor signalling pathways for their ability to activate pro-inflammatory target genes in transient transfection assays and assessed in vivo adjuvant activity by expressing the activators from the DNA backbone of vaccines. We find that a robust increase in the immune response necessitates co-expression of two activators. Accordingly, the combination of tak1 and tram elicits synergistic reporter activation in transient transfection assays. In a mouse model this combination, but not the individual molecules, induced approximately twofold increases in CD8+ T-cell immune responses. These results indicate that optimal immunogenicity may require activation of distinct innate immune signalling pathways. Thus this strategy offers a novel route to the discovery of a new generation of adjuvants.

Real-time detection of multidrug resistant tuberculosis and transmission in England

The World Health Organization estimates that two billion people are infected with tuberculosis, of whom there are 1.5 million deaths per year. Cases of multidrug-resistant tuberculosis are difficult to treat and continue to spread. In Britain, Public Health England and researchers from the University of Oxford are leading international efforts to develop rapid tests using genomics to identify multidrug resistant strains more quickly and identify transmission clusters as new cases emerge.This ...

View project

235

Thank you for registering your interest

We were unable to record your request to register for interest in future opportunities. Please try again and if problems persist contact us at webteam@ndm.ox.ac.uk