Changing Prevalence of Lower Airway Infections in Young Children with Cystic Fibrosis.

Rationale: Historical studies suggest that airway infection in cystic fibrosis initiates with Staphylococcus aureus and Haemophilus influenzae, with later emergence of Pseudomonas aeruginosa. Aspergillus species are regarded as relatively infrequent, late-occurring infections.Objectives: To assess the prevalence and change in prevalence of early lower airway infections in a modern cohort of children with cystic fibrosis.Methods: All infants diagnosed with cystic fibrosis after newborn screening participating in the Australian Respiratory Early Surveillance Team for Cystic Fibrosis (AREST CF) cohort study between 2000 and 2018 were included. Participants prospectively underwent BAL at 3-6 months, 1 year, and annually up to 6 years of age. Lower airway infection prevalence was described. Changes in prevalence patterns were assessed longitudinally using generalized estimating equations controlling for age and repeated visits.Measurements and Main Results: A total of 380 infants underwent 1,759 BALs. The overall prevalence and median age of first acquisition of the most common infections were as follows: S. aureus, 11%, 2.5 years; P. aeruginosa, 8%, 2.4 years; Aspergillus species, 11%, 3.2 years; and H. influenzae, 9%, 3.1 years. During the study, a significant decrease in prevalence of P. aeruginosa (P < 0.001) and S. aureus (P < 0.001) was observed with a significant change toward more aggressive treatment. Prevalence of Aspergillus infections did not significantly change (P = 0.669).Conclusions:Aspergillus species and P. aeruginosa are commonly present in the lower airways from infancy. The decrease in prevalence of P. aeruginosa and S. aureus since 2000, coinciding with a more aggressive therapeutic approach, has resulted in Aspergillus becoming the most commonly isolated pathogen in young children. Further research is warranted to understand the implication of these findings.

Clarithromycin therapy for patients with cystic fibrosis: a randomized controlled trial.

The clinically significant actions of oral azithromycin in modifying progressive cystic fibrosis (CF) lung disease have been well documented. In vitro and clinical data suggests that clarithromycin has immunomodulatory properties similar to other 14-member macrolides, however two previously reported short term, open label trials of clairthromycin in small numbers of patients with CF failed to show significant benefits in modifying lung function or inflammation. We performed an international double blind, cross-over trial in which 63 subjects with CF were studied while receiving either placeo or 500 mg oral clarithromycin twice daily for 5 months, with a 1-month wash-out. The primary efficacy end point was the change in lung function (FEV(1) and FVC) during the clarithromycin treatment period compared to placebo treatment. Secondary efficacy end points included; quality of life, number of pulmonary exacerbations, height and weight, sputum inflammatory mediator content, sputum transportability and surface properties, bacterial flora, nasal potential difference, and breath condensate. No significant difference in either the primary efficacy end point or any secondary end point was seen during the period of clarithromycin treatment compared to those seen during placebo administration. We conclude that clarithromycin is not effective in treating CF lung disease.

Inflammatory responses to individual microorganisms in the lungs of children with cystic fibrosis.

BACKGROUND: We hypothesized that the inflammatory response in the lungs of children with cystic fibrosis (CF) would vary with the type of infecting organism, being greatest with Pseudomonas aeruginosa and Staphylococcus aureus. METHODS: A microbiological surveillance program based on annual bronchoalveolar lavage (BAL) collected fluid for culture and assessment of inflammation was conducted. Primary analyses compared inflammation in samples that grew a single organism with uninfected samples in cross-sectional and longitudinal analyses. RESULTS: Results were available for 653 samples from 215 children with CF aged 24 days to 7 years. A single agent was associated with pulmonary infection (≥10(5) cfu/mL) in 67 BAL samples, with P. aeruginosa (n = 25), S. aureus (n = 17), and Aspergillus species (n = 19) being the most common. These microorganisms were associated with increased levels of inflammation, with P. aeruginosa being the most proinflammatory. Mixed oral flora (MOF) alone was isolated from 165 BAL samples from 112 patients, with 97 of these samples having a bacterial density ≥10(5) cfu/mL, and was associated with increased pulmonary inflammation (P < .001). For patients with current, but not past, infections there was an association with a greater inflammatory response, compared with those who were never infected (P < .05). However, previous infection with S. aureus was associated with a greater inflammatory response in subsequent BAL. CONCLUSIONS: Pulmonary infection with P. aeruginosa, S. aureus, or Aspergillus species and growth of MOF was associated with significant inflammatory responses in young children with CF. Our data support the use of specific surveillance and eradication programs for these organisms. The inflammatory response to MOF requires additional investigation.

Value of serology in predicting Pseudomonas aeruginosa infection in young children with cystic fibrosis.

BACKGROUND: Early detection of Pseudomonas aeruginosa is essential for successful eradication. The accuracy of serum antibodies against specific and multiple P aeruginosa antigens at predicting lower airway infection in young children with cystic fibrosis (CF) was investigated. METHODS: A commercial P aeruginosa multiple antigen (MAg) ELISA and an in-house exotoxin A (ExoA) ELISA were compared in two populations: a discovery population of 76 children (0.1-7.1 years) undergoing annual bronchoalveolar lavage (BAL)-based microbiological surveillance and a test population of 55 children (0.1-5.6 years) participating in the Australasian CF Bronchoalveolar Lavage Trial. RESULTS: In the discovery population, P aeruginosa was cultured from BAL fluid (≥10(5) colony-forming units (cfu)/ml) in 15/76 (19.7%) children (median age 1.88 years). Positive MAg and ExoA serological results were found in 38 (50.0%) and 30 (39.5%) children, respectively. Positive (PPV) and negative (NPV) predictive values for serology at diagnosing P aeruginosa infection (≥10(5) cfu/ml) were 0.14 and 0.99 respectively (MAg assay) and 0.11 and 0.98 (ExoA assay). In the test population, P aeruginosa was cultured from BAL fluid (≥10(5) cfu/ml) in 16/55 (29.1%) children (median age 1.86 years) and from oropharyngeal swabs in 32/36 (88.9%). Positive MAg and ExoA serology was detected in 19 (34.5%) and 33 (60.0%) children, respectively. The PPV and NPV of serology were 0.26 and 0.94 respectively (MAg assay) and 0.19 and 0.98 (ExoA assay) and were marginally higher for oropharyngeal cultures. CONCLUSIONS: Measuring serum antibody responses against P aeruginosa is of limited value for detecting early P aeruginosa infection in young children with CF.

Monocytes from children with clinically stable cystic fibrosis show enhanced expression of Toll-like receptor 4.

SUMMARY: Lung disease in patients with cystic fibrosis (CF) is characterized by recurrent bacterial respiratory infections and intense airway inflammation. Pattern recognition receptors such as Toll-like receptor 2 (TLR2) and TLR4 identify bacterial pathogens and activate the innate immune response. We therefore hypothesized that increased expression of these receptors would be found on circulating immune cells from children with CF. A cohort of 66 young children (median age 3 years) with CF was studied and compared to both healthy controls (n = 14) and children without CF who were being investigated for recurrent respiratory infections (non-CF disease controls; n = 17) of a similar age. Surface expression of TLR2 and TLR4 on peripheral blood monocytes was analyzed using flow cytometry. TLR4 expression was significantly higher in patients with CF compared to healthy controls (P = 0.017) and non-CF disease controls (P = 0.025) but did not vary according to the presence or absence of pulmonary infection with Gram-negative or Gram-positive bacteria (P = 0.387) in the CF group. In contrast, TLR2 expression was similar across all three study groups (P = 0.930). The increased surface expression of TLR4 seen in young children with CF appears to be related to having CF per se and not related to current pulmonary infection.

Identifying peroxidases and their oxidants in the early pathology of cystic fibrosis.

We aimed to determine whether myeloperoxidase (MPO) is the main peroxidase present in the airways of children with cystic fibrosis (CF) and to assess which oxidants it produces and whether they are associated with clinical features of CF. Children with CF (n=54) and without CF (n=16) underwent bronchoscopy and bronchoalveolar lavage (BAL) for assessment of pulmonary infection and inflammation. BAL fluid was analyzed for MPO, halogenated tyrosines as markers of hypohalous acids, thiocyanate, and protein carbonyls. MPO was the only peroxidase detected in BAL samples from children with CF and its concentration was markedly higher than in controls. Levels of 3-chlorotyrosine and 3-bromotyrosine in proteins were higher in the CF group. They correlated with neutrophils and MPO. The concentration of thiocyanate in BAL samples was below 1µM. Protein carbonyl levels correlated with MPO and halogenated tyrosines in patients with CF. Levels of MPO and halogenated tyrosines were higher in children with infections, especially Pseudomonas aeruginosa, and in the presence of respiratory symptoms. They also correlated with the Kanga clinical score. Our findings suggest that MPO produces hypobromous acid as well as hypochlorous acid in the airways of children with CF and that these oxidants are involved in the early pathogenesis of CF.

Bronchiectasis in infants and preschool children diagnosed with cystic fibrosis after newborn screening.

OBJECTIVES: To determine the prevalence of bronchiectasis in young children with cystic fibrosis (CF) diagnosed after newborn screening (NBS) and the relationship of bronchiectasis to pulmonary inflammation and infection. STUDY DESIGN: Children were diagnosed with CF after NBS. Computed tomography and bronchoalveolar lavage were performed with anesthesia (n = 96). Scans were analyzed for the presence and extent of abnormalities. RESULTS: The prevalence of bronchiectasis was 22% and increased with age (P = .001). Factors associated with bronchiectasis included absolute neutrophil count (P = .03), neutrophil elastase concentration (P = .001), and Pseudomonas aeruginosa infection (P = .03). CONCLUSIONS: Pulmonary abnormalities are common in infants and young children with CF and relate to neutrophilic inflammation and infection with P. aeruginosa. Current models of care for infants with CF fail to prevent respiratory sequelae. Bronchiectasis is a clinically relevant endpoint that could be used for intervention trials that commence soon after CF is diagnosed after NBS.

Simultaneous determination of reduced glutathione, glutathione disulphide and glutathione sulphonamide in cells and physiological fluids by isotope dilution liquid chromatography-tandem mass spectrometry.

A stable isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneously quantifying glutathione (GSH), glutathione disulphide (GSSG) and glutathione sulphonamide (GSA) from biological samples. GSA is a selective product of the reaction of GSH with hypochlorous acid and a potential biomarker of myeloperoxidase activity. GSH was detected as the N-ethylmaleimide alkylated adduct, as formation of this species prevented GSH oxidation occurring during sample processing. Synthesised stable isotope analogues were used as internal standards to accurately quantify each target species. The limit of quantification was determined as being 0.1pmol for each species and excellent linearity was observed over relevant concentration ranges for biological samples. Relative standard deviations were <5% for within-day variation and <10% for between-day variation, except at the lower limit of quantification where they remained <20%. Accuracy was between 82% and 113%. We could detect GSA in neutrophils and endothelial cells treated with hypochlorous acid and in bronchoalveolar lavage fluid from children with cystic fibrosis. This is the first time GSA has been quantified in clinical material and suggests it is formed in vivo. The assay can now be used for investigating GSA as a biomarker of myeloperoxidase activity in inflammatory conditions, and is also applicable to measuring GSH:GSSG molar ratios as a general index of oxidative stress.

Lung disease at diagnosis in infants with cystic fibrosis detected by newborn screening.

RATIONALE: The promise of newborn screening (NBS) for cystic fibrosis (CF) has not been fully realized, and the extent of improvement in respiratory outcomes is unclear. We hypothesized that significant lung disease was present at diagnosis. OBJECTIVES: To determine the extent of lung disease in a geographically defined population of infants with CF diagnosed after detection by NBS. METHODS: Fifty-seven infants (median age, 3.6 mo) with CF underwent bronchoalveolar lavage and chest computed tomography (CT) using a three-slice inspiratory and expiratory protocol. MEASUREMENTS AND MAIN RESULTS: Despite the absence of respiratory symptoms in 48 (84.2%) of infants, a substantial proportion had lung disease with bacterial infection detected in 12 (21.1%), including Staphylococcus aureus (n = 4) and Pseudomonas aeruginosa (n = 3); neutrophilic inflammation (41. 4 x 10(3) cells/ml representing 18.7% of total cell count); proinflammatory cytokines, with 44 (77.2%) having detectable IL-8; and 17 (29.8%) having detectable free neutrophil elastase activity. Inflammation was increased in those with infection and respiratory symptoms; however, the majority of those infected were asymptomatic. Radiologic evidence of structural lung disease was common, with 46 (80.7%) having an abnormal CT; 11 (18.6%) had bronchial dilatation, 27 (45.0%) had bronchial wall thickening, and 40 (66.7%) had gas trapping. On multivariate analysis, free neutrophil elastase activity was associated with structural lung disease. Most children with structural lung disease had no clinically apparent lung disease. CONCLUSIONS: These data support the need for full evaluation in infancy and argue for new treatment strategies, especially those targeting neutrophilic inflammation, if the promise of NBS for CF is to be realized.

Innate immune activation and cystic fibrosis.

Since inflammation and infection occur so early in infancy in cystic fibrosis, the function of innate immune defence in cystic fibrosis has been questioned by many investigators. This review aims to summarize the findings relating to the physical, humoral and cellular components of innate immune defence in cystic fibrosis, and highlights the roles of neutrophils, macrophages and epithelial cells in these activities. In addition, recently identified links between antioxidant defences and cystic fibrosis transmembrane conductance regulator (CFTR) function, and how these may impact on innate lung defence, are summarized.

Disease surveillance using bronchoalveolar lavage.

The role of pulmonary infection and inflammation in the pathogenesis of destructive lung disease in cystic fibrosis (CF) is undisputed. The use of bronchoscopy and bronchoalveolar lavage (BAL) has demonstrated that these processes may begin early in life and be present in the absence of overt clinical symptoms. Some children diagnosed following newborn screening can be infected with Pseudomonas aeruginosa in infancy. Studies using BAL have demonstrated a relationship between lower airway inflammation and bacterial load in the lungs; however, inflammation may occur in the absence of obvious current infection. BAL has the potential to provide a greater understanding of the pathogenesis of CF lung disease and microbiological surveillance provides the opportunity for early detection and eradication of P. aeruginosa. Lack of standardization inhibits the ability to compare data from different centres and to optimize treatment strategies. This review discusses the recommendations from a workshop held in early 2007 aimed at achieving a standardized approach to BAL in infants and young children with CF.

Assay for urinary desmosines in a healthy pre-pubertal population using an improved extraction technique.

BACKGROUND: Current evidence indicates that increased desmosine excretion reflects the active inflammatory status of some connective tissue diseases. Our goal was to establish a reliable method of detection and to investigate the normal distribution of urinary desmosine excretion in a healthy pre-pubertal population. METHOD: Urine was collected from healthy volunteers aged four weeks to 12 years old. We modified a published high-performance liquid chromatography (HPLC) method by (a) increasing hydrolysis time and temperature and (b) increasing cellulose column size. RESULTS: Our modified method had small inter- and intra-assay variability, with coefficients of variation of <6.4% and 5.3%, respectively. There was positive correlation between isodesmosine and desmosine (r(2) = 0.91). There was no significant diurnal or day-to-day variability in total desmosine levels. A reference range for healthy pre-pubertal children aged four weeks to 12 years was established. CONCLUSION: The modified HPLC method is reliable with low variability. The technique can now be applied as a non-invasive research or diagnostic tool for children with chronic lung disease.

Lyn-deficient mice develop severe, persistent asthma: Lyn is a critical negative regulator of Th2 immunity.

The etiology of asthma, a chronic inflammatory disorder of the airways, remains obscure, although T cells appear to be central disease mediators. Lyn tyrosine kinase has been implicated as both a facilitator and inhibitor of signaling pathways that play a role in allergic inflammation, although its role in asthma is unclear because Lyn is not expressed in T cells. We show in the present study that Lyn-/- mice develop a severe, persistent inflammatory asthma-like syndrome with lung eosinophilia, mast cell hyperdegranulation, intensified bronchospasm, hyper IgE, and Th2-polarizing dendritic cells. Dendritic cells from Lyn-/- mice have a more immature phenotype, exhibit defective inhibitory signaling pathways, produce less IL-12, and can transfer disease when adoptively transferred into wild-type recipients. Our results show that Lyn regulates the intensity and duration of multiple asthmatic traits and indicate that Lyn is an important negative regulator of Th2 immune responses.

Inflammatory profile in nasal secretions of infants hospitalized with acute lower airway tract infections.

OBJECTIVE: The aim of this study was to determine whether the regulatory immune response (interleukin (IL)-10 response) differed between children hospitalized with acute respiratory infections and wheezing. METHODOLOGY: Infants with signs and symptoms of acute viral respiratory infection, admitted during winter 2000 to Princess Margaret Hospital for Children, Perth, WA, Australia, were enrolled in this study. Nasopharyngeal aspirates were collected in the first 48 h of admission. Total cell count and differential cell counts were assessed. Samples were tested for the presence of respiratory viruses. The concentrations of the anti-inflammatory cytokine IL-10, and pro-inflammatory cytokines IL-8, interferon-gamma, and IL-11 were determined by ELISA. RESULTS: Children with acute bronchiolitis (AB; n = 36), recurrent wheeze (RW; n = 17) and upper respiratory infection (URI; n = 18) were enrolled. Respitory syncytial virus was the most commonly detected virus in all groups. IL-10 concentrations were significantly increased in AB (median, 0.019 ng/mL) when compared to URI (median, 0.006 ng/mL) or to RW (median, 0.007 ng/mL; P < 0.05). Neutrophils were the predominant cells in the cytological analysis in all subjects. CONCLUSION: These data argue that host-response factors are important in determining the clinical phenotype, independent of the causative virus.