Chemical Science Research, Elementary School Children and Their Teachers Are More Closely Related than You May Imagine: The "I Bet You Did Not Know" Project.

Topics associated with the chemical sciences form a significant part of the curriculum in science at the primary school level in the U.K. In this methodology paper, we demonstrate how a wide range of research articles associated with the chemical sciences can be disseminated to an elementary school audience and how children can carry out investigations associated with cutting-edge research in the classroom. We discuss how the Primary Science Teaching Trust's (PSTT's) "I bet you did not know" (IBYDK) articles and their accompanying Teacher Guides benefit children, primary (elementary) school teachers, and other stakeholders including the researchers themselves. We define three types of research articles; ones describing how children can reproduce the research themselves without much adaptation, others where children can mirror the research using similar methods, and some where an analogy can be used to explain the research. We provide exemplars of each type and some preliminary feedback on articles written.

Flipping the Thinking on Equality, Diversity, and Inclusion. Why EDI Is Essential for the Development and Progression of the Chemical Sciences: A Case Study Approach.

All learners have a contribution to make to the development of the Chemical Sciences, be that in novel ways to teach, and their perspectives and contexts, but also in research, both in chemical education and the wider Chemical Sciences. Through four case studies, this paper explores interactions with diverse groups and how this has altered perspectives on both teaching and research. The case studies include work with visually impaired adults, a project bringing together First Peoples in Australia with academics to explore old ways (traditional science) and new ways (modern approaches), primary (elementary) school perspectives on teaching science, and a project in South Africa to connect university and township communities. Not only do these case studies demonstrate the immense value these diverse groups bring to our understanding about how to learn, but they also bring new perspectives on how to view and solve chemical problems.

Correction: Dynamics and impact of homologous recombination on the evolution of Legionella pneumophila.

[This corrects the article DOI: 10.1371/journal.pgen.1006855.].

Dynamics and impact of homologous recombination on the evolution of Legionella pneumophila.

Legionella pneumophila is an environmental bacterium and the causative agent of Legionnaires' disease. Previous genomic studies have shown that recombination accounts for a high proportion (>96%) of diversity within several major disease-associated sequence types (STs) of L. pneumophila. This suggests that recombination represents a potentially important force shaping adaptation and virulence. Despite this, little is known about the biological effects of recombination in L. pneumophila, particularly with regards to homologous recombination (whereby genes are replaced with alternative allelic variants). Using newly available population genomic data, we have disentangled events arising from homologous and non-homologous recombination in six major disease-associated STs of L. pneumophila (subsp. pneumophila), and subsequently performed a detailed characterisation of the dynamics and impact of homologous recombination. We identified genomic "hotspots" of homologous recombination that include regions containing outer membrane proteins, the lipopolysaccharide (LPS) region and Dot/Icm effectors, which provide interesting clues to the selection pressures faced by L. pneumophila. Inference of the origin of the recombined regions showed that isolates have most frequently imported DNA from isolates belonging to their own clade, but also occasionally from other major clades of the same subspecies. This supports the hypothesis that the possibility for horizontal exchange of new adaptations between major clades of the subspecies may have been a critical factor in the recent emergence of several clinically important STs from diverse genomic backgrounds. However, acquisition of recombined regions from another subspecies, L. pneumophila subsp. fraseri, was rarely observed, suggesting the existence of a recombination barrier and/or the possibility of ongoing speciation between the two subspecies. Finally, we suggest that multi-fragment recombination may occur in L. pneumophila, whereby multiple non-contiguous segments that originate from the same molecule of donor DNA are imported into a recipient genome during a single episode of recombination.

Seeding and Establishment of Legionella pneumophila in Hospitals: Implications for Genomic Investigations of Nosocomial Legionnaires' Disease.

Background: Legionnaires' disease is an important cause of hospital-acquired pneumonia and is caused by infection with the bacterium Legionella. Because current typing methods often fail to resolve the infection source in possible nosocomial cases, we aimed to determine whether whole-genome sequencing (WGS) could be used to support or refute suspected links between cases and hospitals. We focused on cases involving a major nosocomial-associated strain, L. pneumophila sequence type (ST) 1. Methods: WGS data from 229 L. pneumophila ST1 isolates were analyzed, including 99 isolates from the water systems of 17 hospitals and 42 clinical isolates from patients with confirmed or suspected hospital-acquired infections, as well as isolates obtained from or associated with community-acquired sources of Legionnaires' disease. Results: Phylogenetic analysis demonstrated that all hospitals from which multiple isolates were obtained have been colonized by 1 or more distinct ST1 populations. However, deep sampling of 1 hospital also revealed the existence of substantial diversity and ward-specific microevolution within the population. Across all hospitals, suspected links with cases were supported with WGS, although the degree of support was dependent on the depth of environmental sampling and available contextual information. Finally, phylogeographic analysis revealed that hospitals have been seeded with L. pneumophila via both local and international spread of ST1. Conclusions: WGS can be used to support or refute suspected links between hospitals and Legionnaires' disease cases. However, deep hospital sampling is frequently required due to the potential coexistence of multiple populations, existence of substantial diversity, and similarity of hospital isolates to local populations.

Development, validation and implementation of a quadruplex real-time PCR assay for identification of potentially toxigenic corynebacteria.

Toxigenic corynebacteria are uncommon in the UK; however, laboratory confirmation by the national reference laboratory can inform public health action according to national guidelines. Standard phenotypic tests for identification and toxin expression of isolates can take from ≥24 to ≥48 h from receipt. To decrease the time to result, a real-time PCR (qPCR) assay was developed for confirmation of both identification of Corynebacterium diphtheriae and Corynebacterium ulcerans/Corynebacterium pseudotuberculosis and detection of the diphtheria toxin gene. Target genes were the RNA polymerase ß-subunit-encoding gene (rpoB) and A-subunit of the diphtheria toxin gene (tox). Green fluorescent protein DNA (gfp) was used as an internal process control. qPCR results were obtained within 3 to 4 h after receipt of isolate. The assay was validated according to published guidelines and demonstrated high diagnostic sensitivity (100 %), high specificity (98-100 %) and positive and negative predictive values of 91 to 100 % and 100 %, respectively, compared to both block-based PCR and the Elek test, together with a greatly reduced time from isolate receipt to reporting. Limitations of the qPCR assay were the inability to distinguish between C. ulcerans and C. pseudotuberculosis and that the presence of the toxin gene as demonstrated by qPCR may not always predict toxin expression. Thus, confirmation of expression of diphtheria toxin is always sought using the phenotypic Elek test. The new qPCR assay was formally introduced as the front-line test for putative toxigenic corynebacteria to inform public health action in England and Wales on 1 April 2014.

Whole genome sequencing of Streptococcus pneumoniae: development, evaluation and verification of targets for serogroup and serotype prediction using an automated pipeline.

Streptococcus pneumoniae typically express one of 92 serologically distinct capsule polysaccharide (cps) types (serotypes). Some of these serotypes are closely related to each other; using the commercially available typing antisera, these are assigned to common serogroups containing types that show cross-reactivity. In this serotyping scheme, factor antisera are used to allocate serotypes within a serogroup, based on patterns of reactions. This serotyping method is technically demanding, requires considerable experience and the reading of the results can be subjective. This study describes the analysis of the S. pneumoniae capsular operon genetic sequence to determine serotype distinguishing features and the development, evaluation and verification of an automated whole genome sequence (WGS)-based serotyping bioinformatics tool, PneumoCaT (Pneumococcal Capsule Typing). Initially, WGS data from 871 S. pneumoniae isolates were mapped to reference cps locus sequences for the 92 serotypes. Thirty-two of 92 serotypes could be unambiguously identified based on sequence similarities within the cps operon. The remaining 60 were allocated to one of 20 'genogroups' that broadly correspond to the immunologically defined serogroups. By comparing the cps reference sequences for each genogroup, unique molecular differences were determined for serotypes within 18 of the 20 genogroups and verified using the set of 871 isolates. This information was used to design a decision-tree style algorithm within the PneumoCaT bioinformatics tool to predict to serotype level for 89/94 (92 + 2 molecular types/subtypes) from WGS data and to serogroup level for serogroups 24 and 32, which currently comprise 2.1% of UK referred, invasive isolates submitted to the National Reference Laboratory (NRL), Public Health England (June 2014-July 2015). PneumoCaT was evaluated with an internal validation set of 2065 UK isolates covering 72/92 serotypes, including 19 non-typeable isolates and an external validation set of 2964 isolates from Thailand (n = 2,531), USA (n = 181) and Iceland (n = 252). PneumoCaT was able to predict serotype in 99.1% of the typeable UK isolates and in 99.0% of the non-UK isolates. Concordance was evaluated in UK isolates where further investigation was possible; in 91.5% of the cases the predicted capsular type was concordant with the serologically derived serotype. Following retesting, concordance increased to 99.3% and in most resolved cases (97.8%; 135/138) discordance was shown to be caused by errors in original serotyping. Replicate testing demonstrated that PneumoCaT gave 100% reproducibility of the predicted serotype result. In summary, we have developed a WGS-based serotyping method that can predict capsular type to serotype level for 89/94 serotypes and to serogroup level for the remaining four. This approach could be integrated into routine typing workflows in reference laboratories, reducing the need for phenotypic immunological testing.

Multiple major disease-associated clones of Legionella pneumophila have emerged recently and independently.

Legionella pneumophila is an environmental bacterium and the leading cause of Legionnaires' disease. Just five sequence types (ST), from more than 2000 currently described, cause nearly half of disease cases in northwest Europe. Here, we report the sequence and analyses of 364 L. pneumophila genomes, including 337 from the five disease-associated STs and 27 representative of the species diversity. Phylogenetic analyses revealed that the five STs have independent origins within a highly diverse species. The number of de novo mutations is extremely low with maximum pairwise single-nucleotide polymorphisms (SNPs) ranging from 19 (ST47) to 127 (ST1), which suggests emergences within the last century. Isolates sampled geographically far apart differ by only a few SNPs, demonstrating rapid dissemination. These five STs have been recombining recently, leading to a shared pool of allelic variants potentially contributing to their increased disease propensity. The oldest clone, ST1, has spread globally; between 1940 and 2000, four new clones have emerged in Europe, which show long-distance, rapid dispersal. That a large proportion of clinical cases is caused by recently emerged and internationally dispersed clones, linked by convergent evolution, is surprising for an environmental bacterium traditionally considered to be an opportunistic pathogen. To simultaneously explain recent emergence, rapid spread and increased disease association, we hypothesize that these STs have adapted to new man-made environmental niches, which may be linked by human infection and transmission.

Evaluation of an Optimal Epidemiological Typing Scheme for Legionella pneumophila with Whole-Genome Sequence Data Using Validation Guidelines.

Sequence-based typing (SBT), analogous to multilocus sequence typing (MLST), is the current "gold standard" typing method for investigation of legionellosis outbreaks caused by Legionella pneumophila However, as common sequence types (STs) cause many infections, some investigations remain unresolved. In this study, various whole-genome sequencing (WGS)-based methods were evaluated according to published guidelines, including (i) a single nucleotide polymorphism (SNP)-based method, (ii) extended MLST using different numbers of genes, (iii) determination of gene presence or absence, and (iv) a kmer-based method. L. pneumophila serogroup 1 isolates (n = 106) from the standard "typing panel," previously used by the European Society for Clinical Microbiology Study Group on Legionella Infections (ESGLI), were tested together with another 229 isolates. Over 98% of isolates were considered typeable using the SNP- and kmer-based methods. Percentages of isolates with complete extended MLST profiles ranged from 99.1% (50 genes) to 86.8% (1,455 genes), while only 41.5% produced a full profile with the gene presence/absence scheme. Replicates demonstrated that all methods offer 100% reproducibility. Indices of discrimination range from 0.972 (ribosomal MLST) to 0.999 (SNP based), and all values were higher than that achieved with SBT (0.940). Epidemiological concordance is generally inversely related to discriminatory power. We propose that an extended MLST scheme with ∼50 genes provides optimal epidemiological concordance while substantially improving the discrimination offered by SBT and can be used as part of a hierarchical typing scheme that should maintain backwards compatibility and increase discrimination where necessary. This analysis will be useful for the ESGLI to design a scheme that has the potential to become the new gold standard typing method for L. pneumophila.

Rapid investigation of cases and clusters of Legionnaires' disease in England and Wales using direct molecular typing.

Legionella pneumophila is the leading cause of Legionnaires' disease, a severe pneumonia that can occur as sporadic cases or point-source outbreaks affecting multiple patients. The infection is acquired by inhalation of aerosols from contaminated water systems. In order to identify the probable source and prevent further cases, clinical and environmental isolates are compared using phenotypic and genotypic methods. Typically up to 10 days are required to isolate L. pneumophila prior to the application of standard typing protocols. A rapid protocol using a real-time PCR specific for L. pneumophila and serogroup 1, combined with nested direct molecular typing, was adopted by Public Health England in 2012 to reduce reporting time for preliminary typing results. This rapid protocol was first used to investigate an outbreak that occurred in July/August 2012 and due to the positive feedback from that investigation, it was subsequently applied to other incidents in England and Wales where faster typing results would have aided incident investigation. We present here results from seven incidents that occurred between July 2012 and June 2015 where the use of this rapid approach provided preliminary characterization of the infecting strain in an average 1.58 days (SD 1.01) after sample receipt in contrast to 9.53 days (SD 3.73) when standard protocols were applied.

Use of a serotype-specific urine immunoassay to determine the course of a hospital outbreak of Streptococcus pneumoniae complicated by influenza A.

INTRODUCTION: An outbreak of Streptococcus pneumoniae (pneumococcal) infection complicated by concomitant influenza A on an elderly care ward was detected. CASE PRESENTATION: Thirteen patients with hospital-acquired respiratory infections were investigated during the course of the outbreak investigation. Six had a positive BinaxNOW S. pneumoniae urinary antigen test and two patients had culture-confirmed pneumococcal bacteraemia and a positive urine antigen test. Five patients gave positive influenza A PCR results of which two were also positive for S. pneumoniae antigen. CONCLUSION: The concurrence of influenza and pneumococcal infections made tracking the course of the infection difficult. This case study shows how the use of a sensitive, S. pneumoniae serotype-specific urine antigen assay, in the absence of cultured isolates, helped determine whether patients were infected with the same pneumococcal serotype. This was particularly useful when additional respiratory symptoms were seen following the administration of chemoprophylaxis.

Whooping cough in school age children presenting with persistent cough in UK primary care after introduction of the preschool pertussis booster vaccination: prospective cohort study.

OBJECTIVE: To estimate the prevalence and clinical severity of whooping cough (pertussis) in school age children presenting with persistent cough in primary care since the introduction and implementation of the preschool pertussis booster vaccination. DESIGN: Prospective cohort study (November 2010 to December 2012). SETTING: General practices in Thames Valley, UK. PARTICIPANTS: 279 children aged 5 to 15 years who presented in primary care with a persistent cough of two to eight weeks' duration. Exclusion criteria were cough likely to be caused by a serious underlying medical condition, known immunodeficiency or immunocompromise, participation in another clinical research study, and preschool pertussis booster vaccination received less than one year previously. MAIN OUTCOME MEASURES: Evidence of recent pertussis infection based on an oral fluid anti-pertussis toxin IgG titre of at least 70 arbitrary units. Cough frequency was measured in six children with laboratory confirmed pertussis. RESULTS: 56 (20%, 95% confidence interval 16% to 25%) children had evidence of recent pertussis infection, including 39 (18%, 13% to 24%) of 215 children who had been fully vaccinated. The risk of pertussis was more than three times higher (21/53; 40%, 26% to 54%) in children who had received the preschool pertussis booster vaccination seven years or more previously than in those who had received it less than seven years previously (20/171; 12%, 7% to 17%). The risk of pertussis was similar between children who received five and three component preschool pertussis booster vaccines (risk ratio for five component vaccine 1.14, 0.64 to 2.03). Four of six children in whom cough frequency was measured coughed more than 400 times in 24 hours. CONCLUSIONS: Pertussis can still be found in a fifth of school age children who present in primary care with persistent cough and can cause clinically significant cough in fully vaccinated children. These findings will help to inform consideration of the need for an adolescent pertussis booster vaccination in the United Kingdom. STUDY REGISTRATION: UK Clinical Research Network portfolio ID 8361.

Oral fluid testing for pertussis, England and wales, june 2007-august 2009.

Existing pertussis surveillance systems tend to underidentify less severe cases among older children and adults. For routine follow-up of notified, nonconfirmed, clinically diagnosed pertussis cases, use of an oral fluid test was pilot tested in England and Wales during June 2007-August 2009. During that period, 1,852 cases of pertussis were confirmed by established laboratory methods and another 591 by oral fluid testing only. Although introduction of serologic testing in 2002 led to the greatest increase in ascertainment of pertussis, oral fluid testing increased laboratory ascertainment by 32% overall; maximal increase (124%) occurred among children 5-9 years of age. Patients whose pertussis was confirmed by oral fluid testing were least likely to be hospitalized, suggesting that milder community cases were being confirmed by this method. Oral fluid testing is an easily administered, noninvasive surveillance tool that could further our understanding of pertussis epidemiology and thereby contribute to decisions on vaccination strategies.

Pneumococcal carriage in children and adults two years after introduction of the thirteen valent pneumococcal conjugate vaccine in England.

BACKGROUND/AIMS: In April 2010 the 7-valent pneumococcal conjugate vaccine (PCV7) was replaced by the 13-valent PCV. We investigated pneumococcal carriage in children eligible for PCV7 or PCV13 and their household contacts. METHODS: Eligible families in Hertfordshire and Gloucester were identified and a nasopharyngeal swab obtained from consenting household members between July 2012 and March 2013. Samples were cultured for Streptococcus pneumoniae and serotyped by standard methods. For each serotype the ratio of its prevalence in invasive pneumococcal disease (IPD) to its carriage prevalence (case:carrier ratio, CCR) was calculated. Results were compared with previous carriage studies in 2001/2002 and 2008/2009, before and after PCV7 introduction. RESULTS: 217 households were included. Among <5-year olds 47.7% (95% confidence interval 41.8-53.5) were carrying a pneumococcus compared with 51.0% (95% CI: 44.0-58.0) in 2008/2009 and 48.4% (95% CI: 44.1-52.7) in 2001/2002. The odds of carrying a PCV7 serotype was significantly reduced in 2008/2009 (0.07, 95% CI: 0.03-0.16) and 2012/2013 (0.01 95% CI: 0.00-0.07) relative to 2001/2002, while the odds of carrying any of the extra six PCV13 serotypes increased after PCV7 introduction (1.38, 95%CI: 0.73-2.59) but declined significantly after PCV13 introduction (0.05, 95%CI: 0.01-0.37). The CCRs for the frequently carried serotypes were relatively low, with the highest CCR observed for serotypes 7F, 19A, 3, 8, and 33F. Across the three carriage studies, CCR estimates were stable for nearly all serotypes. CONCLUSION: Carriage of additional PCV13 serotypes has rapidly reduced post-PCV13 introduction in both vaccinated and unvaccinated individuals with a continued decline in transmission of PCV7 serotypes. Carriage rates in children remain unchanged, but the low CCRs of replacing serotypes would be expected to further reduce overall IPD across all age groups.

Montelukast for postinfectious cough in adults: a double-blind randomised placebo-controlled trial.

BACKGROUND: Postinfectious cough is common in primary care, but has no proven effective treatments. Cysteinyl leukotrienes are involved in the pathogenesis of postinfectious cough and whooping cough (pertussis). We investigated the effectiveness of montelukast, a cysteinyl leukotriene receptor antagonist, in the treatment of postinfectious cough. METHODS: In this randomised, placebo-controlled trial, non-smoking adults aged 16-49 years with postinfectious cough of 2-8 weeks' duration were recruited from 25 general practices in England. Patients were tested for pertussis (oral fluid anti-pertussis toxin IgG) and randomly assigned (1:1) to montelukast 10 mg daily or image-matched placebo for 2 weeks. Patients chose whether to continue study drug for another 2 weeks. The randomisation sequence was computer-generated and stratified by general practice. Patients, health-care professionals, and researchers were masked to treatment allocation. Effectiveness was assessed with the Leicester Cough Questionnaire to measure changes in cough-specific quality of life; the primary outcomes were changes in total score between baseline and two follow-up stages (2 weeks and 4 weeks). The primary analysis was by intention to treat with imputation by last observation carried forward. Recruitment closed on Sept 21, 2012, and follow-up has been completed. This trial is registered with EudraCT (2010-019647-19), UKCRN Portfolio (ID 8360), and ClinicalTrials.gov (NCT01279668). FINDINGS: From April 13, 2011, to Sept 21, 2012, we randomly assigned 276 patients to montelukast (n=137) or placebo (n=139). 70 (25%) patients had laboratory-confirmed pertussis. Improvements in cough-specific quality of life occurred in both groups after 2 weeks (montelukast: mean 2·7, 95% CI 2·2-3·3; placebo: 3·6, 2·9-4·3), but the difference between groups did not meet the minimum clinically important difference of 1·3 (mean difference -0·9, -1·7 to -0·04, p=0·04). This difference was not statistically significant in any sensitivity analyses. After 2 weeks, 192 of 259 participants from whom data were available elected to continue study drug (99 [77%] of 129 participants on montelukast; 93 [72%] of 130 on placebo). After 4 weeks, there were no significant between-group differences in cough-specific quality of life improvement (montelukast: 5·2, 4·5-5·9; placebo: 5·9, 5·1-6·7; mean difference -0·5, -1·5 to 0·6, p=0·38) or adverse event rates (21 (15%) of 137 patients on montelukast reported one or more adverse events; 31 (22%) of 139 on placebo; p=0·14). The most common adverse events reported were increased mucus production (montelukast, n=6; placebo, n=2), gastrointestinal disturbance (montelukast, n=3; placebo, n=5), and headache (montelukast, n=2; placebo, n=6). One serious adverse event was reported (placebo, n=1), which was unrelated to study drug (shortness of breath and throat tightness after severe coughing bouts). INTERPRETATION: Montelukast is not an effective treatment for postinfectious cough. However, the burden of postinfectious cough in primary care is high, making it an ideal setting for future antitussive treatment trials. FUNDING: National Institute for Health Research School for Primary Care Research, UK.

Comparison of the Legionella pneumophila population structure as determined by sequence-based typing and whole genome sequencing.

BACKGROUND: Legionella pneumophila is an opportunistic pathogen of humans where the source of infection is usually from contaminated man-made water systems. When an outbreak of Legionnaires' disease caused by L. pneumophila occurs, it is necessary to discover the source of infection. A seven allele sequence-based typing scheme (SBT) has been very successful in providing the means to attribute outbreaks of L. pneumophila to a particular source or sources. Particular sequence types described by this scheme are known to exhibit specific phenotypes. For instance some types are seen often in clinical cases but are rarely isolated from the environment and vice versa. Of those causing human disease some types are thought to be more likely to cause more severe disease. It is possible that the genetic basis for these differences are vertically inherited and associated with particular genetic lineages within the population. In order to provide a framework within which to test this hypothesis and others relating to the population biology of L. pneumophila, a set of genomes covering the known diversity of the organism is required. RESULTS: Firstly, this study describes a means to group L. pneumophila strains into pragmatic clusters, using a methodology that takes into consideration the genetic forces operating on the population. These clusters can be used as a standardised nomenclature, so those wishing to describe a group of strains can do so. Secondly, the clusters generated from the first part of the study were used to select strains rationally for whole genome sequencing (WGS). The data generated was used to compare phylogenies derived from SBT and WGS. In general the SBT sequence type (ST) accurately reflects the whole genome-based genotype. Where there are exceptions and recombination has resulted in the ST no longer reflecting the genetic lineage described by the whole genome sequence, the clustering technique employed detects these sequence types as being admixed, indicating their mixed inheritance. CONCLUSIONS: We conclude that SBT is usually a good proxy for the genetic lineage described by the whole genome, and therefore utility of SBT is still suitable until the technology and economics of high throughput sequencing reach the point where routine WGS of L. pneumophila isolates for outbreak investigation is feasible.

Modelling anti-pertussis toxin IgG antibody decay following primary and preschool vaccination with an acellular pertussis vaccine in UK subjects using a modified oral fluid assay.

Recent vaccination with pertussis vaccine can confound serological and oral fluid (OF) assays targeting anti-pertussis toxin (anti-PT) IgG antibodies as a marker of recent infection. This study sought to establish the minimum potentially confounding time period based on experimental data to assist interpretation from such samples submitted from UK subjects for pertussis diagnosis. Anti-PT IgG antibody response and decay were measured post-vaccination using a modified OF IgG antibody-capture ELISA (GACELISA). Data were obtained from 72 infants after the third acellular pertussis vaccine dose in the primary schedule (4 months of age) and from 119 children after the single dose at preschool age (3 years 4 months to 5 years 8 months of age). Specimens were taken at approximately 1 month intervals for 9 months post-primary immunization (third dose) and 13 months post-preschool booster (PSB). The modified GACELISA demonstrated a sensitivity of 52/56 (92.9 %: 95 % CI 82.7-98.0) and a specificity of 120/128 (93.8 %: 95 % CI 88.0-97.3) and showed good agreement with the National Reference Laboratory standard anti-PT IgG serum ELISA (rank correlation = 0.80) and the original OF assay (rank correlation = 0.79). Modelling of the decline in antibody titres showed a reduction of 54 % and 34 % for each doubling of time after day 14 for the post-third primary dose and post-PSB subjects, respectively. These data suggest that the minimum confounding time period is approximately 300 days for samples obtained post-primary immunization and at least 3 years for samples submitted from UK children following immunization with the PSB. These data will greatly assist the interpretation of single high diagnostic anti-PT IgG titres by allowing an estimate of the positive predictive value, when the number of days post-immunization and prevalence are known or assumed.

Molecular diagnostics and the public health management of legionellosis.

In 2009-2010, we investigated four legionella cases notified over an 8-month period in two adjacent villages in South East England. Molecular techniques enabled us to conclude that three of the cases had distinct infections. The absence of an adequate respiratory sample in one case necessitated epidemiological investigations to exclude a potential common environmental source of further infections. One of the cases had spent a part of their incubation period in a country in South East Asia. DNA-sequence-based typing of their isolate showed it to be of the Legionella pneumophila serogroup 1 (LP1) DNA-sequence type (ST) 481. Intriguingly, the only other two ST 481 isolates in the European Working Group for Legionella Infections database were among Dutch travellers to the same country in 2003 and 2006. This case makes clear the value of molecular diagnostics and the importance of obtaining adequate clinical specimens. The potential future uses for typing data are discussed.

First outbreak of nosocomial Legionella infection in term neonates caused by a cold mist ultrasonic humidifier.

BACKGROUND: To date, all descriptions of legionellosis in neonates have emerged from a small number of isolated case reports in newborns with unusually severe pneumonia. In December 2008, a large outbreak of Legionella infection occurred in term neonates in Cyprus, providing new information on the epidemiological and clinical features of Legionellosis in this age group. METHODS: An environmental investigation was performed at a small private hospital where the infected neonates were delivered. The medical records of the infected neonates were retrospectively reviewed to obtain clinical data on presentation, complications, and course of disease. RESULTS: Nine of the 32 (28%) newborns who were exposed to the contaminated source at the private nursery were infected with Legionella. Six subjects had pulmonary infiltrates, but in 3 cases there were no abnormal radiological findings and clinical presentation was mild. In 4 neonates, pulmonary infiltrates at presentation were bilateral and extensive and 3 died, conferring a mortality rate of 50% in subjects with pulmonary infiltrates and an overall mortality of 33.3%. Legionella pneumophila serogroup 3 was recovered in neonatal biological samples, although in some patients there was implication of a second strain, serogroup 1. It was determined that the neonates were infected while in the nursery at the private hospital by aerosol produced by a recently installed cold-mist humidifier that was filled with contaminated water. CONCLUSIONS: Use of humidifiers in nursery units must be avoided as the risk of disseminating Legionella in neonates is very high. In neonates legionellosis should be suspected when signs of infection first appear and take an unusual course, even when no pulmonary infiltrates appear.

A pilot study of rapid whole-genome sequencing for the investigation of a Legionella outbreak.

OBJECTIVES: Epidemiological investigations of Legionnaires' disease outbreaks rely on the rapid identification and typing of clinical and environmental Legionella isolates in order to identify and control the source of infection. Rapid bacterial whole-genome sequencing (WGS) is an emerging technology that has the potential to rapidly discriminate outbreak from non-outbreak isolates in a clinically relevant time frame. METHODS: We performed a pilot study to determine the feasibility of using bacterial WGS to differentiate outbreak from non-outbreak isolates collected during an outbreak of Legionnaires' disease. Seven Legionella isolates (three clinical and four environmental) were obtained from the reference laboratory and sequenced using the Illumina MiSeq platform at Addenbrooke's Hospital, Cambridge. Bioinformatic analysis was performed blinded to the epidemiological data at the Wellcome Trust Sanger Institute. RESULTS: We were able to distinguish outbreak from non-outbreak isolates using bacterial WGS, and to confirm the probable environmental source. Our analysis also highlighted constraints, which were the small number of Legionella pneumophila isolates available for sequencing, and the limited number of published genomes for comparison. CONCLUSIONS: We have demonstrated the feasibility of using rapid WGS to investigate an outbreak of Legionnaires' disease. Future work includes building larger genomic databases of L pneumophila from both clinical and environmental sources, developing automated data interpretation software, and conducting a cost-benefit analysis of WGS versus current typing methods.