Multiple breath washout in bronchiectasis clinical trials:is it feasible?

Background: Evaluation of Multiple Breath Washout (MBW) set-up including staff training, certification and central “over-reading” for data quality control is essential to determine the feasibility of MBW in future bronchiectasis studies. Aims: To assess the outcomes of a MBW training, certification and central over-reading programme. Methods: MBW training and certification was conducted in European sites collecting LCI data in the BronchUK clinimetrics and/or i-BEST-1 studies. The blended training programme included the use of an eLearning tool and a 1-day face-to-face session. Sites submitted MBW data to trained central over-readers who determined validity and quality. Results: Thirteen training days were delivered to 56 participants from 22 sites. 18/22 (82%) were MBW naïve. Participant knowledge and confidence increased significantly (p<0.001). By the end of the study recruitment, 15/22 sites (68%) had completed certification with a mean (range) time since training of 6.2 (3-14) months. In the BronchUK clinimetrics study, 468/589 (79%) tests met45 the quality criteria following central over-reading, compared with 137/236 (58%) tests in the i-BEST-1 study. Conclusions: LCI is feasible in a bronchiectasis multicentre clinical trial setting however, consideration of site experience in terms of training as well as assessment of skill drift and the need for re-training may be important to reduce time to certification and optimise data quality. Longer times to certification, a higher percentage of naive sites and patients with worse lung function may have contributed to the lower success rate in the i-BEST-1 study

Characterisation of eppin function: expression and activity in the lung

Eppin is a serine protease inhibitor expressed in male reproductive tissues. In this study we have demonstrated novel sites of eppin expression in myeloid and epithelial cell lines with further confirmation in primary myeloid cell types. Using immunohistochemistry and Western blotting, eppin was detected in the lungs of patients with Acute Respiratory Distress Syndrome and Cystic Fibrosis lung disease. Expression of eppin in monocytic cells was unaffected by stimulation with TLR agonists, cytokine stimulation and hormone receptor agonist stimulation. However, upregulated expression and secretion of eppin was observed following treatment of monocytes with epidermal growth factor (EGF). Incubation of recombinant eppin with monocytic cells resulted in significant inhibition of lipopolysaccharide (LPS)-induced chemokine production. Furthermore, eppin inhibited LPS- induced NF-κB activation by a mechanism which involved accumulation of phosphorylated IκBα. In an in vivo model of lung inflammation induced by LPS, eppin administration resulted in decreased recruitment of neutrophils to the lung with a concomitant reduction in the levels of the neutrophil chemokine MIP-2. Overall, these results suggest a role for eppin outside of the reproductive tract and that eppin may have a role in the innate immuneresponse in the lung

Predominant pathogen competition and core microbiota divergence in chronic airway infection

© 2015 International Society for Microbial Ecology All rights reserved. Chronic bacterial lung infections associated with non-cystic fibrosis bronchiectasis represent a substantial and growing health-care burden. Where Pseudomonas aeruginosa is the numerically dominant species within these infections, prognosis is significantly worse. However, in many individuals, Haemophilus influenzae predominates, a scenario associated with less severe disease. The mechanisms that determine which pathogen is most abundant are not known. We hypothesised that the distribution of H. influenzae and P. aeruginosa would be consistent with strong interspecific competition effects. Further, we hypothesised that where P. aeruginosa is predominant, it is associated with a distinct 'accessory microbiota' that reflects a significant interaction between this pathogen and the wider bacterial community. To test these hypotheses, we analysed 16S rRNA gene pyrosequencing data generated previously from 60 adult bronchiectasis patients, whose airway microbiota was dominated by either P. aeruginosa or H. influenzae. The relative abundances of the two dominant species in their respective groups were not significantly different, and when present in the opposite pathogen group the two species were found to be in very low abundance, if at all. These findings are consistent with strong competition effects, moving towards competitive exclusion. Ordination analysis indicated that the distribution of the core microbiota associated with each pathogen, readjusted after removal of the dominant species, was significantly divergent (analysis of similarity (ANOSIM), R=0.07, P=0.019). Taken together, these findings suggest that both interspecific competition and also direct and/or indirect interactions between the predominant species and the wider bacterial community may contribute to the predominance of P. aeruginosa in a subset of bronchiectasis lung infections

Lung function and microbiota diversity in cystic fibrosis

Abstract: Background: Chronic infection and concomitant airway inflammation is the leading cause of morbidity and mortality for people living with cystic fibrosis (CF). Although chronic infection in CF is undeniably polymicrobial, involving a lung microbiota, infection surveillance and control approaches remain underpinned by classical aerobic culture-based microbiology. How to use microbiomics to direct clinical management of CF airway infections remains a crucial challenge. A pivotal step towards leveraging microbiome approaches in CF clinical care is to understand the ecology of the CF lung microbiome and identify ecological patterns of CF microbiota across a wide spectrum of lung disease. Assessing sputum samples from 299 patients attending 13 CF centres in Europe and the USA, we determined whether the emerging relationship of decreasing microbiota diversity with worsening lung function could be considered a generalised pattern of CF lung microbiota and explored its potential as an informative indicator of lung disease state in CF. Results: We tested and found decreasing microbiota diversity with a reduction in lung function to be a significant ecological pattern. Moreover, the loss of diversity was accompanied by an increase in microbiota dominance. Subsequently, we stratified patients into lung disease categories of increasing disease severity to further investigate relationships between microbiota characteristics and lung function, and the factors contributing to microbiota variance. Core taxa group composition became highly conserved within the severe disease category, while the rarer satellite taxa underpinned the high variability observed in the microbiota diversity. Further, the lung microbiota of individual patient were increasingly dominated by recognised CF pathogens as lung function decreased. Conversely, other bacteria, especially obligate anaerobes, increasingly dominated in those with better lung function. Ordination analyses revealed lung function and antibiotics to be main explanators of compositional variance in the microbiota and the core and satellite taxa. Biogeography was found to influence acquisition of the rarer satellite taxa. Conclusions: Our findings demonstrate that microbiota diversity and dominance, as well as the identity of the dominant bacterial species, in combination with measures of lung function, can be used as informative indicators of disease state in CF. BBFJdPr3cu-jH3LTAhe361Video Abstrac

Culture Enriched Molecular Profiling of the Cystic Fibrosis Airway Microbiome

The microbiome of the respiratory tract, including the nasopharyngeal and oropharyngeal microbiota, is a dynamic community of microorganisms that is highly diverse. The cystic fibrosis (CF) airway microbiome refers to the polymicrobial communities present in the lower airways of CF patients. It is comprised of chronic opportunistic pathogens (such as Pseudomonas aeruginosa) and a variety of organisms derived mostly from the normal microbiota of the upper respiratory tract. The complexity of these communities has been inferred primarily from culture independent molecular profiling. As with most microbial communities it is generally assumed that most of the organisms present are not readily cultured. Our culture collection generated using more extensive cultivation approaches, reveals a more complex microbial community than that obtained by conventional CF culture methods. To directly evaluate the cultivability of the airway microbiome, we examined six samples in depth using culture-enriched molecular profiling which combines culture-based methods with the molecular profiling methods of terminal restriction fragment length polymorphisms and 16S rRNA gene sequencing. We demonstrate that combining culture-dependent and culture-independent approaches enhances the sensitivity of either approach alone. Our techniques were able to cultivate 43 of the 48 families detected by deep sequencing; the five families recovered solely by culture-independent approaches were all present at very low abundance (<0.002% total reads). 46% of the molecular signatures detected by culture from the six patients were only identified in an anaerobic environment, suggesting that a large proportion of the cultured airway community is composed of obligate anaerobes. Most significantly, using 20 growth conditions per specimen, half of which included anaerobic cultivation and extended incubation times we demonstrate that the majority of bacteria present can be cultured

Partitioning core and satellite taxa from within cystic fibrosis lung bacterial communities

Cystic fibrosis (CF) patients suffer from chronic bacterial lung infections that lead to death in the majority of cases. The need to maintain lung function in these patients means that characterising these infections is vital. Increasingly, culture-independent analyses are expanding the number of bacterial species associated with CF respiratory samples; however, the potential significance of these species is not known. Here, we applied ecological statistical tools to such culture-independent data, in a novel manner, to partition taxa within the metacommunity into core and satellite species. Sputa and clinical data were obtained from 14 clinically stable adult CF patients. Fourteen rRNA gene libraries were constructed with 35 genera and 82 taxa, identified in 2139 bacterial clones. Shannon–Wiener and taxa-richness analyses confirmed no undersampling of bacterial diversity. By decomposing the distribution using the ratio of variance to the mean taxon abundance, we partitioned objectively the species abundance distribution into core and satellite species. The satellite group comprised 67 bacterial taxa from 33 genera and the core group, 15 taxa from 7 genera (including Pseudomonas (1 taxon), Streptococcus (2), Neisseria (2), Catonella (1), Porphyromonas (1), Prevotella (5) and Veillonella (3)], the last four being anaerobes). The core group was dominated by Pseudomonas aeruginosa. Other recognised CF pathogens were rare. Mantel and partial Mantel tests assessed which clinical factors influenced the composition observed. CF transmembrane conductance regulator genotype and antibiotic treatment correlated with all core taxa. Lung function correlated with richness. The clinical significance of these core and satellite species findings in the CF lung is discussed

In vitro activity of Cefiderocol against Gram-negative pathogens isolated from people with cystic fibrosis and bronchiectasis

ObjectivesGram-negative pathogens causing respiratory infection in people with cystic fibrosis and bronchiectasis are becoming progressively more resistant to conventional antibiotics. Although cefiderocol is licenced for the treatment of infections due to Gram-negative organisms, there are limited data on the activity of cefiderocol against pathogens associated with chronic respiratory diseases. The aim of this study was to determine the susceptibility of Gram-negative pathogens from cystic fibrosis and bronchiectasis to cefiderocol and comparator antibiotics.MethodsMinimal inhibitory concentrations (MICs) of cefiderocol and 15 comparator antibiotics were determined by broth microdilution against 300 respiratory isolates: Burkholderia spp., Stenotrophomonas spp., Achromobacter spp., Ralstonia spp. and Pandoraea spp., and used to calculate the MIC of each antibiotic required to inhibit 50% (MIC50) and 90% (MIC90) of isolates.ResultsThe MIC50 and MIC90 of cefiderocol for all 300 isolates tested was 0.25 and 32 mg/L, with 232 (77.3%) isolates having an MIC value ≤2 mg/L. In addition, cefiderocol demonstrated excellent activity against Stenotrophomonas spp. and Achromobacter spp. isolates, with 86.7% and 87.2%, respectively, exhibiting an MIC of 2 mg/L. Tigecycline also demonstrated good activity against all isolates with an MIC50 of &lt;0.5 mg/L.ConclusionsThese in vitro data demonstrated that cefiderocol had greater activity than most comparator antibiotics and could be an alternative treatment option for respiratory infection caused by these pathogens that has not responded to first-line therapy

Invisible and displaced dark matter signatures at Belle II

Many dark matter models generically predict invisible and displaced signatures at Belle II, but even striking events may be missed by the currently implemented search programme because of inefficient trigger algorithms. Of particular interest are final states with a single photon accompanied by missing energy and a displaced pair of electrons, muons, or hadrons. We argue that a displaced vertex trigger will be essential to achieve optimal sensitivity at Belle II. To illustrate this point, we study a simple but well-motivated model of thermal inelastic dark matter in which this signature naturally occurs and show that otherwise inaccessible regions of parameter space can be tested with such a search. We also evaluate the sensitivity of single-photon searches at BaBar and Belle II to this model and provide detailed calculations of the relic density target

Responsabilidade civil de provedores de internet

O tema do presente trabalho versa sobre a responsabilidade civil de provedores de Internet, mais especificamente sobre os provedores de backbone, de acesso, de hospedagem e de conteúdo. A Internet é hoje um dos principais meios de comunicação, sendo utilizada para a troca de informações, entretenimento, execução de negócios, aquisição de produtos e serviços etc. Nesse contexto, surge também o impacto do uso dessa ferramenta sobre os tradicionais valores, tais como a liberdade, a privacidade e relações consumeristas. Pela pesquisa, analisando as atividades realizadas pelos provedores de Internet, pretendeu-se verificar responsabilidade civil pelos danos causados por ilícitos praticados por seus próprios atos, bem como por atos de seus usuários e de terceiros que, ao utilizar os serviços dos provedores, venham a causar danos a outrem. A partir da apreciação conjunta de doutrina e jurisprudências e com base, principalmente, nos ordenamentos jurídicos constantes no Código Civil e Código de Defesa do Consumidor, dada a inexistência de orientação legislativa específica sobre a matéria, conclui-se que a responsabilidade civil dos provedores de Internet por seus próprios atos decorre da natureza da atividade por eles exercida e das cláusulas contratuais estabelecidas entre eles e seus consumidores ou tomadores de serviço. Nesses casos, a reparação dos danos causados funda-se nos princípios estabelecidos pelo Código Civil e Código de Defesa do Consumidor, imputando-se aos provedores a responsabilidade civil objetiva. Quanto aos danos causados por seus usuários ou por terceiros, a responsabilidade civil dos provedores dependerá também da natureza da atividade por eles exercida, do dever jurídico violado e do controle exercido pelo provedor sobre o conteúdo veiculado na Internet. Via de regra, os provedores de Internet não assumem a responsabilidade pelos danos causados por seus usuários ou terceiros, pois a fiscalização do conteúdo não é atividade inerente aos serviços por eles prestados. No entanto, se notificados acerca do conteúdo ilícito e não tomarem providências para removê-lo ou cessar o acesso do usuário que esteja utilizando os serviços de conexão para praticar o ato ilícito, terão responsabilidade subjetiva baseada em tal omissão. Especificamente em relação ao provedor de conteúdo, detendo este poder de controle sobre o material divulgado, responderá de forma solidária com autor da informação, efetivo causador do dano. Não havendo este controle, haverá responsabilidade se, tendo conhecimento do ilícito praticado, deixar o provedor de bloquear o acesso ao conteúdo danoso disponibilizado. Nesses casos, o provedor terá responsabilidade subjetiva e responderá solidariamente com o autor do dan