The impact of plasma 25-hydroxyvitamin D on pulmonary function and exercise physiology in cystic fibrosis: A multicentre retrospective study.

BACKGROUND: A 25-hydroxyvitamin D (25OHD) may exert immunomodulatory effects on respiratory health, which may translate to improvements in exercise physiology. Thus, we aimed to investigate whether plasma 25OHD is associated with lung function and aerobic fitness in people with cystic fibrosis (pwCF). METHODS: A multicentre retrospective review of pwCF (> 9 years old) attending the Royal Hospital for Sick Children (Edinburgh) or Wessex CF-Unit (Southampton) was performed between July 2017 and October 2019. Demographic and clinical data were collected. Plasma 25OHD measured closest in time to clinical cardiopulmonary exercise testing and/or spirometry [forced expiratory volume (FEV1 )% predicted] was recorded. Pancreatic insufficiency was diagnosed based on faecal elastase of < 100 µg g-1 . We performed multiple-regression analysis with aerobic fitness outcomes [peak oxygen uptake (VO2 peak )] and FEV1 % predicted as primary outcomes. RESULTS: Ninety pwCF [mean ± SD age: 19.1 ± 8.6 years, 54 (60%) children, 48 (53%) males and 88 (98%) Caucasian] were included. 25OHD deficiency and insufficiency was 15 (17%) and 44 (49%), respectively. 25OHD deficiency and insufficiency was significantly associated with pancreatic insufficiency (χ2  = 4.8, p = 0.02). Plasma 25OHD was not significantly associated with FEV1 % predicted (r2  = 0.06, p = 0.42, 95% CI = -0.09 to 0.19) or VO2 peak (r2  = 0.04, p = 0.07, 95% CI = -011 to 0.005) in all pwCF. However, 25OHD was significantly associated with both FEV1 % (r2  = 0.15, p = 0.02, 95% CI = 1.99-2.64) and VO2 peak (r2  = 0.13, p = 0.05, 95% CI = -0.26 to -0.005) in the paediatric cohort. CONCLUSIONS: We showed that 25OHD is associated with improved lung function and aerobic fitness in children and adolescents with CF. Mechanistic and high-quality prospective studies including both lung function and aerobic fitness as primary outcomes are now warranted.

Contributions of Composition and Interactions to Bacterial Respiration Are Reliant on the Phylogenetic Similarity of the Measured Community.

Bacterial diversity underpins many ecosystem functions; however, the impact of within-species variation on the relationship between diversity and function remains unclear. Processes involving strain differentiation, such as niche radiation, are often overlooked in studies that focus on phylogenetic variation. This study used bacterial isolates assembled in two comparable microcosm experiments to test how species variation affected ecosystem function. We compared the relationship between diversity and activity (CO2 production) in increasingly diverse multispecies microcosms and with multiple ecotypes of a single species. The bacteria used were isolated from a low-diversity environment and are species of potential clinical significance such as Pseudomonas aeruginosa. All isolates were profiled for single carbon source utilisation. These data showed an increased breadth of resource use in the multiple ecotypes when compared to the mixed-species. The study observed significantly increasing respiration in more complex mixed-species assemblages, which was not observed when ecotypes of a single species were combined. We further demonstrate that the variation observed in the bacterial activity was due to the roles of each of the constituent isolates; between different species, the interactions between the isolates drove the variation in activity, whilst in single species, assemblage variation was due to which isolates were present. We conclude that both between- and within-species variations play different roles in community function, although through different mechanisms, and should be included in models of changing diversity and ecosystem functioning.

Respiratory microbiota: addressing clinical questions, informing clinical practice.

Over the last decade, technological advances have revolutionised efforts to understand the role played by microbes in airways disease. With the application of ever more sophisticated techniques, the literature has become increasingly inaccessible to the non-specialist reader, potentially hampering the translation of these gains into improvements in patient care. In this article, we set out the key principles underpinning microbiota research in respiratory contexts and provide practical guidance on how best such studies can be designed, executed and interpreted. We examine how an understanding of the respiratory microbiota both challenges fundamental assumptions and provides novel clinical insights into lung disease, and we set out a number of important targets for ongoing research.

A quality-of-life measure for adults with primary ciliary dyskinesia: QOL-PCD.

Primary ciliary dyskinesia (PCD) is characterised by chronic suppurative lung disease, rhino-sinusitis, hearing impairment and sub-fertility. We have developed the first multidimensional measure to assess health-related quality of life (HRQoL) in adults with PCD (QOL-PCD).Following a literature review and expert panel meeting, open-ended interviews with patients investigated the impact of PCD on HRQoL in the UK and North America (n=21). Transcripts were content analysed to derive saturation matrices. Items were rated for relevance by patients (n=49). Saturation matrices, relevance scores, literature review, evaluation of existing measures, and expert opinion contributed to development of a preliminary questionnaire. The questionnaire was refined following cognitive interviews (n=18).Open-ended interviews identified a spectrum of issues unique to adults with PCD. Saturation matrices confirmed comprehensive coverage of content. QOL-PCD includes 48 items covering the following seven domains: Physical Functioning, Emotional Functioning, Treatment Burden, Respiratory and Sinus Symptoms, Ears and Hearing, Social Functioning, and Vitality and Health Perceptions. Cognitive testing confirmed that content was comprehensive and the items were well-understood by respondents.Content validity and cognitive testing supported the items and structure. QOL-PCD has been translated into other languages and is awaiting psychometric testing.

Republished: Respiratory microbiota: addressing clinical questions, informing clinical practice.

Over the last decade, technological advances have revolutionised efforts to understand the role played by microbes in airways disease. With the application of ever more sophisticated techniques, the literature has become increasingly inaccessible to the non-specialist reader, potentially hampering the translation of these gains into improvements in patient care. In this article, we set out the key principles underpinning microbiota research in respiratory contexts and provide practical guidance on how best such studies can be designed, executed and interpreted. We examine how an understanding of the respiratory microbiota both challenges fundamental assumptions and provides novel clinical insights into lung disease, and we set out a number of important targets for ongoing research.

Time between collection and storage significantly influences bacterial sequence composition in sputum samples from cystic fibrosis respiratory infections.

Spontaneously expectorated sputum is traditionally used as the sampling method for the investigation of lower airway infections. While guidelines exist for the handling of these samples for culture-based diagnostic microbiology, there is no comparable consensus on their handling prior to culture-independent analysis. The increasing incorporation of culture-independent approaches in diagnostic microbiology means that it is of critical importance to assess potential biases. The aim of this study was to assess the impact of delayed freezing on culture-independent microbiological analyses and to identify acceptable parameters for sample handling. Sputum samples from eight adult cystic fibrosis (CF) patients were collected and aliquoted into sterile Bijou bottles. Aliquots were stored at room temperature before being frozen at -80 °C for increasing intervals, up to a 72-h period. Samples were treated with propidium monoazide to distinguish live from dead cells prior to DNA extraction, and 16S rRNA gene pyrosequencing was used to characterize their bacterial compositions. Substantial variation was observed in samples with high-diversity bacterial communities over time, whereas little variation was observed in low-diversity communities dominated by recognized CF pathogens, regardless of time to freezing. Partitioning into common and rare species demonstrated that the rare species drove changes in similarity. The percentage abundance of anaerobes over the study significantly decreased after 12 h at room temperature (P = 0.008). Failure to stabilize samples at -80 °C within 12 h of collection results in significant changes in the detected community composition.

Complexity, temporal stability, and clinical correlates of airway bacterial community composition in primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a genetic disease characterized by abnormalities in ciliary function, leading to compromised airway clearance and chronic bacterial infection of the upper and lower airways. The compositions of these infections and the relationships between their characteristics and disease presentation are poorly defined. We describe here the first systematic culture-independent evaluation of lower airway bacteriology in PCD. Thirty-three airway samples (26 from sputum, 7 from bronchoalveolar lavage [BAL] fluid) were collected from 24 PCD patients aged 4 to 73 years. 16S rRNA quantitative PCR and pyrosequencing were used to determine the bacterial loads and community compositions of the samples. Bacterial loads, which ranged from 1.3 × 10(4) to 5.2 × 10(9) CFU/ml, were positively correlated with age (P = 0.002) but not lung function. An analysis of ∼7,000 16S rRNA sequences per sample identified bacterial species belonging to 128 genera. The concurrently collected paired samples showed high bacterial community similarity. The mean relative abundance of the dominant genera was 64.5% (standard deviation [SD], 24.5), including taxa reported through standard diagnostic microbiology (members of the genera Pseudomonas, Haemophilus, and Streptococcus) and those requiring specific ex vivo growth conditions (members of the genera Prevotella and Porphyromonas). The significant correlations observed included a positive relationship between Pseudomonas aeruginosa relative abundance and age and a negative relationship between P. aeruginosa relative abundance and lung function. Members of the genus Ralstonia were also found to contribute substantially to the bacterial communities in a number of patients. Follow-up samples from a subset of patients revealed high levels of bacterial community temporal stability. The detailed microbiological characterization presented here provides a basis for the reassessment of the clinical management of PCD airway infections.

Long-term cultivation-independent microbial diversity analysis demonstrates that bacterial communities infecting the adult cystic fibrosis lung show stability and resilience.

BACKGROUND: Culture-independent analysis of the respiratory secretions of people with cystic fibrosis (CF) has identified many bacterial species not previously detected using culture in this context. However, little is known about their clinical significance or persistence in CF airways. METHODS: The authors characterised the viable bacterial communities in the sputum collected from 14 patients at monthly intervals over 1 year using a molecular community profiling technique-terminal restriction fragment length polymorphism. Clinical characteristics were also collected, including lung function and medications. Ecological community measures were determined for each sample. Microbial community change over time within subjects was defined using ecological analytical tools, and these measures were compared between subjects and to clinical features. RESULTS: Bacterial communities were stable within subjects over time but varied between subjects, despite similarities in clinical course. Antibiotic therapy temporarily perturbed these communities which generally returned to pretreatment configurations within 1 month. Species usually considered CF pathogens and those not previously regarded as such exhibited similar patterns of persistence. Less diverse sputum bacterial communities were correlated to lung disease severity and relative abundance of Pseudomonas aeruginosa. CONCLUSION: Whilst not true in all cases, the microbial communities that chronically infect the airways of patients with CF can vary little over a year despite antibiotic perturbation. The species present tended to vary more between than within subjects, suggesting that each CF airway infection is unique, with relatively stable and resilient bacterial communities. The inverse relationship between community richness and disease severity is similar to findings reported in other mucosal infections.

The disease-specific clinical trial network for primary ciliary dyskinesia: PCD-CTN.

Primary ciliary dyskinesia (PCD) is a rare genetic disorder characterised by impaired mucociliary clearance leading to irreversible lung damage. In contrast to other rare lung diseases like cystic fibrosis (CF), there are only few clinical trials and limited evidence-based treatments. Management is mainly based on expert opinions and treatment is challenging due to a wide range of clinical manifestations and disease severity. To improve clinical and translational research and facilitate development of new treatments, the clinical trial network for PCD (PCD-CTN) was founded in 2020 under the framework of the European Reference Network (ERN)-LUNG PCD Core. Applications from European PCD sites interested in participating in the PCD-CTN were requested. Inclusion criteria consisted of patient numbers, membership of ERN-LUNG PCD Core, use of associated standards of care, experience in PCD and/or CF clinical research, resources to run clinical trials, good clinical practice (GCP) certifications and institutional support. So far, applications from 22 trial sites in 18 European countries have been approved, including >1400 adult and >1600 paediatric individuals with PCD. The PCD-CTN is headed by a coordinating centre and consists of a steering and executive committee, a data safety monitoring board and committees for protocol review, training and standardisation. A strong association with patient organisations and industrial companies are further cornerstones. All participating trial sites agreed on a code of conduct. As CTNs from other diseases have demonstrated successfully, this newly formed PCD-CTN operates to establish evidence-based treatments for this orphan disease and to bring new personalised treatment approaches to patients.

Analysis of the bacterial communities present in lungs of patients with cystic fibrosis from American and British centers.

The aim of this study was to determine whether geographical differences impact the composition of bacterial communities present in the airways of cystic fibrosis (CF) patients attending CF centers in the United States or United Kingdom. Thirty-eight patients were matched on the basis of clinical parameters into 19 pairs comprised of one U.S. and one United Kingdom patient. Analysis was performed to determine what, if any, bacterial correlates could be identified. Two culture-independent strategies were used: terminal restriction fragment length polymorphism (T-RFLP) profiling and 16S rRNA clone sequencing. Overall, 73 different terminal restriction fragment lengths were detected, ranging from 2 to 10 for U.S. and 2 to 15 for United Kingdom patients. The statistical analysis of T-RFLP data indicated that patient pairing was successful and revealed substantial transatlantic similarities in the bacterial communities. A small number of bands was present in the vast majority of patients in both locations, indicating that these are species common to the CF lung. Clone sequence analysis also revealed that a number of species not traditionally associated with the CF lung were present in both sample groups. The species number per sample was similar, but differences in species presence were observed between sample groups. Cluster analysis revealed geographical differences in bacterial presence and relative species abundance. Overall, the U.S. samples showed tighter clustering with each other compared to that of United Kingdom samples, which may reflect the lower diversity detected in the U.S. sample group. The impact of cross-infection and biogeography is considered, and the implications for treating CF lung infections also are discussed.

The implications of dysglycaemia on aerobic exercise and ventilatory function in cystic fibrosis.

BACKGROUND: The development of cystic fibrosis (CF)-related diabetes (CFRD) in paediatric groups is associated with a reduced aerobic fitness. However, this has yet to be investigated in adults with more severe lung disease. METHODS: Cardiopulmonary exercise and glycaemic control tests were retrospectively analysed in 46 adults with CF (age: 26.9 y [range: 16.3-66.5 y]; forced expiratory volume in 1s: 65.3% [range: 26.8-105.7%]; 26 males), diagnosed with CFRD (n = 19), impaired glucose tolerance (IGT; n = 8) or normal glucose tolerance (NGT; n = 19). RESULTS: Maximal oxygen uptake (V˙O2max) was reduced in adults with IGT and CFRD compared to their age- and gender-matched counterparts with NGT (p < 0.05); however, there was no difference when lung function was included as a covariate (all p > 0.05). V˙O2max was greater in adults who experienced post-reactive hypoglycaemia vs. NGT without hypoglycaemia (p < 0.05). The frequency of ventilatory limitation (84%, 63% and 37%, respectively; p < 0.05) but not ventilation-perfusion mismatch (42%, 38% and 16%, respectively; p > 0.05), was greater with CFRD and IGT vs. NGT. There was also no difference in arterial oxygen saturation changes between groups (p > 0.05). Gender and body mass index were significant predictors of V˙O2max (adjusted R2 = 0.37, p < 0.01), but glycaemic control did not explain additional variance (p > 0.05). CONCLUSIONS: Adults with CF-related dysglycaemia had a reduced V˙O2max compared to age- and gender-matched counterparts, due to a greater degree of CF lung disease in these populations.

Circulating biomarkers of antioxidant status and oxidative stress in people with cystic fibrosis: A systematic review and meta-analysis.

INTRODUCTION: Oxidative stress may play an important role in the pathophysiology of cystic fibrosis (CF). This review aimed to quantify CF-related redox imbalances. METHODS: Systematic searches of the Medline, CINAHL, CENTRAL and PsycINFO databases were conducted. Mean content of blood biomarkers from people with clinically-stable CF and non-CF controls were used to calculate the standardized mean difference (SMD) and 95% confidence intervals (95% CI). RESULTS: Forty-nine studies were eligible for this review including a total of 1792 people with CF and 1675 controls. Meta-analysis revealed that protein carbonyls (SMD: 1.13, 95% CI: 0.48 to 1.77), total F2-isoprostane 8-iso-prostaglandin F2α (SMD: 0.64, 95% CI: 0.23 to 1.05) and malondialdehyde (SMD: 1.34, 95% CI: 0.30 to 2.39) were significantly higher, and vitamins A (SMD: -0.66, 95% CI -1.14 to -0.17) and E (SMD: -0.74, 95% CI: -1.28 to -0.20), ß-carotene (SMD: -1.80, 95% CI: -2.92 to -0.67), lutein (SMD: -1.52, 95% CI: -1.83 to -1.20) and albumin (SMD: -0.98, 95% CI: -1.68 to -0.27) were significantly lower in the plasma or serum of people with CF versus controls. CONCLUSIONS: This systematic review and meta-analysis found good evidence for reduced antioxidant capacity and elevated oxidative stress in people with clinically-stable CF.

Lung function and microbiota diversity in cystic fibrosis.

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. Video Abstract.

Macrorheology of cystic fibrosis, chronic obstructive pulmonary disease & normal sputum.

BACKGROUND: Prior microrheologic assessments of selected, microlitre plugs of cystic fibrosis (CF) sputum suggest no intrinsic rheologic abnormality. However, such analyses may not be representative of CF sputum as a whole. We therefore reassessed this question using whole sputum macrorheology. Additionally, we wished to further explore the relationships between sputum rheology, inflammation and infection. METHODS: Dynamic oscillatory macrorheometry was performed on whole expectorated sputum from stable adults with CF (n = 18) and COPD (n = 12) and induced sputum from normal controls (n = 7). Concomitant sputum inflammatory mediator levels were measured in CF and COPD samples. Sputum collected from CF subjects (n = 6) at commencement and completion of intravenous antibiotic therapy for an infective exacerbation was also assessed. RESULTS: CF sputum neutrophil elastase activity (NE) was significantly related to degree of sputum purulence (p = 0.049) and correlated significantly with measures of sputum viscoelasticity (r = 0.696, p = 0.008 for storage modulus G' at 9 Hz). There were significant differences in viscoelasticity between subject groups when samples were compared irrespective of appearance/degree of sputum purulence. However, the macrorheology of mucoid CF sputum did not differ from normal sputum (eg median (range) G' at 9 Hz 2.25 (0.79, 3.26) vs 2.04 (1.4,4.6) Pa, p = 1). In contrast, mucoid COPD samples demonstrated significantly greater viscoelasticity (G' at 9 Hz 4.5 (2.4, 23) Pa) than sputum from both CF (p = 0.048) & normal subjects (p = 0.009). Antibiotic therapy during exacerbations was associated with significant reductions in CF sputum viscoelasticity, with mean (SD) G' at 9 Hz decreasing from 28.5 (11.5) Pa at commencement to 6.4 (4.6) Pa on day 7 (p = 0.01). CONCLUSION: The macrorheologic properties of whole, mucoid CF sputum are not different from normal, confirming the results of prior microrheologic studies. Instead, CF sputum viscoelasticity is related to secondary infection, decreases with intravenous antibiotic therapy and correlates with inflammation. In contrast, COPD sputum demonstrates inherently greater viscoelasticity, providing a novel target for potential therapeutic interventions.

Studying bacteria in respiratory specimens by using conventional and molecular microbiological approaches.

BACKGROUND: Drawing from previous studies, the traditional routine diagnostic microbiology evaluation of samples from chronic respiratory conditions may provide an incomplete picture of the bacteria present in airways disease. Here, the aim was to determine the extent to which routine diagnostic microbiology gave a different assessment of the species present in sputa when analysed by using culture-independent assessment. METHODS: Six different media used in routine diagnostic microbiology were inoculated with sputum from twelve patients. Bacterial growth on these plates was harvested and both RNA and DNA extracted. DNA and RNA were also extracted directly from the same sample of sputum. All nucleic acids served as templates for PCR and reverse transcriptase-PCR amplification of "broad range" bacterial 16S rRNA gene regions. The regions amplified were separated by Terminal Restriction Fragment Length Polymorphism (T-RFLP) profiling and compared to assess the degree of overlap between approaches. RESULTS: A mean of 16.3 (SD 10.0) separate T-RF band lengths in the profiles from each sputum sample by Direct Molecular Analysis, with a mean of 8.8 (SD 5.8) resolved by DNA profiling and 13.3 (SD 8.0) resolved by RNA profiling. In comparison, 8.8 (SD 4.4) T-RF bands were resolved in profiles generated by Culture-derived Molecular Analysis. There were a total of 184 instances of T-RF bands detected in the direct sputum profiles but not in the corresponding culture-derived profiles, representing 83 different T-RF band lengths. Amongst these were fifteen instances where the T-RF band represented more than 10% of the total band volume (with a mean value of 23.6%). Eight different T-RF band lengths were resolved as the dominant band in profiles generated directly from sputum. Of these, only three were detected in profiles generated from the corresponding set of cultures. CONCLUSION: Due to their focus on isolation of a small group of recognised pathogens, the use of culture-dependent methods to analyse samples from chronic respiratory infections can provide a restricted understanding of the bacterial species present. The use of a culture-independent molecular approach here identifies that there are many bacterial species in samples from CF and COPD patients that may be clinically relevant.

Assessing the diagnostic importance of nonviable bacterial cells in respiratory infections.

Identification of bacteria in clinical samples is fundamental to combating infections. Modern molecular genetic approaches exploit nucleic acids signals from clinical samples. However, DNA-derived signals can originate from nonviable bacterial cells and, therefore, generate data that could be misinterpreted. Terminal restriction fragment length polymorphism profiling of cystic fibrosis sputum samples was combined with propidium monoazide (PMA) photo-induced cross-linking. PMA is highly membrane impermeant and is excluded from viable bacteria but readily penetrates dead cells. Exposure to a light source renders DNA in permeable cells incapable of contributing to polymerase chain reaction. PMA treatment was shown to effectively prevent dead bacteria, spiked into sputum samples, from contributing to profiles. Comparison of treated and untreated clinical samples indicated that dead bacterial cells significantly bias untreated profiles. These findings highlight the significant contribution that nonviable bacteria can make to DNA-based diagnostic analysis of clinical samples while providing a simple and effective means of avoiding such bias.

Cardiopulmonary exercise testing with supramaximal verification produces a safe and valid assessment of V̇o2max in people with cystic fibrosis: a retrospective analysis.

The validity and safety of using supramaximal verification (Smax) to confirm a maximal effort during cardiopulmonary exercise testing (CPET) in people with cystic fibrosis (CF) and/or those with severe disease has been questioned. Therefore, this study aimed to investigate these concerns in children, adolescents, and adults with mild-to-severe CF lung disease. Retrospective analysis of 17 pediatric and 28 adult participants with CF [age range: 9.2-62.9 y; forced expiratory volume in 1 s: 66.7% (range: 29.9%-102.3%); 30 men] who completed a routine ramp-incremental cycling test to determine peak oxygen uptake (V̇o2peak) was studied. Maximal oxygen uptake (V̇o2max) was subsequently confirmed by Smax at 110% of peak power output. All participants satisfied the criteria to verify a maximal effort during CPET. However, Smax-V̇o2peak exceeded ramp-V̇o2peak in 3/14 (21.4%) of pediatric and 6/28 (21.4%) adult exercise tests. A valid measurement of V̇o2max was attained in 85.7% of pediatric and 96.4% of adult exercise tests, as Smax-V̇o2peak did not exceed ramp-V̇o2peak by >9%. Adults ( n = 9) experienced a ≥5% reduction in arterial O2 saturation during CPET, 4 during both the ramp and Smax, 3 during only the ramp, and 2 during only Smax. Smax did not significantly worsen perceived breathing effort, chest tightness, throat narrowing, or exertion compared with ramp-incremental testing. Given the clinical importance of aerobic fitness in people with CF, incorporating Smax is recommended to provide a safe and valid measure of V̇o2max in children, adolescents, and adults who span the spectrum of CF disease severity. NEW & NOTEWORTHY Incorporating supramaximal verification into cardiopulmonary exercise testing protocols did not increase the frequency of adverse events or perceived discomfort versus a single-phase incremental exercise test in people with mild-to-severe cystic fibrosis. Furthermore, a valid measure of maximal oxygen uptake (V̇o2max) was obtained from 85.7% of pediatric and 96.4% of adult exercise tests, whereas peak oxygen uptake underestimated aerobic fitness in comparison with V̇o2max in 21.4% of cases (by up to 24.4%).

Respiratory microbiota resistance and resilience to pulmonary exacerbation and subsequent antimicrobial intervention.

Pulmonary symptoms in cystic fibrosis (CF) begin in early life with chronic lung infections and concomitant airway inflammation leading to progressive loss of lung function. Gradual pulmonary function decline is interspersed with periods of acute worsening of respiratory symptoms known as CF pulmonary exacerbations (CFPEs). Cumulatively, CFPEs are associated with more rapid disease progression. In this study multiple sputum samples were collected from adult CF patients over the course of CFPEs to better understand how changes in microbiota are associated with CFPE onset and management. Data were divided into five clinical periods: pre-CFPE baseline, CFPE, antibiotic treatment, recovery, and post-CFPE baseline. Samples were treated with propidium monoazide prior to DNA extraction, to remove the impact of bacterial cell death artefacts following antibiotic treatment, and then characterised by 16S rRNA gene-targeted high-throughput sequencing. Partitioning CF microbiota into core and rare groups revealed compositional resistance to CFPE and resilience to antibiotics interventions. Mixed effects modelling of core microbiota members revealed no significant negative impact on the relative abundance of Pseudomonas aeruginosa across the exacerbation cycle. Our findings have implications for current CFPE management strategies, supporting reassessment of existing antimicrobial treatment regimens, as antimicrobial resistance by pathogens and other members of the microbiota may be significant contributing factors.

Bacterial activity in cystic fibrosis lung infections.

BACKGROUND: Chronic lung infections are the primary cause of morbidity and mortality in Cystic Fibrosis (CF) patients. Recent molecular biological based studies have identified a surprisingly wide range of hitherto unreported bacterial species in the lungs of CF patients. The aim of this study was to determine whether the species present were active and, as such, worthy of further investigation as potential pathogens. METHODS: Terminal Restriction Fragment Length Polymorphism (T-RFLP) profiles were generated from PCR products amplified from 16S rDNA and Reverse Transcription Terminal Restriction Fragment Length Polymorphism (RT-T-RFLP) profiles, a marker of metabolic activity, were generated from PCR products amplified from 16S rRNA, both extracted from the same CF sputum sample. To test the level of activity of these bacteria, T-RFLP profiles were compared to RT-T-RFLP profiles. RESULTS: Samples from 17 individuals were studied. Parallel analyses identified a total of 706 individual T-RF and RT-T-RF bands in this sample set. 323 bands were detected by T-RFLP and 383 bands were detected by RT-T-RFLP (statistically significant; P < or = 0.001). For the group as a whole, 145 bands were detected in a T-RFLP profile alone, suggesting metabolically inactive bacteria. 205 bands were detected in an RT-T-RFLP profile alone and 178 bands were detected in both, suggesting a significant degree of metabolic activity. Although Pseudomonas aeruginosa was present and active in many patients, a low occurrence of other species traditionally considered to be key CF pathogens was detected. T-RFLP profiles obtained for induced sputum samples provided by healthy individuals without CF formed a separate cluster indicating a low level of similarity to those from CF patients. CONCLUSION: These results indicate that a high proportion of the bacterial species detected in the sputum from all of the CF patients in the study are active. The widespread activity of bacterial species in these samples emphasizes the potential importance of these previously unrecognized species within the CF lung.

Implications of multiple freeze-thawing on respiratory samples for culture-independent analyses.

BACKGROUND: Best practice when performing culture-independent microbiological analysis of sputum samples involves their rapid freezing and storage at -80°C. However, accessing biobanked collections can mean that material has been passed through repeated freeze-thaw cycles. The aim of this study was to determine the impact of these cycles on microbial community profiles. METHODS: Sputum was collected from eight adults with cystic fibrosis, and each sample was subjected to six freeze-thaw cycles. Following each cycle, an aliquot was removed and treated with propidium monoazide (PMA) prior to DNA extraction and 16S rRNA gene pyrosequencing. RESULTS: The impact of freeze-thaw cycles was greatest on rare members of the microbiota, with variation beyond that detected with within-sample repeat analysis observed after three cycles. CONCLUSION: Four or more freeze thaw cycles result in a significant distortion of microbiota profiles from CF sputum.