Molecular detection of CF lung pathogens:current status and future potential

Molecular diagnostic tests, based on the detection and identification of nucleic acids in human biological samples, are increasingly employed in the diagnosis of infectious diseases and may be of future benefit to CF microbiology services. Our growing understanding of the complex polymicrobial nature of CF airway infection has highlighted current and likely future shortcomings in standard diagnostic practices. Failure to detect fastidious or slow growing microbes and misidentification of newly emerging pathogens could potentially be addressed using culture-independent molecular technologies with high target specificity. This review considers existing molecular diagnostic tests in the context of the key requirements for an envisaged CF microbiology focussed assay. The issues of assay speed, throughput, detection of multiple pathogens, data interpretation and antimicrobial susceptibility testing are discussed.

The impact of personalised therapies on respiratory medicine

Stratified approaches to treating disease are very attractive, as efficacy is maximised by identifying responders using a companion diagnostic or by careful phenotyping. This approach will spare non-responders form potential side-effects. This has been pioneered in oncology where single genes or gene signatures indicate tumours that will respond to specific chemotherapies. Stratified approaches to the treatment of asthma with biological therapies are currently being extensively studied. In cystic fibrosis (CF), therapies have been developed that are targeted at specific functional classes of mutations. Ivacaftor, the first of such therapies, potentiates dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) protein Class III mutations and is now available in the USA and some European countries. Pivotal studies in patients with a G551D mutation, the most common Class III mutation, have demonstrated significant improvements in clinically important outcomes such as spirometry and exacerbations. Sweat chloride was significantly reduced demonstrating a functional effect on the dysfunctional CFTR protein produced by the G551D mutation. Symptom scores are also greatly improved to a level that indicates that this is a transformational treatment for many patients. This stratified approach to the development of therapies based on the functional class of the mutations in CF is likely to lead to new drugs or combinations that will correct the basic defect in many patients with CF. © ERS 2013.

Interventions for enhancing adherence to treatment in adults with bronchiectasis

Background: Bronchiectasis is characterised by a widening of the airways, leading to excess mucus production and recurrent infection. It is more prevalent in women and those in middle age. Many patients with bronchiectasis do not adhere to treatments (medications, exercise and airway clearance) prescribed for their condition. The best methods to change these adherence behaviours have not been identified.
Objectives: To assess the effects of interventions to enhance adherence to any aspect of treatment in adults with bronchiectasis in terms of adherence and health outcomes, such as pulmonary exacerbations, health-related quality of life and healthcare costs.
Search methods:We searched the Cochrane Airways Gr oup Specialised Register (CAGR), which contains trial reports identified through systematic searches of CENTRAL, MEDLINE, EMBASE, CINAHL, AMED and PsycINFO, from inception to October 2015.
Selection criteria: We planned to include randomised controlled trials (RCTs) of adults with bronchiectasis that compared any intervention aimed at enhancing adherence versus no intervention, usual care or another adherence intervention. We excluded studies of those who had bronchiectasis due to cystic fibrosis.
Data collection and analysis: Two review authors (AMcC and ET) independently screened titles, abstracts and full-texts of identified studies.
Main results: Searches retrieved 36 studies reported in 37 articles; no eligible studies were identified.
Author's Conclusions: We did not identify any studies that assessed the effect of interventions to enhance adherence to treatment in bronchiectasis. Adequately powered, well-designed trials of adherence interventions for bronchiectasis are needed.

A Burkholderia cenocepacia gene encoding a non-functional tyrosine phosphatase is required for the delayed maturation of the bacteria-containing vacuoles in macrophages

Burkholderia cenocepacia infects patients with cystic fibrosis. We have previously shown that B. cenocepacia can survive in macrophages within membrane vacuoles (BcCVs) that preclude fusion with the lysosome. The bacterial factors involved in B. cenocepacia intracellular survival are not fully elucidated. We report here that deletion of BCAM0628, encoding a predicted low-molecular weight protein tyrosine phosphatase (LMW-PTP) that is restricted to B. cenocepacia strains of the transmissible ET-12 clone, accelerates the maturation of the BcCVs. Compared to parental strain and deletion mutants in other LMW-PTPs that are widely conserved in Burkholderia species, a greater proportion of BcCVs containing the BCAM0628 mutant were targeted to the lysosome. Accelerated BcCV maturation was not due to reduced intracellular viability since BCAM0628 survived and replicated in macrophages similarly to the parental strain. Therefore, BCAM0628 was referred to as dpm (delayed phagosome maturation). We provide evidence that the Dpm protein is secreted during growth in vitro and upon macrophage infection. Dpm secretion requires an N-terminal signal peptide. Heterologous expression of Dpm in B. multivorans confers to this bacterium a similar phagosomal maturation delay as found with B. cenocepacia. We demonstrate that Dpm is an inactive phosphatase, suggesting that its contribution to phagosomal maturation arrest must be unrelated to tyrosine phosphatase activity.

Identification of the Flagellin Glycosylation System in Burkholderia cenocepacia and the Contribution of Glycosylated Flagellin to Evasion of Human Innate Immune Responses

Burkholderia cenocepacia is an opportunistic pathogen threatening patients with cystic fibrosis. Flagella are required for biofilm formation, as well as adhesion to and invasion of epithelial cells. Recognition of flagellin via the Toll-like receptor 5 (TLR5) contributes to exacerbate B. cenocepacia-induced lung epithelial inflammatory responses. In this study, we report that B. cenocepacia flagellin is glycosylated on at least 10 different sites with a single sugar, 4,6-dideoxy-4-(3-hydroxybutanoylamino)-d-glucose. We have identified key genes that are required for flagellin glycosylation, including a predicted glycosyltransferase gene that is linked to the flagellin biosynthesis cluster and a putative acetyltransferase gene located within the O-antigen lipopolysaccharide cluster. Another O-antigen cluster gene, rmlB, which is required for flagellin glycan and O-antigen biosynthesis, was essential for bacterial viability, uncovering a novel target against Burkholderia infections. Using glycosylated and nonglycosylated purified flagellin and a cell reporter system to assess TLR5-mediated responses, we also show that the presence of glycan in flagellin significantly impairs the inflammatory response of epithelial cells. We therefore suggest that flagellin glycosylation reduces recognition of flagellin by host TLR5, providing an evasive strategy to infecting bacteria.

Dendrons with spermine surface groups as potential building blocks for nonviral vectors in gene therapy

This paper investigates a series of dendrons based on the Newkome dendritic scaffold that displays a naturally occurring polyamine (spermine) on their surface. These dendrons have previously been shown to interact with DNA in a generation dependent manner with the more highly branched dendrons exhibiting a strong multivalency effect for the spermine surface groups. In this paper, we investigate the ability of these dendrons to transfect DNA into cells (human breast carcinoma cells, MDA-MB-231, and murine myoblast cells, C2C12) as determined by the luciferase assay. Although the dendrons are unable to transfect DNA in their own right, they are capable of delivering DNA in vitro when administered with chloroquine, which assists with escape from endocytic vesicles. The cytotoxicity of the dendrons was determined using the XTT assay, and it was shown that the dendrons were nontoxic either alone or in the presence of DNA. However, when administered with DNA and chloroquine, the most highly branched dendron did exhibit some cytotoxicity. This paper elucidates the relationship between in vitro transfection efficiency and toxicity. While transfection efficiencies are modest, the low toxicity of the dendrons, both in their own right, and in the presence of DNA, provides encouragement that this type of building block, which has a relatively high affinity for DNA, will provide a useful starting point for the further synthetic development of more effective gene transfection agents.

A Functional Variant of Elafin With Improved Anti-inflammatory Activity for Pulmonary Inflammation

Elafin is a serine protease inhibitor produced by epithelial and immune cells with anti-inflammatory properties. Research has shown that dysregulated protease activity may elicit proteolytic cleavage of elafin, thereby impairing the innate immune function of the protein. The aim of this study was to generate variants of elafin (GG- and QQ-elafin) that exhibit increased protease resistance while retaining the biological properties of wild-type (WT) elafin. Similar to WT-elafin, GG- and QQ-elafin variants retained antiprotease activity and susceptibility to transglutaminase-mediated fibronectin cross-linking. However, in contrast to WT-elafin, GG- and QQ-elafin displayed significantly enhanced resistance to degradation when incubated with bronchoalveolar lavage fluid from patients with cystic fibrosis. Intriguingly, both variants, particularly GG-elafin, demonstrated improved lipopolysaccharide (LPS) neutralization properties in vitro. In addition, GG-elafin showed improved anti-inflammatory activity in a mouse model of LPS-induced acute lung inflammation. Inflammatory cell infiltration into the lung was reduced in lungs of mice treated with GG-elafin, predominantly neutrophilic infiltration. A reduction in MCP-1 levels in GG-elafin treated mice compared to the LPS alone treatment group was also demonstrated. GG-elafin showed increased functionality when compared to WT-elafin and may be of future therapeutic relevance in the treatment of lung diseases characterized by a protease burden.

Care Planning in Children and Young Peoples Nursing

Care Planning in Children and Young People's Nursing addresses a selection of the most common concerns that arise when planning care for infants, children and young people within the hospital and community setting. Clear and detailed, this text reflects both the uniqueness and diversity of contemporary children's nursing and utilizes images and case studies to provide a holistic insight into the practice of care planning through the reporting of best available evidence and current research, policy and education.

Divided into sections for ease of reference, Care Planning in Children and Young People’s Nursing explores both the theory and practice of care planning. Chapters on the principles of care planning include issues such as managing risk, safeguarding children, ethical and legal implications, integrated care pathways, interprofessional assessment, and invaluable parent perspectives. Additional chapters on the application of planning care examine the practical aspects of a wide range of specific conditions including cystic fibrosis, obesity, cardiac/renal failure and HIV/AIDS. Each chapter is interactive, with questions, learning activities and points for discussion creating an engaging and enquiry-based learning approach.

Care Planning in Children and Young People’s Nursing is a definitive resource, reflecting innovative practice which is suitable for undergraduate and postgraduate nurse education.

Developing an international Pseudomonas aeruginosa reference panel

Pseudomonas aeruginosa is a major opportunistic pathogen in cystic fibrosis (CF) patients and causes a wide range of infections among other susceptible populations. Its inherent resistance to many antimicrobials also makes it difficult to treat infections with this pathogen. Recent evidence has highlighted the diversity of this species, yet despite this, the majority of studies on virulence and pathogenesis focus on a small number of strains. There is a pressing need for a P. aeruginosa reference panel to harmonize and coordinate the collective efforts of the P. aeruginosa research community. We have collated a panel of 43 P. aeruginosa strains that reflects the organism's diversity. In addition to the commonly studied clones, this panel includes transmissible strains, sequential CF isolates, strains with specific virulence characteristics, and strains that represent serotype, genotype or geographic diversity. This focussed panel of P. aeruginosa isolates will help accelerate and consolidate the discovery of virulence determinants, improve our understanding of the pathogenesis of infections caused by this pathogen, and provide the community with a valuable resource for the testing of novel therapeutic agents.

Pseudomonas aeruginosa exhibits frequent recombination, but only a limited association between genotype and ecological setting

Pseudomonas aeruginosa is an opportunistic pathogen and an important cause of infection, particularly amongst cystic fibrosis (CF) patients. While specific strains capable of patient-to-patient transmission are known, many infections appear to be caused by unique and unrelated strains. There is a need to understand the relationship between strains capable of colonising the CF lung and the broader set of P. aeruginosa isolates found in natural environments. Here we report the results of a multilocus sequence typing (MLST)-based study designed to understand the genetic diversity and population structure of an extensive regional sample of P. aeruginosa isolates from South East Queensland, Australia. The analysis is based on 501 P. aeruginosa isolates obtained from environmental, animal and human (CF and non-CF) sources with particular emphasis on isolates from the Lower Brisbane River and isolates from CF patients obtained from the same geographical region. Overall, MLST identified 274 different sequence types, of which 53 were shared between one or more ecological settings. Our analysis revealed a limited association between genotype and environment and evidence of frequent recombination. We also found that genetic diversity of P. aeruginosa in Queensland, Australia was indistinguishable from that of the global P. aeruginosa population. Several CF strains were encountered frequently in multiple ecological settings; however, the most frequently encountered CF strains were confined to CF patients. Overall, our data confirm a non-clonal epidemic structure and indicate that most CF strains are a random sample of the broader P. aeruginosa population. The increased abundance of some CF strains in different geographical regions is a likely product of chance colonisation events followed by adaptation to the CF lung and horizontal transmission among patients.

Identification of Pseudomonas aeruginosa by a duplex real-time polymerase chain reaction assay targeting the ecfX and the gyrB genes

Phenotypic identification of Gram-negative bacteria from respiratory specimens of patients with cystic fibrosis carries a high risk of misidentification. Molecular identification techniques that use single-gene targets are also susceptible to error, including cross-reaction issues with other Gram-negative organisms. In this study, we have designed a Pseudomonas aeruginosa duplex real-time polymerase chain reaction (PCR) (PAduplex) assay targeting the ecfX and the gyrB genes. The PAduplex was evaluated against a panel of 91 clinical and environmental isolates that were presumptively identified as P. aeruginosa. The results were compared with those obtained using a commercial biochemical identification kit and several other P. aeruginosa PCR assays. The results showed that the PAduplex assay is highly suitable for routine identification of P. aeruginosa isolates from clinical or environmental samples. The 2-target format provides simultaneous confirmation of P. aeruginosa identity where both the ecfX and gyrB PCR reactions are positive and may also reduce the potential for false negatives caused by sequence variation in primer or probe targets.

Combination aerosol therapy to treat Burkholderia cepacia complex

Burkholderia cepacia infection in cystic fibrosis (CF) patients is associated with significant morbidity and mortality, yet no definitive treatment is currently available. This report describes a new approach to treat B. cepacia infection in CF patients, using a combination of amiloride and tobramycin aerosols. Four adults with the typical clinical syndrome of CF were recruited after repeated positive sputum cultures for B. cepacia. Aerosols of amiloride and tobramycin were given three times daily for 1-6 months, and repeated sputum cultures were collected to assess efficacy. Three of the four patients treated with the combined therapy eradicated B. cepacia from their sputum cultures for at least 2 yrs, and there were no adverse events. This novel combination may provide a new therapeutic option for Burkholderia cepacia infections. Furthermore, the strategy of combining antibiotics with ion transport agents may have ramifications for the treatment of other multi-resistant organisms.

THE CULTURE MICROBIOME IN THE LUNGS OF PATIENTS WITH COPD

Introduction and Aims: Previous studies have shown that the lungs of Cystic Fibrosis (CF) and bronchiectasis (BE, not caused by CF) patients are colonised by a range of aerobic and anaerobic bacteria. As bacteria are also implicated in the pathogenesis and progression of chronic obstructive pulmonary disease (COPD), this study aimed to determine the culture microbiome of the COPD airways.

Methods: Samples were collected from 13 stable COPD patients during routine bronchoscopy. Bronchial washings were taken at a single location in the right middle lobe by flushing and removing 30 ml of sterile saline. Samples were cultured under strict anaerobic conditions with bacteria detected by plating on both selective and non-selective agar media and quantified by total viable count (TVC). Identification of the cultured bacteria was performed by amplification and subsequent sequencing of the 16sRNA gene.

Results: Mean FEV1 was 1.36 (range 0.84–2.26, mean per cent predicted FEV1, 54%), and the mean ratio (FEV1/FVC) was 51%. Bacteria were detected in 12/13 samples (92%) with bacteria from the genera Streptococcus [12/13 samples, 92%; mean (range) TVC 9.62×105 cfu/ml (1.50×103–1.42×107)] and Haemophilus [4/13 samples, 31%; mean (range) 6.40×104 cfu/ml (2.20×103–1.60×105)] most frequently detected. Anaerobic bacteria primarily from the genera Prevotella [8/13 samples, 62%; mean (range) TVC 1.12×104 cfu/ml (1.30×103–4.20×104)] and Veillonella [5/13 samples, 38%; mean (range) TVC 1.29×105 cfu/ml (4.20×103–3.60×105)] were also detected. Pseudomonas and Moraxella were not detected in any samples.

Conclusions: Our results show that bacteria from the genera Streptococcus, Haemophilus, Prevotella and Veillonella are frequently present the airways of patients suffering from COPD. Taking account of the dilutional effect of the bronchial wash procedure and extrapolating to allow comparison with sputum data in our laboratory for CF and BE, the relative load of bacteria from the genera Streptococcus, Prevotella and Veillonella is similar in these three airway diseases. The potential role of these bacteria in the progression and pathogenesis of COPD requires further investigation.