Standards for the care of people with cystic fibrosis (CF); recognising and addressing CF health issues.

This is the third in a series of four papers updating the European Cystic Fibrosis Society (ECFS) standards for the care of people with CF. This paper focuses on recognising and addressing CF health issues. The guidance was produced with wide stakeholder engagement, including people from the CF community, using an evidence-based framework. Authors contributed sections, and summary statements which were reviewed by a Delphi consultation. Monitoring and treating airway infection, inflammation and pulmonary exacerbations remains important, despite the widespread availability of CFTR modulators and their accompanying health improvements. Extrapulmonary CF-specific health issues persist, such as diabetes, liver disease, bone disease, stones and other renal issues, and intestinal obstruction. These health issues require multidisciplinary care with input from the relevant specialists. Cancer is more common in people with CF compared to the general population, and requires regular screening. The CF life journey requires mental and emotional adaptation to psychosocial and physical challenges, with support from the CF team and the CF psychologist. This is particularly important when life gets challenging, with disease progression requiring increased treatments, breathing support and potentially transplantation. Planning for end of life remains a necessary aspect of care and should be discussed openly, honestly, with sensitivity and compassion for the person with CF and their family. CF teams should proactively recognise and address CF-specific health issues, and support mental and emotional wellbeing while accompanying people with CF and their families on their life journey.

Future therapies for cystic fibrosis.

We are currently witnessing transformative change for people with cystic fibrosis with the introduction of small molecule, mutation-specific drugs capable of restoring function of the defective protein, cystic fibrosis transmembrane conductance regulator (CFTR). However, despite being a single gene disorder, there are multiple cystic fibrosis-causing genetic variants; mutation-specific drugs are not suitable for all genetic variants and also do not correct all the multisystem clinical manifestations of the disease. For many, there will remain a need for improved treatments. Those patients with gene variants responsive to CFTR modulators may have found these therapies to be transformational; research is now focusing on safely reducing the burden of symptom-directed treatment. However, modulators are not available in all parts of the globe, an issue which is further widening existing health inequalities. For patients who are not suitable for- or do not have access to- modulator drugs, alternative approaches are progressing through the trials pipeline. There will be challenges encountered in design and implementation of these trials, for which the established global CF infrastructure is a major advantage. Here, the Cystic Fibrosis National Research Strategy Group of the UK NIHR Respiratory Translational Research Collaboration looks to the future of cystic fibrosis therapies and consider priorities for future research and development.

Analysis of Neuropsychiatric Diagnoses After Montelukast Initiation.

Importance: The evidence base for the association between montelukast and adverse neuropsychiatric outcomes is mixed and inconclusive. Several methodological limitations have been identified in the evidence base on the safety of montelukast in observational studies. Objective: To investigate the association between new montelukast exposure and 1-year incident neuropsychiatric diagnoses with improved precision and control for baseline confounders. Design, Setting, and Participants: This propensity score-matched cohort study was conducted using electronic health records from 2015 to 2019 in the TriNetX Analytics Network patient repository of more than 51 million patients from 56 health care organizations, mainly in the US. Included patients were those aged 15 to 64 years at index prescription for montelukast or for control prescription who had a history of asthma or allergic rhinitis. After propensity score matching for various baseline confounders, including comorbidities and dispensed prescription medicines, we included 154 946 patients, of whom 77 473 individuals were exposed to montelukast. Patients were followed up for 12 months. Data were analyzed from June through November 2021. Exposures: New dispensed prescription for leukotriene receptor antagonist montelukast or control medication. Main Outcomes and Measures: Incident neuropsychiatric diagnoses at 12 months identified using International Statistical Classification of Diseases, Tenth Revision, Clinical Modification (ICD-10-CM) codes. Results: There were 72 490 patients with asthma (44 726 [61.7%] women; mean [SD] age at index prescription, 35 [15] years) and 82 456 patients with allergic rhinitis (54 172 [65.7%] women; mean [SD] age at index prescription, 40 [14] years). In patients exposed to montelukast, the odds ratio [OR] for any incident neuropsychiatric outcome was 1.11 (95% CI, 1.04-1.19) in patients with asthma and 1.07 (95% CI, 1.01-1.14) in patients with allergic rhinitis compared with patients who were unexposed. The highest OR was for anxiety disorders (OR, 1.21; 95% CI, 1.05-1.20) among patients with asthma exposed to montelukast and insomnia (OR, 1.15; 95% CI, 1.05-1.27) among patients with allergic rhinitis exposed to montelukast. Conclusions and Relevance: This study found that patients with asthma or allergic rhinitis had increased odds of adverse neuropsychiatric outcomes after montelukast initiation. These findings suggest that clinicians should consider monitoring potential adverse mental health symptoms during montelukast treatment, particularly in individuals with a history of mental health or sleep problems.

The lung microbiota in children with cystic fibrosis captured by induced sputum sampling.

BACKGROUND: Spatial topography of the cystic fibrosis (CF) lung microbiota is poorly understood in childhood. How best to sample the respiratory tract in children for microbiota analysis, and the utility of microbiota profiling in clinical management of early infection remains unclear. By comparison with bronchoalveolar lavage (BAL), we assessed the ability of induced sputum (IS) sampling to characterise the lower airway microbiota. METHODS: Sample sets from IS and two or three matched BAL compartments were obtained for microbiota analysis as part of the CF-Sputum Induction Trial (UKCRN_14615, ISRCTNR_12473810). Microbiota profiles and pathogen detection were compared between matched samples. RESULTS: Twenty-eight patients, aged 1.1-17.7 years, provided 30 sample sets. Within-patient BAL comparisons revealed spatial heterogeneity in 8/30 (27%) sample sets indicating that the lower airway microbiota from BAL is frequently compartmentalised in children with CF. IS samples closely resembled one or more matched BAL compartments in 15/30 (50%) sets, and were related in composition in a further 9/30 (30%). IS detected 86.2% of the Top 5 genera found across matched BAL samples. The sensitivity of IS to detect specific CF-pathogens identified in matched BAL samples at relative abundance ≥5% varied between 43 and 100%, with negative predictive values between 73 and 100%. CONCLUSIONS: Spatial heterogeneity of the lower airway microbiota was observed in BAL samples and presents difficulties for consistent lung sampling. IS captured a microbiota signature representative of the lower airway in 80% of cases, and is a straightforward, non-invasive intervention that can be performed frequently to aid pathogen diagnosis and understand microbiota evolution in children with CF.

Phenotypic and Genotypic Adaptations in Pseudomonas aeruginosa Biofilms following Long-Term Exposure to an Alginate Oligomer Therapy.

Chronic Pseudomonas aeruginosa lung infections in cystic fibrosis (CF) evolve to generate environmentally adapted biofilm communities, leading to increased patient morbidity and mortality. OligoG CF-5/20, a low-molecular-weight inhaled alginate oligomer therapy, is currently in phase IIb/III clinical trials in CF patients. Experimental evolution of P. aeruginosa in response to OligoG CF-5/20 was assessed using a bead biofilm model allowing continuous passage (45 days; ∼245 generations). Mutants isolated after OligoG CF-5/20 treatment typically had a reduced biofilm-forming ability and altered motility profile. Genotypically, OligoG CF-5/20 provided no selective pressure on genomic mutations within morphotypes. Chronic exposure to azithromycin, a commonly prescribed antibiotic in CF patients, with or without OligoG CF-5/20 in the biofilm evolution model also had no effect on rates of resistance acquisition. Interestingly, however, cross-resistance to other antibiotics (e.g., aztreonam) was reduced in the presence of OligoG CF-5/20. Collectively, these findings show no apparent adverse effects from long-term exposure to OligoG CF-5/20, instead resulting in both fewer colonies with multidrug resistance (MDR)-associated phenotypes and improved antibiotic susceptibility of P. aeruginosaIMPORTANCE The emergence of multidrug-resistant (MDR) pathogens within biofilms in the cystic fibrosis lung results in increased morbidity. An inhalation therapy derived from alginate, OligoG CF-5/20, is currently in clinical trials for cystic fibrosis patients. OligoG CF-5/20 has been shown to alter sputum viscoelasticity, disrupt mucin polymer networks, and disrupt MDR pseudomonal biofilms. Long-term exposure to inhaled therapeutics may induce selective evolutionary pressures on bacteria within the lung biofilm. Here, a bead biofilm model with repeated exposure of P. aeruginosa to OligoG CF-5/20 (alone and in combination with azithromycin) was conducted to study these long-term effects and characterize the phenotypic and genotypic adaptations which result. These findings, over 6 weeks, show that long-term use of OligoG CF-5/20 does not lead to extensive mutational changes and may potentially decrease the pathogenicity of the bacterial biofilm and improve the susceptibility of P. aeruginosa to other classes of antibiotics.

Cystic fibrosis newborn screening: the importance of bloodspot sample quality.

OBJECTIVE: Wales has an immunoreactive trypsin (IRT)-DNA cystic fibrosis (CF) newborn screening (NBS) programme. Most CF NBS false negative cases are due to an IRT concentration below the screening threshold. The accuracy of IRT results is dependent on the quality of the dried bloodspot (DBS) sample. The aim of this study was to determine the cause of false negative cases in CF NBS and their relationship to DBS quality. DESIGN: Longitudinal birth cohort. SETTING: Wales 1996-2016. PATIENTS: Children with CF. INTERVENTIONS: Identification of all CF patients with triangulation of multiple data sources to detect false negative cases. MAIN OUTCOME MEASURES: False negative cases. RESULTS: Over 20 years, 673 952 infants were screened and 239 were diagnosed with CF (incidence 1:2819). The sensitivity of the programme was 0.958, and positive predictive value was 0.476. Eighteen potential false negatives were identified, of whom eight were excluded: four screened outside Wales, two had complex comorbidities, no identified cystic fibrosis transmembrane conductance regulator (CFTR) variants on extended analysis and thus not considered to have CF and two were diagnosed after their 16th birthday. Of the 10 false negatives, 9 had a low DBS IRT and at least one common CFTR variant and thus should have received a sweat test under the programme. DBS cards were available for inspection for five of the nine false negative cases-all were classified as small/insufficient or poor quality. CONCLUSIONS: The majority of false negatives had a low bloodspot IRT, and this was associated with poor quality DBS. The optimal means to improve the sensitivity of our CF NBS programme would be to improve DBS sample quality.

Detecting respiratory infection in children with cystic fibrosis: Cough swab, sputum induction or bronchoalveolar lavage.

Young children with cystic fibrosis (CF) are generally very well, cough free and non-productive, and are often incapable of spontaneously expectorating sputum even if actively coughing during an exacerbation. Obtaining a meaningful airway sample for microbiological analysis is therefore problematic, yet essential if lower airway infection is to be detected and adequately treated. Recently there has been increasing interest in the use of sputum-induction in young children with CF, as a simple, cost effective, well tolerated and frequently repeatable approach to sampling the lower airway, and the relative merits of this approach to bacterial sampling are discussed. Culture-independent microbiology has increased our understanding of the respiratory microbiota and has challenged the current paradigm of "single pathogen causes disease". Understanding how to diagnose infection using these new, highly sensitive technologies will be important. How we should best intervene to optimise, manipulate and prevent disruption of the respiratory microbiota is likely to greatly influence how we manage infection in the future.

The CF-Sputum Induction Trial (CF-SpIT) to assess lower airway bacterial sampling in young children with cystic fibrosis: a prospective internally controlled interventional trial.

BACKGROUND: Pathogen surveillance is challenging but crucial in children with cystic fibrosis-who are often non-productive of sputum even if actively coughing-because infection and lung disease begin early in life. The role of sputum induction as a diagnostic tool for infection has not previously been systematically addressed in young children with cystic fibrosis. We aimed to assess the pathogen yield from sputum induction compared with that from cough swab and single-lobe, two-lobe, and six-lobe bronchoalveolar lavage. METHODS: This prospective internally controlled interventional trial was done at the Children's Hospital for Wales (Cardiff, UK) in children with cystic fibrosis aged between 6 months and 18 years. Samples from cough swab, sputum induction, and single-lobe, two-lobe, and six-lobe bronchoalveolar lavage were matched for within-patient comparisons. Primary outcomes were comparative pathogen yield between sputum induction and cough swab for stage 1, and between sputum induction, and single-lobe, two-lobe, and six-lobe bronchoalveolar lavage for stage 2. Data were analysed as per protocol. This study is registered with the UK Clinical Research Network (14615) and with the International Standard Randomised Controlled Trial Network Registry (12473810). FINDINGS: Between Jan 23, 2012, and July 4, 2017, 124 patients were prospectively recruited to the trial and had 200 sputum induction procedures for stage 1. 167 (84%) procedures were successful and the procedure was well tolerated. Of the 167 paired samples, 63 (38%) sputum-induction samples were pathogen positive compared with 24 (14%) cough swabs (p<0·0001; odds ratio [OR] 7·5; 95% CI 3·19-17·98). More pathogens were isolated from sputum induction than cough swab (79 [92%] of 86 vs 27 [31%] of 86; p<0·0001). For stage 2, 35 patients had a total of 41 paired sputum-induction and bronchoalveolar lavage procedures. Of the 41 paired samples, 28 (68%) were positive for at least one of the concurrent samples. 39 pathogens were isolated. Sputum induction identified 27 (69%) of the 39 pathogens, compared with 22 (56%; p=0·092; OR 3·3, 95% CI 0·91-12·11) on single-lobe, 28 (72%; p=1·0; OR 1·1, 95% CI 0·41-3·15) on two-lobe, and 33 (85%; p=0·21; OR 2·2, 95% CI 0·76-6·33) on six-lobe bronchoalveolar lavage. INTERPRETATION: Sputum induction is superior to cough swab for pathogen detection, is effective at sampling the lower airway, and is a credible surrogate for bronchoalveolar lavage in symptomatic children. A substantial number of bronchoscopies could be avoided if sputum induction is done first and pathogens are appropriately treated. Both sputum induction and six-lobe bronchoalveolar lavage provide independent, sizeable gains in pathogen detection compared with the current gold-standard two-lobe bronchoalveolar lavage. We propose that sputum induction and six-lobe bronchoalveolar lavage combined are used as standard of care for comprehensive lower airway pathogen detection in children with cystic fibrosis. FUNDING: Health and Care Research Wales-Academic Health Science Collaboration and Wellcome Trust Institutional Strategic Support Fund.

Differential susceptibility of Dectin-1 isoforms to functional inactivation by neutrophil and fungal proteases.

Patients with cystic fibrosis (CF) experience chronic or recurrent bacterial and fungal lung infections. Many patients with CF cannot effectively clear Aspergillus from their lungs. This may result in IgE sensitization and the development of allergic bronchopulmonary aspergillosis, or invasive infections, such as Aspergillus bronchitis. Lung disease in patients with CF is associated with neutrophil-dominated inflammation and elevated levels of the serine protease, neutrophil elastase (NE). Various C-type lectin-like receptors (CLRs), including Dectin-1 and Dectin-2, are involved in the immune response to Aspergillus. Here, we show that purified NE cleaves Dectin-1 in an isoform-specific manner. Bronchoalveolar lavage fluid from patients with CF, which contains high NE activity, induces Dectin-1 cleavage. Similarly, filtrate from a protease-producing strain of Aspergillus fumigatus induces isoform-specific cleavage of Dectin-1. Dectin-1 knockout (KO) cells and NE-treated cells demonstrated reduced phagocytosis of zymosan, a fungal cell wall preparation. In addition, NE cleaves 2 other CLRs, Dectin-2 and Mincle, and fungal-induced cytokine production was reduced in Dectin-1 KO cells, Dectin-2 KO cells, and NE-treated cells. Thus, Dectin-1 and Dectin-2 cleavage by NE and/or A. fumigatus-derived proteases results in an aberrant antifungal immune response that likely contributes to disease pathology in patients with CF.-Griffiths, J. S., Thompson, A., Stott, M., Benny, A., Lewis, N. A., Taylor, P. R., Forton, J., Herrick, S., Orr, S. J., McGreal, E. P. Differential susceptibility of Dectin-1 isoforms to functional inactivation by neutrophil and fungal proteases.

A Low-Molecular-Weight Alginate Oligosaccharide Disrupts Pseudomonal Microcolony Formation and Enhances Antibiotic Effectiveness.

In chronic respiratory disease, the formation of dense, 3-dimensional "microcolonies" by Pseudomonas aeruginosa within the airway plays an important role in contributing to resistance to treatment. An in vitro biofilm model of pseudomonal microcolony formation using artificial-sputum (AS) medium was established to study the effects of low-molecular-weight alginate oligomers (OligoG CF-5/20) on pseudomonal growth, microcolony formation, and the efficacy of colistin. The studies employed clinical cystic fibrosis (CF) isolates (n = 3) and reference nonmucoid and mucoid multidrug-resistant (MDR) CF isolates (n = 7). Bacterial growth and biofilm development and disruption were studied using cell viability assays and image analysis with scanning electron and confocal laser scanning microscopy. Pseudomonal growth in AS medium was associated with increased ATP production (P < 0.05) and the formation (at 48 h) of discrete (>10-µm) microcolonies. In conventional growth medium, colistin retained an ability to inhibit growth of planktonic bacteria, although the MIC was increased (0.1 to 0.4 µg/ml) in AS medium compared to Mueller-Hinton (MH) medium. In contrast, in an established-biofilm model in AS medium, the efficacy of colistin was decreased. OligoG CF-5/20 (≥2%) treatment, however, induced dose-dependent biofilm disruption (P < 0.05) and led to colistin retaining its antimicrobial activity (P < 0.05). While circular dichroism indicated that OligoG CF-5/20 did not change the orientation of the alginate carboxyl groups, mass spectrometry demonstrated that the oligomers induced dose-dependent (>0.2%; P < 0.05) reductions in pseudomonal quorum-sensing signaling. These findings reinforce the potential clinical significance of microcolony formation in the CF lung and highlight a novel approach to treat MDR pseudomonal infections.

AUDIT OF OXYGEN PRESCRIBING IN A CHILDREN'S HOSPITAL.

AIM: To audit oxygen prescribing in a children's hospital following the introduction of a new paediatric medication chart, which incorporates an oxygen prescription section. METHOD: In June 2015 a 1-day snapshot audit was carried out across all wards in the children's hospital. All patients receiving oxygen on that day were included:▸ The audit was repeated in July 2015.▸ The standards for the audit were set at 100% in accordance with our local guidelines.1 ▸ All patients receiving oxygen should have a prescription. Of these:▸ All patients should have target saturations identified.▸ All patients should have an administration device identified.▸ All patients should have a nurse signature on the chart within the last 12 hrs. RESULTS: In June, 13 patients were receiving oxygen on the audit day. 0/14 had a prescription.In July, 18 patients were receiving oxygen on the audit day. (14 critical care, 4 medicine).4/18 had an oxygen prescription (22%). These were all medical patients. Of these, 4 patients had a target saturation identified (100%), 1 had a device prescribed (25%), and 4 had a nurse signature within the last 12 hrs (100%). CONCLUSION: The initial audit showed no compliance with either local or national guidance for oxygen prescribing.1 2 The re-audit showed improved prescribing on the medical wards but not within critical care. The new paediatric medication chart was launched early in 2015, along with a training package for doctors, nurses and pharmacists. This was in response to the National Patient Safety Agency (NPSA) rapid response report on oxygen safety in hospitals.3 There was a gap between the training and the new charts being available which may have led to the poor results in the first audit. Increased awareness of the charts and the initial audit results probably helped improve prescribing in the re-audit. For medical patients, prescribing and monitoring was good, although device was infrequently prescribed. Critical care have not engaged with the new chart and oxygen prescription process. Although the British Thoracic Society guidelines indicate that oxygen for adult patients must be prescribed, these do not currently cover critical care or children under 16 years.2 There are guidelines for children in development which are likely to advocate the same. This could be another reason why there is no prescribing in critical care.Patient numbers were small in this snapshot audit which could limit its validity. Future work will include re-audit in our hospital and audit across the whole region where the new charts have been introduced.

Induced sputum in young healthy children with cystic fibrosis.

Young children with CF are often asymptomatic and non-productive, yet CF lung disease occurs early in life. Cough swabs are used routinely to sample bacteria from the CF respiratory tract in non-productive healthy children; bronchoscopy is used to definitively sample the lower airway, but is an invasive procedure. Induced sputum is a non-invasive approach to sampling the lower airway. The article concentrates on how well it is tolerated in children, how successful it is in identifying respiratory pathogens, and how it may be important in routine surveillance if 16S technology is to be used in the clinical forum.

TLR9 polymorphisms in African populations: no association with severe malaria, but evidence of cis-variants acting on gene expression.

BACKGROUND: During malaria infection the Toll-like receptor 9 (TLR9) is activated through induction with plasmodium DNA or another malaria motif not yet identified. Although TLR9 activation by malaria parasites is well reported, the implication to the susceptibility to severe malaria is not clear. The aim of this study was to assess the contribution of genetic variation at TLR9 to severe malaria. METHODS: This study explores the contribution of TLR9 genetic variants to severe malaria using two approaches. First, an association study of four common single nucleotide polymorphisms was performed on both family- and population-based studies from Malawian and Gambian populations (n>6000 individual). Subsequently, it was assessed whether TLR9 expression is affected by cis-acting variants and if these variants could be mapped. For this work, an allele specific expression (ASE) assay on a panel of HapMap cell lines was carried out. RESULTS: No convincing association was found with polymorphisms in TLR9 for malaria severity, in either Gambian or Malawian populations, using both case-control and family based study designs. Using an allele specific expression assay it was observed that TLR9 expression is affected by cis-acting variants, these results were replicated in a second experiment using biological replicates. CONCLUSION: By using the largest cohorts analysed to date, as well as a standardized phenotype definition and study design, no association of TLR9 genetic variants with severe malaria was found. This analysis considered all common variants in the region, but it is remains possible that there are rare variants with association signals. This report also shows that TLR9 expression is potentially modulated through cis-regulatory variants, which may lead to differential inflammatory responses to infection between individuals.

Susceptibility to sequelae of human ocular chlamydial infection associated with allelic variation in IL10 cis-regulation.

Trachoma, an infectious disease of the conjunctiva caused by Chlamydia trachomatis, causes scarring and blindness in some infected individuals but not others. In an African community where trachoma is endemic, we have previously identified an IL10 haplotype that is associated with increased risk of scarring complications. Here we examine the hypothesis that the risk haplotype (H-RISK) affects levels of IL10 expression in the conjunctiva during active trachoma infection. To overcome potential genetic and environmental confounders we used the method of allele-specific quantification, which involved identifying subjects in the community who had active trachoma and were also heterozygous for the H-RISK. We find that there is allelic variation in cis-regulation of IL10 in the conjunctiva during active trachoma, with the H-RISK generating relatively more IL10 transcripts than other haplotypes in this population (average difference in IL10 allelic transcripts in the conjunctiva of heterozygous individuals infected with C. trachomatis of 23% (95% confidence interval: 14-32%, P < 0.0001). These findings provide a plausible functional explanation for the observed genetic association, and support the hypothesis that an excessive IL10 response to C. trachomatis infection is a risk factor for scarring and blindness.

Validating discovered Cis-acting regulatory genetic variants: application of an allele specific expression approach to HapMap populations.

BACKGROUND: Localising regulatory variants that control gene expression is a challenge for genome research. Several studies have recently identified non-coding polymorphisms associated with inter-individual differences in gene expression. These approaches rely on the identification of signals of association against a background of variation due to other genetic and environmental factors. A complementary approach is to use an Allele-Specific Expression (ASE) assay, which is more robust to the effects of environmental variation and trans-acting genetic factors. METHODOLOGY/PRINCIPAL FINDINGS: Here we apply an ASE method which utilises heterozygosity within an individual to compare expression of the two alleles of a gene in a single cell. We used individuals from three HapMap population groups and analysed the allelic expression of genes with cis-regulatory regions previously identified using total gene expression studies. We were able to replicate the results in five of the six genes tested, and refined the cis- associated regions to a small number of variants. We also showed that by using multi-populations it is possible to refine the associated cis-effect DNA regions. CONCLUSIONS/SIGNIFICANCE: We discuss the efficacy and drawbacks of both total gene expression and ASE approaches in the discovery of cis-acting variants. We show that the ASE approach has significant advantages as it is a cleaner representation of cis-acting effects. We also discuss the implication of using different populations to map cis-acting regions and the importance of finding regulatory variants which contribute to human phenotypic variation.