Cell-free DNA methylation in the clinical management of lung cancer.

The clinical use of cell-free DNA (cfDNA) methylation in managing lung cancer depends on its ability to differentiate between malignant and healthy cells, assign methylation changes to specific tissue sources, and elucidate opportunities for targeted therapy. From a technical standpoint, cfDNA methylation analysis is primed as a potential clinical tool for lung cancer screening, early diagnosis, prognostication, and treatment, pending the outcome of elaborate validation studies. Here, we discuss the current state of the art in cfDNA methylation analysis, examine the unique features and limitations of these new methods in a clinical context, propose two models for applying cfDNA methylation data for lung cancer screening, and discuss future research directions.

Pseudomonas aeruginosa is oxygen-deprived during infection in cystic fibrosis lungs, reducing the effectiveness of antibiotics.

Pseudomonas aeruginosa infects the lungs of patients with cystic fibrosis. Sputum expectorated from the lungs of patients contains low levels of oxygen, indicating that P. aeruginosa may be oxygen-deprived during infection. During in vitro growth under oxygen-limiting conditions, a P. aeruginosa reference strain increases expression of a cytochrome oxidase with a high affinity for oxygen, and of nitrate and nitrite reductases that enable it to use nitrate instead of oxygen during respiration. Here, we quantified transcription of the genes encoding these three enzymes in sputum samples from 18 infected patients, and in bacteria isolated from the sputum samples and grown in aerobic and anaerobic culture. In culture, expression of all three genes was increased by averages of 20- to 500-fold in anaerobically grown bacteria compared with those grown aerobically, although expression levels varied greatly between isolates. Expression of the same genes in sputum was similar to that of the corresponding bacteria in anaerobic culture. The isolated bacteria were less susceptible to tobramycin and ciprofloxacin, two widely used anti-pseudomonal antibiotics, when grown anaerobically than when grown aerobically. Our findings show that P. aeruginosa experiences oxygen starvation during infection in cystic fibrosis, reducing the effectiveness of antibiotic treatment.

Exercise and motivational text messaging to support physical activity behaviour change in a population with obstructive sleep apnoea: a feasibility study.

Introduction Patients with obstructive sleep apnoea (OSA) commonly present in primary care. Increasing physical activity reduces symptoms and severity of OSA. Low motivation is a barrier to physical activity in adults with OSA. Aim To investigate the feasibility and acceptability of an exercise and personalised text messaging programme to enhance motivation and support physical activity behaviour change in adults with OSA. Methods Participants were recruited from the local Sleep Clinic. Exclusion criteria were unstable angina, and/or poorly controlled hypertension. The intervention comprised three groups, who received either individual exercise prescription, personalised text messages or both over a 24-week period. Participants were allocated to one of the three groups. The primary outcome was feasibility of study design including participant recruitment and retention. Secondary outcomes were a change in 6-min walk distance and exercise self-efficacy over time. Results Thirty participants were recruited, 17 male and 13 female, with a mean age of 54.6 years. The study design appears feasible and the outcome measures used were acceptable to participants. Recruitment and retention rates were lower than anticipated. A trend towards increased functional exercise capacity was identified in all three groups, along with a corresponding increase in exercise self-efficacy over time. Discussion Exercise and personalised text messaging both appear to offer an acceptable and feasible means to increase physical activity in adults with OSA. A larger scale trial may provide justification for physiotherapist input to support patients with OSA to address physical inactivity.

Genome evolution drives transcriptomic and phenotypic adaptation in Pseudomonas aeruginosa during 20 years of infection.

The opportunistic pathogen Pseudomonas aeruginosa chronically infects the lungs of patients with cystic fibrosis (CF). During infection the bacteria evolve and adapt to the lung environment. Here we use genomic, transcriptomic and phenotypic approaches to compare multiple isolates of P. aeruginosa collected more than 20 years apart during a chronic infection in a CF patient. Complete genome sequencing of the isolates, using short- and long-read technologies, showed that a genetic bottleneck occurred during infection and was followed by diversification of the bacteria. A 125 kb deletion, an 0.9 Mb inversion and hundreds of smaller mutations occurred during evolution of the bacteria in the lung, with an average rate of 17 mutations per year. Many of the mutated genes are associated with infection or antibiotic resistance. RNA sequencing was used to compare the transcriptomes of an earlier and a later isolate. Substantial reprogramming of the transcriptional network had occurred, affecting multiple genes that contribute to continuing infection. Changes included greatly reduced expression of flagellar machinery and increased expression of genes for nutrient acquisition and biofilm formation, as well as altered expression of a large number of genes of unknown function. Phenotypic studies showed that most later isolates had increased cell adherence and antibiotic resistance, reduced motility, and reduced production of pyoverdine (an iron-scavenging siderophore), consistent with genomic and transcriptomic data. The approach of integrating genomic, transcriptomic and phenotypic analyses reveals, and helps to explain, the plethora of changes that P. aeruginosa undergoes to enable it to adapt to the environment of the CF lung during a chronic infection.

Perceived versus objective sleep quality in long-stay hospitalised older adults-a mixed methods study.

BACKGROUND: Hospital inpatients experience substantial sleep problems that have been linked with worse health outcomes, poor quality of life and the post-hospital syndrome. However, little is known about assessing sleep issues in older hospitalised patients. OBJECTIVE: To conduct an in-depth investigation on hospitalised older adults' sleep challenges and methods of sleep assessment. DESIGN: Cross-sectional observational study. SETTING: Public hospital inpatient unit. SUBJECTS: Long-stay hospitalised older adults. METHODS: Data were collected using validated sleep questionnaires, actigraphy devices and qualitative interviews. Quantitative data were analysed with descriptive statistics, multiple logistic regression and Cohen's Kappa. Qualitative data were analysed with qualitative content analysis; findings compared to the quantitative assessments. RESULTS: We collected data on 33 older long-stay hospital inpatients, who were mean (SD) 80.2(7.4) years old, 57.6% female and were hospitalised following stroke, medical illness and orthopaedic fracture. Mean (SD) total sleep time and actigraphic sleep efficiency were 480.6(73.6) minutes and 81.5(11.2)%, respectively. About, 57.6% were poor sleepers (Pittsburgh Sleep Quality Index [PSQI]) and 30.8% had indicators of clinical depression/low quality of life (WHO-5 well-being index). Three main themes were identified: "sleep assessment"; "factors that affect sleep"; "expectations of sleep". Bad sleepers were more likely to feel a lack of control over their sleep, while good sleepers spoke about the ability to adjust and accept their circumstances. CONCLUSIONS: We found high levels of sleep problems and identified substantial discrepancies between the validated sleep questionnaire and qualitative response data. Our findings indicate that standard assessment tools, such as PSQI, may not be suitable to assess sleep in hospitalised older adults and call for further investigations to build more appropriate methods. Further exploring psychological factors and expectations could potentially lead to novel interventions to improve sleep in this setting.

Physical activity behaviour and barriers to activity in adults at high risk of obstructive sleep apnoea.

INTRODUCTION Physical inactivity is a risk factor for disease severity among people with obstructive sleep apnoea. AIM To determine physical activity levels in patients at risk of obstructive sleep apnoea and explore their perceptions about barriers to participation in physical activity. METHODS This was a cross-sectional observational study. Eligible participants were adults with symptoms of obstructive sleep apnoea hypopnea syndrome and Epworth Sleepiness Scale score ≥11, awaiting prioritisation for a diagnostic overnight sleep study at the local sleep clinic. Sixty participants (mean age±standard deviation: 51±12 years) each attended an individual appointment. Anthropometric measurements were taken and standardised questionnaires regarding quality of life, physical activity behaviour and perceptions of physical activity were completed. RESULTS Over one-third of the cohort did not meet World Health Organization guidelines for weekly physical activity. Hypertension, type 2 diabetes and obesity were also more prevalent in this subgroup. Low motivation and pain were commonly reported barriers to activity in participants not meeting the physical activity guidelines. Overall, 53 (88%) participants stated they would like to be more active. DISCUSSION Physical inactivity represents an additional risk factor for adults at high risk of obstructive sleep apnoea. Lack of motivation and pain were the most commonly perceived barriers to participation in activity. Physical activity interventions tailored to the individual, and including a motivational component, need to be included as integral components of management to reduce cardiovascular and metabolic risk factors more effectively in this group.

Biallelic variants in EFEMP1 in a man with a pronounced connective tissue phenotype.

Connective tissue disorders are a spectrum of diseases that affect the integrity of tissues including skin, vasculature, and joints. They are often caused by variants that disrupt genes encoding components of extracellular matrix (ECM). The fibulin glycoproteins are ECM proteins important for integrity of tissues including dermis, retina, fascia, and vasculature. The fibulin family consists of seven members (fibulins-1 to -7) and is defined by a fibulin-type domain at the C-terminus. The family is associated with human diseases, for instance a variant in FBLN1, encoding fibulin-1, is associated with synpolydactyly, while one in EFEMP1, encoding fibulin-3, causes Doyne honeycomb degeneration of the retina. Loss-of-function of fibulins-4 and -5 causes cutis laxa, while variants in fibulins-5 and -6 are associated with age-related macular degeneration. Of note, EFEMP1 is not currently associated with any connective tissue disorder. Here we show biallelic loss-of-function variants in EFEMP1 in an individual with multiple and recurrent abdominal and thoracic herniae, myopia, hypermobile joints, scoliosis, and thin translucent skin. Fibroblasts from this individual express significantly lower EFEMP1 transcript than age-matched control cells. A skin biopsy, visualised using light microscopy, showed normal structure and abundance of elastic fibres. The phenotype of this individual is remarkably similar to the Efemp1 knockout mouse model that displays multiple herniae with premature aging and scoliosis. We conclude that loss of EFEMP1 function in this individual is the cause of a connective tissue disorder with a novel combination of phenotypic features, and can perhaps explain similar, previously reported cases in the literature.

Genomic and phenotypic comparison of environmental and patient-derived isolates of Pseudomonas aeruginosa suggest that antimicrobial resistance is rare within the environment.

Patient-derived isolates of the opportunistic pathogen Pseudomonas aeruginosa are frequently resistant to antibiotics due to the presence of sequence variants in resistance-associated genes. However, the frequency of antibiotic resistance and of resistance-associated sequence variants in environmental isolates of P. aeruginosa has not been well studied. Antimicrobial susceptibility testing (ciprofloxacin, ceftazidime, meropenem, tobramycin) of environmental (n=50) and cystic fibrosis (n=42) P. aeruginosa isolates was carried out. Following whole genome sequencing of all isolates, 25 resistance-associated genes were analysed for the presence of likely function-altering sequence variants. Environmental isolates were susceptible to all antibiotics with one exception, whereas patient-derived isolates had significant frequencies of resistance to each antibiotic and a greater number of likely resistance-associated genetic variants. These findings indicate that the natural environment does not act as a reservoir of antibiotic-resistant P. aeruginosa, supporting a model in which antibiotic susceptible environmental bacteria infect patients and develop resistance during infection.

Phase 1 trial of intrapleural LTI-01; single chain urokinase in complicated parapneumonic effusions or empyema.

BACKGROUND: Current dosing of intrapleural fibrinolytic therapy (IPFT) in adults with complicated parapneumonic effusion (CPE) / empyema is empiric, as dose-escalation trials have not previously been conducted. We hypothesized that LTI-01 (scuPA), which is relatively resistant to PA inhibitor-1 (PAI-1), would be well-tolerated. METHODS: This was an open-label, dose-escalation trial of LTI-01 IPFT at 50,000-800,000 IU daily for up to 3 days in adults with loculated CPE/empyema and failed pleural drainage. The primary objective was to evaluate safety and tolerability, and secondary objectives included assessments of processing and bioactivity of scuPA in blood and pleural fluid (PF), and early efficacy. RESULTS: LTI-01 was well tolerated with no bleeding, treatment-emergent adverse events or surgical referrals (n=14 subjects). uPA antigen increased in PFs at 3 hours after LTI-01 (p<0.01) but not in plasma. PF saturated active PAI-1, generated PAI-1-resistant bioactive complexes, increased PA and fibrinolytic activities and D-dimers. There was no systemic fibrinogenolysis, nor increments in plasma D-dimer. Decreased pleural opacities occurred in all but one subject. Both subjects receiving 800,000 IU required two doses to relieve pleural sepsis, with two other subjects similarly responding at lower doses. CONCLUSION: LTI-01 IPFT was well-tolerated at these doses with no safety concerns. Bioactivity of LTI-01 IPFT was confirmed, limited to PFs where its processing simulated that previously reported in preclinical studies. Preliminary efficacy signals including reduction of pleural opacity were observed.

Treatable traits can be identified in a severe asthma registry and predict future exacerbations.

BACKGROUND AND OBJECTIVE: A new taxonomic and management approach, termed treatable traits, has been proposed for airway diseases including severe asthma. This study examined whether treatable traits could be identified using registry data and whether particular treatable traits were associated with future exacerbation risk. METHODS: The Australasian Severe Asthma Web-Based Database (SAWD) enrolled 434 participants with severe asthma and a comparison group of 102 participants with non-severe asthma. Published treatable traits were mapped to registry data fields and their prevalence was described. Participants were characterized at baseline and every 6 months for 24 months. RESULTS: In SAWD, 24 treatable traits were identified in three domains: pulmonary, extrapulmonary and behavioural/risk factors. Patients with severe asthma expressed more pulmonary and extrapulmonary treatable traits than non-severe asthma. Allergic sensitization, upper-airway disease, airflow limitation, eosinophilic inflammation and frequent exacerbations were common in severe asthma. Ten traits predicted exacerbation risk; among the strongest were being prone to exacerbations, depression, inhaler device polypharmacy, vocal cord dysfunction and obstructive sleep apnoea. CONCLUSION: Treatable traits can be assessed using a severe asthma registry. In severe asthma, patients express more treatable traits than non-severe asthma. Traits may be associated with future asthma exacerbation risk demonstrating the clinical utility of assessing treatable traits.

Expression of Pseudomonas aeruginosa Antibiotic Resistance Genes Varies Greatly during Infections in Cystic Fibrosis Patients.

The lungs of individuals with cystic fibrosis (CF) become chronically infected with Pseudomonas aeruginosa that is difficult to eradicate by antibiotic treatment. Two key P. aeruginosa antibiotic resistance mechanisms are the AmpC ß-lactamase that degrades ß-lactam antibiotics and MexXYOprM, a three-protein efflux pump that expels aminoglycoside antibiotics from the bacterial cells. Levels of antibiotic resistance gene expression are likely to be a key factor in antibiotic resistance but have not been determined during infection. The aims of this research were to investigate the expression of the ampC and mexX genes during infection in patients with CF and in bacteria isolated from the same patients and grown under laboratory conditions. P. aeruginosa isolates from 36 CF patients were grown in laboratory culture and gene expression measured by reverse transcription-quantitative PCR (RT-qPCR). The expression of ampC varied over 20,000-fold and that of mexX over 2,000-fold between isolates. The median expression levels of both genes were increased by the presence of subinhibitory concentrations of antibiotics. To measure P. aeruginosa gene expression during infection, we carried out RT-qPCR using RNA extracted from fresh sputum samples obtained from 31 patients. The expression of ampC varied over 4,000-fold, while mexX expression varied over 100-fold, between patients. Despite these wide variations, median levels of expression of ampC in bacteria in sputum were similar to those in laboratory-grown bacteria. The expression of mexX was higher in sputum than in laboratory-grown bacteria. Overall, our data demonstrate that genes that contribute to antibiotic resistance can be highly expressed in patients, but there is extensive isolate-to-isolate and patient-to-patient variation.

Effect of Obstructive Sleep Apnea Treatment on Renal Function in Patients with Cardiovascular Disease.

RATIONALE: Obstructive sleep apnea (OSA) is associated with impaired renal function, but uncertainty exists over whether OSA treatment can influence renal outcomes. OBJECTIVES: To determine the effects of continuous positive airway pressure (CPAP) on renal function in subjects with coexisting OSA and cardiovascular disease. METHODS: This was a substudy of the international SAVE (Sleep Apnea Cardiovascular Endpoints) trial, in which 2,717 patients with moderate to severe OSA and established coronary or cerebrovascular disease were randomized to receive either CPAP plus usual care or usual care alone. Renal function and adverse renal events were compared between the CPAP (n = 102) and usual care (n = 98) groups. Glomerular filtration rate was estimated at randomization and at the end of follow-up, and the urinary albumin-to-creatinine ratio was measured at study exit. MEASUREMENTS AND MAIN RESULTS: In 200 substudy participants (mean age, 64 yr; median, 4% oxygen desaturation index; 20 events/h; mean estimated glomerular filtration rate at baseline, 82 ml/min/1.73 m2), the median (interquartile range) changes in estimated glomerular filtration rate (ml/min/1.73 m2/yr) were -1.64 (-3.45 to -0.740) in the CPAP group and -2.30 (-4.53 to -0.71) in the usual care group (P = 0.21) after a median of 4.4 years. There were no between-group differences in end-of-study urinary albumin-to-creatinine ratio or in the occurrence of serious renal or urinary adverse events during the trial. The level of CPAP adherence did not influence the findings. CONCLUSIONS: CPAP treatment of OSA in patients with cardiovascular disease does not alter renal function or the occurrence of renal adverse events. Clinical trial registered with www.clinicaltrials.gov (NCT00738179).

Effect of Inhaled ß2-Agonist on Exhaled Nitric Oxide in Chronic Obstructive Pulmonary Disease.

The fractional exhaled nitric oxide measured at an expiratory flow of 50mL/s (FENO50) is a marker of airway inflammation, and high levels are associated with greater response to steroid treatment. In asthma, FENO50 increases with bronchodilation and decreases with bronchoconstriction, the latter potentially causing an underestimate of the degree of airway inflammation when asthma worsens. It is unknown whether the same effect occurs in chronic obstructive lung disease (COPD). Likewise, it is not known whether changes in airway calibre in COPD patients alter flow-independent parameters describing pulmonary nitric oxide exchange, such as the maximal flux of nitric oxide (NO) from the proximal airway compartment (J'awNO) and the distal airway/alveolar concentration of NO (CANO). We recruited 24 patients with COPD and performed FENO analysis at multiple expiratory flows before and after treatment with inhaled ß2-agonist bronchodilator therapy. For the 21 patients analysed, FENO50 rose from 17.1 (1.4) ppb (geometric mean (geometric SD)) at baseline, to 19.3 (1.3) ppb after bronchodilator therapy, an increase of 2.2 ppb (95% CI, 0.7-3.6; P = 0.005). There were non-significant changes in flow-independent NO parameters. The change in FENO50 correlated positively with the change in J'awNO (rs = 0.67, P < 0.001; rs = 0.62, P = 0.002 before and after correction for axial back-diffusion respectively) following bronchodilation. Inhaled bronchodilator therapy can increase exhaled nitric oxide measurements in COPD. The standardisation of inhaled bronchodilator therapy before FENO analysis in COPD patients should therefore be considered in both research and clinical settings.

Intrapleural tissue plasminogen activator and deoxyribonuclease for pleural infection. An effective and safe alternative to surgery.

RATIONALE: Intrapleural tissue plasminogen activator (tPA)/deoxyribonuclease (DNase) therapy for pleural infection given at the time of diagnosis has been shown to significantly improve radiological outcomes. Published cases are limited to only a single randomized controlled trial and a few case reports. OBJECTIVES: Multinational observation series to evaluate the pragmatic "real-life" application of tPA/DNase treatment for pleural infection in a large cohort of unselected patients. METHODS: All patients from eight centers who received intrapleural tPA/DNase for pleural infection between January 2010 and September 2013 were included. Measured outcomes included treatment success at 30 days, volume of pleural fluid drained, improvement in radiographic pleural opacity and inflammatory markers, need for surgery, and adverse events. MEASUREMENTS AND MAIN RESULTS: Of 107 patients treated, the majority (92.3%) were successfully managed without the need for surgical intervention. No patients died as a result of pleural infection. Most patients (84%) received tPA/DNase more than 24 hours after failing to respond to initial conservative management with antibiotics and thoracostomy. tPA/DNase increased fluid drained from a median of 250 ml (interquartile range [IQR], 100-654) in the 24 hours preceding commencement of intrapleural therapy to 2,475 ml (IQR 1,800-3,585) in the 72 hours following treatment initiation (P < 0.05). We observed a corresponding clearance of pleural opacity on chest radiographs from a median of 35% (IQR 25-31) to 14% (7-28) of the hemithorax (P < 0.001), as well as significant reduction in C-reactive protein (P < 0.05). Pain necessitating escalation of analgesia occurred in 19.6% patients, and nonfatal bleeding occurred in 1.8%. CONCLUSIONS: This large series of patients who received intrapleural tPA/DNase therapy provides important evidence that the treatment is effective and safe, especially as a "rescue therapy" in patients who do not initially respond to antibiotics and thoracostomy drainage.