[Show abstract][Hide abstract] ABSTRACT: Estimation of immunological and microbiological diversity is vital to our understanding of infection and the immune response. For instance, what is the diversity of the T cell repertoire? These questions are partially addressed by high-throughput sequencing techniques that enable identification of immunological and microbiological "species" in a sample. Estimators of the number of unseen species are needed to estimate population diversity from sample diversity. Here we test five widely used non-parametric estimators, and develop and validate a novel method, DivE, to estimate species richness and distribution. We used three independent datasets: (i) viral populations from subjects infected with human T-lymphotropic virus type 1; (ii) T cell antigen receptor clonotype repertoires; and (iii) microbial data from infant faecal samples. When applied to datasets with rarefaction curves that did not plateau, existing estimators systematically increased with sample size. In contrast, DivE consistently and accurately estimated diversity for all datasets. We identify conditions that limit the application of DivE. We also show that DivE can be used to accurately estimate the underlying population frequency distribution. We have developed a novel method that is significantly more accurate than commonly used biodiversity estimators in microbiological and immunological populations.
[Show abstract][Hide abstract] ABSTRACT: Neisseria meningitidis is a commensal of humans that can colonise the nasopharyngeal epithelium for weeks to months, and occasionally invades to cause life-threatening septicaemia and meningitis. Comparatively little is known about meningococcal gene expression during colonisation beyond those first few hours. In this study, the transcriptome of adherent serogroup B N. meningitidis strain MC58 was determined at intervals during prolonged co-cultivation with confluent monolayers of the human respiratory epithelial cell line 16HBE14. At different time points up to 21 days, 7 - 14% of the meningococcal genome was found to be differentially regulated. The transcriptome of adherent meningococci obtained after 4 hours of co-culture was markedly different from that obtained after prolonged co-cultivation (24 hours, 96 hours and 21 days). Genes persistently up-regulated during prolonged co-cultivation included three (hfq, misR/phoP and lrp) encoding global regulatory proteins. Many genes encoding known adhesins involved in epithelial adherence were up-regulated, including those of a novel locus encoding epithelial adhesive function (NMB0342 - NMB0348). Sixteen genes (including porA, porB, rmpM and fbpA) encoding proteins previously identified by their immunoreactivity to sera from individuals colonised long term with serogroup B meningococci were also up-regulated during prolonged co-cultivation, indicating that our system models growth conditions in vivo during the commensal state. Surface-expressed proteins down-regulated in the nasopharynx (and so less subject to selection pressure) but upregulated in the bloodstream (and so vulnerable to antibody-mediated bactericidal activity) should be interesting candidate vaccine antigens, and in this study three new proteins fulfilling these criteria have been identified - NMB0497, NMB0866 and NMB1882.
Infection and immunity 08/2013; · 4.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The NadA adhesin is a major component of 4CMenB, a novel vaccine to prevent meningococcus B (MenB) infection. Under in vitro growth conditions nadA is repressed by the regulator NadR and poorly expressed, resulting in inefficient killing of MenB strains by anti-NadA antibodies. Interestingly, sera from children infected with strains that express low levels of NadA in laboratory growth, nevertheless recognize the NadA antigen, suggesting that during infection NadA expression may be different from that observed in vitro. In a strain panel covering a range of NadA levels, repression was relieved through deleting nadR. All nadR(-) strains expressed high levels of NadA and were efficiently killed by sera from subjects immunized with 4CMenB. A selected MenB strain, NGP165, mismatched for other vaccine antigens, is not killed by sera from immunized infants when grown in vitro. However, in an in vivo passive protection model the same sera effectively protected infant rats from bacteremia with NGP165. Furthermore, we identify a novel HPA derivative, reported by others to be produced during inflammation, which induces expression of NadA in vitro leading to efficient antibody-mediated killing. Finally, using bioluminescent reporters, nadA expression in the infant rat model is induced in vivo at three hours post infection. Our results suggest that during infectious disease, NadR repression is alleviated due to niche-specific signals, resulting in high levels of NadA expression from any nadA(+) strain and therefore efficient killing by anti-NadA antibodies elicited by the 4CMenB vaccine.
Infection and immunity 12/2012; · 4.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An emergent clone of Haemophilus influenzae biogroup aegyptius (Hae) is responsible for outbreaks of Brazilian purpuric fever (BPF). First recorded in Brazil in 1984, the so-called BPF clone of Hae caused a fulminant disease that started with conjunctivitis but developed into septicemic shock; mortality rates were as high as 70%. To identify virulence determinants, we conducted a pan-genomic analysis. Sequencing of the genomes of the BPF clone strain F3031 and a noninvasive conjunctivitis strain, F3047, and comparison of these sequences with 5 other complete H. influenzae genomes showed that >77% of the F3031 genome is shared among all H. influenzae strains. Delineation of the Hae accessory genome enabled characterization of 163 predicted protein-coding genes; identified differences in established autotransporter adhesins; and revealed a suite of novel adhesins unique to Hae, including novel trimeric autotransporter adhesins and 4 new fimbrial operons. These novel adhesins might play a critical role in host-pathogen interactions.
[Show abstract][Hide abstract] ABSTRACT: Public concern about an unsubstantiated link between MMR vaccine and autism stemmed from a 1998 paper by Dr Andrew Wakefield and colleagues, and the substantial media coverage which that work attracted. Though the Wakefield paper is now discredited and an MMR-autism link has never been demonstrated empirically, this concern has manifested in over a decade of suboptimal MMR uptake. Few qualitative studies have explored parents' MMR decision-making since uptake began to improve in 2004. This study updates and adds methodological rigour to the evidence base.
24 mothers planning to accept, postpone or decline the first MMR dose (MMR1) for their 11-36 month-old children, described their decision-making in semi-structured interviews. Mothers were recruited via General Practice, parents' groups/online forums, and chain referral. MMR1 status was obtained from General Practice records 6 months post-interview. Interview transcripts were coded and interpreted using a modified Grounded Theory approach.
Five themes were identified: MMR vaccine and controversy; Social and personal consequences of MMR decision; Health professionals and policy; Severity and prevalence of measles, mumps and rubella infections; Information about MMR and alternatives. Results indicated that MMR1 acceptors were sympathetic toward Wakefield as a person, but universally rejected his study which sparked the controversy; parents opting for single vaccines expressed the sense that immune overload is not a consideration but that not all three components of MMR are warranted by disease severity; and MMR1 rejectors openly criticised other parents' MMR decisions and decision-making.
This study corroborated some previous qualitative work but indicated that the shrinking group of parents now rejecting MMR comprises mainly those with more extreme and complex anti-immunisation views, whilst parents opting for single vaccines may use second-hand information about the controversy. In response, policymakers and practitioners should revise their expectations of today's MMR decision-makers, and their methods for supporting them.
[Show abstract][Hide abstract] ABSTRACT: This article addresses key questions frequently asked by researchers conducting systematic reviews in patient safety. This discipline is relatively young, and asks complex questions about complex aspects of health care delivery and experience, therefore its studies are typically methodologically heterogeneous, non-randomized and complex; but content rich and highly relevant to practice. Systematic reviews are increasingly necessary to drive forward practice and research in this area, but the data do not always lend themselves to 'standard' review methodologies. This accessible 'how-to' article demonstrates that data diversity need not preclude high-quality systematic reviews. It draws together information from published guidelines and experience within our multidisciplinary patient safety research group to provide entry-level advice for the clinician-researcher new to systematic reviewing, to non-biomedical research data or to both. It offers entry-level advice, illustrated with detailed practical examples, on defining a research question, creating a comprehensive search strategy, selecting articles for inclusion, assessing study quality, extracting data, synthesizing data and evaluating the impact of your review. The article concludes with a comment on the vital role of robust systematic reviews in the continuing advancement of the patient safety field.
Journal of Evaluation in Clinical Practice 02/2012; 18(1):172-81. · 1.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The 16S rRNA gene is conserved across all bacteria and as such is routinely targeted in PCR surveys of bacterial diversity. PCR primer design aims to amplify as many different 16S rRNA gene sequences from as wide a range of organisms as possible, though there are no suitable 100% conserved regions of the gene, leading to bias. In the gastrointestinal tract, bifidobacteria are a key genus, but are often under-represented in 16S rRNA surveys of diversity. We have designed modified, 'bifidobacteria-optimised' universal primers, which we have demonstrated detection of bifidobacterial sequence present in DNA mixtures at 2% abundance, the lowest proportion tested. Optimisation did not compromise the detection of other organisms in infant faecal samples. Separate validation using fluorescence in situ hybridisation (FISH) shows that the proportions of bifidobacteria detected in faecal samples were in agreement with those obtained using 16S rRNA based pyrosequencing. For future studies looking at faecal microbiota, careful selection of primers will be key in order to ensure effective detection of bifidobacteria.
PLoS ONE 01/2012; 7(3):e32543. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Neisseria meningitidis is a nasopharyngeal commensal of humans which occasionally invades the blood to cause septicaemia. The transcriptome of N. meningitidis strain MC58 grown in human blood for up to 4 hours was determined and around 10% of the genome was found to be differentially regulated. The nuo, pet and atp operons, involved in energy metabolism, were up-regulated, while many house-keeping genes were down-regulated. Genes encoding protein chaperones and proteases, involved in the stress response; complement resistant genes encoding enzymes for LOS sialylation and biosynthesis; and fHbp (NMB1870) and nspA (NMB0663), encoding vaccine candidates, were all up-regulated. Genes for glutamate uptake and metabolism, and biosynthesis of purine and pyrimidine were also up-regulated. Blood grown meningococci are under stress and undergo a metabolic adaptation and energy conservation strategy. The localisation of four putative outer membrane proteins encoded by genes found to be up-regulated in blood was assessed by FACS using polyclonal mouse antisera, and one (NMB0390) showed evidence of surface expression, supporting its vaccine candidacy.
PLoS ONE 01/2012; 7(6):e39718. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Phenotypic and genetic variation in bacteria can take bewilderingly complex forms even within a single genus. One of the most intriguing examples of this is the genus Neisseria, which comprises both pathogens and commensals colonizing a variety of body sites and host species, and causing a range of disease. Complex relatedness among both named species and previously identified lineages of Neisseria makes it challenging to study their evolution. Using the largest publicly available collection of bacterial sequence data in combination with a population genetic analysis and experiment, we probe the contribution of inter-species recombination to neisserial population structure, and specifically whether it is more common in some strains than others. We identify hybrid groups of strains containing sequences typical of more than one species. These groups of strains, typical of a fuzzy species, appear to have experienced elevated rates of inter-species recombination estimated by population genetic analysis and further supported by transformation experiments. In particular, strains of the pathogen Neisseria meningitidis in the fuzzy species boundary appear to follow a different lifestyle, which may have considerable biological implications concerning distribution of novel resistance elements and meningococcal vaccine development. Despite the strong evidence for negligible geographical barriers to gene flow within the population, exchange of genetic material still shows directionality among named species in a non-uniform manner.
Journal of The Royal Society Interface 11/2011; 9(71):1208-15. · 4.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Herd immunity is important in the effectiveness of conjugate polysaccharide vaccines against encapsulated bacteria. A large multicenter study investigated the effect of meningococcal serogroup C conjugate vaccine introduction on the meningococcal population.
Carried meningococci in individuals aged 15-19 years attending education establishments were investigated before and for 2 years after vaccine introduction. Isolates were characterized by multilocus sequence typing, serogroup, and capsular region genotype and changes in phenotypes and genotypes assessed.
A total of 8462 meningococci were isolated from 47 765 participants (17.7%). Serogroup prevalence was similar over the 3 years, except for decreases of 80% for serogroup C and 40% for serogroup 29E. Clonal complexes were associated with particular serogroups and their relative proportions fluctuated, with 12 statistically significant changes (6 up, 6 down). The reduction of ST-11 complex serogroup C meningococci was probably due to vaccine introduction. Reasons for a decrease in serogroup 29E ST-254 meningococci (from 1.8% to 0.7%) and an increase in serogroup B ST-213 complex meningococci (from 6.7% to 10.6%) were less clear.
Natural fluctuations in carried meningococcal genotypes and phenotypes a can be affected by the use of conjugate vaccines, and not all of these changes are anticipatable in advance of vaccine introduction.
The Journal of Infectious Diseases 10/2011; 204(7):1046-53. · 5.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Because meningitis and septicaemia caused by Neisseria meningitidis are major public health problems worldwide, the design of a broadly protective vaccine remains a priority. Type IV pili (Tfp) are surface-exposed filaments playing a key role in pathogenesis in a variety of bacterial species, including N. meningitidis, that have demonstrated vaccine potential. Unfortunately, in the meningococcus, the major pilus subunit PilE usually undergoes extensive antigenic variation and is therefore not suitable as a vaccine component. However, we have recently shown that N. meningitidis Tfp contain low abundance subunits PilX, PilV and ComP, collectively called minor pilins, that are highly conserved and modulate Tfp-linked functions key to pathogenesis. This prompted us to examine the vaccine potential of these proteins by assessing whether sera directed against them have bactericidal properties and/or are able to interfere with Tfp-linked functions. Here we show that minor pilin proteins are recognized by sera of patients convalescent from meningococcal disease and that antibodies directed against some of them can selectively interfere with Tfp-linked functions. This shows that, despite their apparent inability to elicit bactericidal antibodies, minor pilins might have vaccine potential.
[Show abstract][Hide abstract] ABSTRACT: The aim of the meeting was to consider the latest advances in meningitis, covering epidemiology, pathogenic mechanisms, host-interactive biology and vaccines in a variety of bacteria, fungi and protozoa that cause meningitis. The program was comprised of speakers from the UK, as well as international presenters, who had been invited and offered selected papers. Owing to space limitations, only the four bacteria with multiple invited speakers will be considered here.
[Show abstract][Hide abstract] ABSTRACT: Parents' attitudes toward MMR vaccine and measles, mumps and rubella infections relate to their child's MMR status, therefore improving these attitudes is central to improving current suboptimal MMR uptake. However, no study has yet combined evidence-based, comprehensive and psychometrically validated assessment of these attitudes with reliable objective MMR status data, in order to identify through multivariate analyses the strongest attitudinal predictors of MMR uptake for interventions to target. The present study fills this lacuna by developing and testing a robust evidence-based MMR attitudes measurement instrument.
Cross-sectional self-administered postal/telephone questionnaire with objective behavioural outcome.
535 parents of children aged 5-18 in London and north-west England, UK (response rate 18.1%). Recruitment via Primary Care Trust records, age-stratified purposive sample with suboptimally immunised cases oversampled.
Parents' responses to evidence-based measurement instrument comprising 20 attitude/previous behaviour items (collapsing to 5 scales) and 7 demographic items, and their children's PCT-recorded 5th birthday status for MMR dose 1 (on-time, late or none) and MMR dose 2 (on-time or none).
The attitudes measurement instrument was psychometrically robust: content valid, and demonstrating good or acceptable internal consistency (Cronbach's alpha=0.55-0.75 for all scales), test-retest reliability (Pearson's correlation >0.60-0.80, p<0.01 to <0.001 for all scales and 11 individual items), concurrent/construct validity (t-tests for difference between MMR status groups p<0.05 for four scales and thirteen individual items), and predictive/criterion validity (OR=0.66, 95% confidence interval=0.48-0.92 to OR=1.97, 95% CI=1.18-3.31 for three scales and five individual items). Black and minority ethnicity (OR=1.94, 95% CI=1.15-3.30 to OR=4.15, 95% CI=2.40-7.19), positive MMR attitudes (OR=1.63, 95% CI=1.00-2.66 to OR=1.97, 95% CI=1.18-1.31), and positive social attitudes (OR=1.64, 95% CI=1.23-2.40 to OR=1.72, 95% CI=1.13-2.38) independently predicted uptake for both MMR doses. MMR status groups differed most strongly on preference for single measles, mumps and rubella vaccines (6-9% variance in status explained), previous MMR acceptance/rejection (5-9%), and wishing to protect others through vaccinating one's own child (6-8%).
The measurement instrument is robust on multiple validity and reliability dimensions, and is appropriate for use in research and practice as a tool for designing and evaluating interventions. Parents appear to act in line with their attitudes toward MMR vaccine, though attitudes toward measles infection bore little relation to MMR uptake. This study indicates populations and attitudes to be prioritized in MMR uptake improvement interventions.
[Show abstract][Hide abstract] ABSTRACT: Continued suboptimal measles-mumps-rubella (MMR) vaccine uptake has re-established measles epidemic risk, prompting a UK catch-up campaign in 2008-09 for children who missed MMR doses at scheduled age. Predictors of vaccine uptake during catch-ups are poorly understood, however evidence from routine schedule uptake suggests demographics and attitudes may be central. This work explored this hypothesis using a robust evidence-based measure.
Cross-sectional self-administered questionnaire with objective behavioural outcome.
365 UK parents, whose children were aged 5-18 years and had received <2 MMR doses before the 2008-09 UK catch-up started.
Parents' attitudes and demographics, parent-reported receipt of invitation to receive catch-up MMR dose(s), and catch-up MMR uptake according to child's medical record (receipt of MMR doses during year 1 of the catch-up).
Perceived social desirability/benefit of MMR uptake (OR = 1.76, 95% CI = 1.09-2.87) and younger child age (OR = 0.78, 95% CI = 0.68-0.89) were the only independent predictors of catch-up MMR uptake in the sample overall. Uptake predictors differed by whether the child had received 0 MMR doses or 1 MMR dose before the catch-up. Receipt of catch-up invitation predicted uptake only in the 0 dose group (OR = 3.45, 95% CI = 1.18-10.05), whilst perceived social desirability/benefit of MMR uptake predicted uptake only in the 1 dose group (OR = 9.61, 95% CI = 2.57-35.97). Attitudes and demographics explained only 28% of MMR uptake in the 0 dose group compared with 61% in the 1 dose group.
Catch-up MMR invitations may effectively move children from 0 to 1 MMR doses (unimmunised to partially immunised), whilst attitudinal interventions highlighting social benefits of MMR may effectively move children from 1 to 2 MMR doses (partially to fully immunised). Older children may be best targeted through school-based programmes. A formal evaluation element should be incorporated into future catch-up campaigns to inform their continuing improvement.
PLoS ONE 01/2011; 6(5):e19381. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Both Neisseria meningitidis and the closely related bacterium Neisseria lactamica colonise human nasopharyngeal mucosal surface, but only N. meningitidis invades the bloodstream to cause potentially life-threatening meningitis and septicaemia. We have hypothesised that the two neisserial species differentially modulate host respiratory epithelial cell gene expression reflecting their disease potential. Confluent monolayers of 16HBE14 human bronchial epithelial cells were exposed to live and/or dead N. meningitidis (including capsule and pili mutants) and N. lactamica, and their transcriptomes were compared using whole genome microarrays. Changes in expression of selected genes were subsequently validated using Q-RT-PCR and ELISAs. Live N. meningitidis and N. lactamica induced genes involved in host energy production processes suggesting that both bacterial species utilise host resources. N. meningitidis infection was associated with down-regulation of host defence genes. N. lactamica, relative to N. meningitidis, initiates up-regulation of proinflammatory genes. Bacterial secreted proteins alone induced some of the changes observed. The results suggest N. meningitidis and N. lactamica differentially regulate host respiratory epithelial cell gene expression through colonisation and/or protein secretion, and that this may contribute to subsequent clinical outcomes associated with these bacteria.
PLoS ONE 01/2011; 6(10):e26130. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: APXIVA is an RTX toxin of Actinobacillus pleuropneumoniae that is a candidate antigen to differentiate infected from vaccinated animals (DIVA). Insertion of ISApl1 into the apxIVA gene is known to compromise an APXIVA-based DIVA approach, as is potentially a TGG to TGA mutation in the apxIVA gene. ISApl1 was found in 63/349 (18.1%) A. pleuropneumoniae isolates from England and Wales including serovars 2, 3, 6-8 and 12. No ISApl1 insertions into apxIVA were found. Only two serovar 3 isolates contained the TGG to TGA mutation. We conclude that an ApxIVA-based DIVA approach would potentially be viable in England and Wales.
[Show abstract][Hide abstract] ABSTRACT: The last few years has seen a rapid increase in the use of surface enhanced laser desorption ionisation–time of flight (SELDI, SELDI-TOF) mass spectrometry, at the core of which are ProteinChip arrays, to search for biomarkers of infectious disease. Reasons include the greater reproducibility of current technology (instrument and ProteinChip arrays), the availability of increasingly powerful analytical tools and the appreciation of confounding variables (e.g. sample preparation and study design). Biomarkers have been sought for diagnosis of infection, disease progression, prediction of those at high risk from other disease as the result of infection (e.g. cancer) and to increase understanding of infectious disease processes. SELDI has been used for biomarker discovery in humans and animals as a result of infection by bacteria, viruses and parasites. The molecular identification of the discriminatory peptides/proteins allows the development or utilisation of cheaper simpler tests such as ELISAs. In some instances, SELDI has been used to measure biomarkers for infectious disease where conventional tests are unavailable or of insufficient sensitivity and specificity. It is likely that there will be increased use of SELDI, particularly if it is combined with parallel developments in sample preparation, allowing the detection of less abundant and potentially more informative peptides/proteins.
[Show abstract][Hide abstract] ABSTRACT: 2009 H1N1 influenza A ("swine flu") vaccine has been offered to healthy UK children aged 6 months-5 years since December 2009, though around 50% of parents plan to reject the vaccine. This study examined whether such parents exhibit omission bias (preference for errors arising from inaction over errors arising from action). One-hundred and forty-two parents completed an online questionnaire in which they rated (a) probability of occurrence, (b) symptoms and (c) duration of a hypothetical disease and a hypothetical vaccine adverse event (VAE). Almost all attributes were rated significantly less favourably when relating to VAE than to disease (p<0.01 for 17 of 22 outcomes), despite the attributes being objectively identical. These data suggest that any vaccine is at a disadvantage in many parents' consciousness in comparison with the infection itself, and that minor safety concerns could have disproportionately detrimental effects on vaccine uptake. Behavioural science offers strategies to ameliorate the impact of this bias and these should be explored further.
[Show abstract][Hide abstract] ABSTRACT: Suboptimal childhood vaccination uptake results in disease outbreaks, and in developed countries is largely attributable to parental choice. To inform evidence-based interventions, we conducted a systematic review of factors underlying parental vaccination decisions. Thirty-one studies were reviewed. Outcomes and methods are disparate, which limits synthesis; however parents are consistently shown to act in line with their attitudes to combination childhood vaccinations. Vaccine-declining parents believe that vaccines are unsafe and ineffective and that the diseases they are given to prevent are mild and uncommon; they mistrust their health professionals, Government and officially-endorsed vaccine research but trust media and non-official information sources and resent perceived pressure to risk their own child's safety for public health benefit. Interventions should focus on detailed decision mechanisms including disease-related anticipated regret and perception of anecdotal information as statistically representative. Self-reported vaccine uptake, retrospective attitude assessment and unrepresentative samples limit the reliability of reviewed data - methodological improvements are required in this area.