16S rRNA-Based Microbiota Profiling Assists Conventional Culture Analysis of Airway Samples from Pediatric Cystic Fibrosis Patients.

16S-based sequencing provides broader information on the respiratory microbial community than conventional culturing. However, it (often) lacks species- and strain-level information. To overcome this issue, we used 16S rRNA-based sequencing results from 246 nasopharyngeal samples obtained from 20 infants with cystic fibrosis (CF) and 43 healthy infants, which were all 0 to 6 months old, and compared them to both standard (blind) diagnostic culturing and a 16S-sequencing-informed "targeted" reculturing approach. Using routine culturing, we almost uniquely detected Moraxella catarrhalis, Staphylococcus aureus, and Haemophilus influenzae (42%, 38%, and 33% of samples, respectively). Using the targeted reculturing approach, we were able to reculture 47% of the top-5 operational taxonomical units (OTUs) in the sequencing profiles. In total, we identified 60 species from 30 genera with a median of 3 species per sample (range, 1 to 8). We also identified up to 10 species per identified genus. The success of reculturing the top-5 genera present from the sequencing profile depended on the genus. In the case of Corynebacterium being in the top 5, we recultured them in 79% of samples, whereas for Staphylococcus, this value was only 25%. The success of reculturing was also correlated with the relative abundance of those genera in the corresponding sequencing profile. In conclusion, revisiting samples using 16S-based sequencing profiles to guide a targeted culturing approach led to the detection of more potential pathogens per sample than conventional culturing and may therefore be useful in the identification and, consequently, treatment of bacteria considered relevant for the deterioration or exacerbation of disease in patients like those with CF. IMPORTANCE Early and effective treatment of pulmonary infections in cystic fibrosis is vital to prevent chronic lung damage. Although microbial diagnostics and treatment decisions are still based on conventional culture methods, research is gradually focusing more on microbiome and metagenomic-based approaches. This study compared the results of both methods and proposed a way to combine the best of both worlds. Many species can relatively easily be recultured based on the 16S-based sequencing profile, and it provides more in-depth information about the microbial composition of a sample than that obtained through routine (blind) diagnostic culturing. Still, well-known pathogens can be missed by both routine diagnostic culture methods as well as by targeted reculture methods, sometimes even when they are highly abundant, which may be a consequence of either sample storage conditions or antibiotic treatment at the time of sampling.

Development of the gut microbiota in early life: The impact of cystic fibrosis and antibiotic treatment.

OBJECTIVES: Patients with Cystic Fibrosis (CF) suffer from pancreatic insufficiency, lipid malabsorption and gastrointestinal complaints, next to progressive pulmonary disease. Altered mucosal homoeostasis due to malfunctioning chloride channels results in an adapted microbial composition of the gastrointestinal and the respiratory tract. Additionally, antibiotic treatment has the potential to distort resident microbial communities dramatically. This study aims to investigate early life development of the gut microbial community composition of children with CF compared to healthy infants and to study the independent effects of antibiotics taking into account other clinical and lifestyle factors. STUDY DESIGN: Faecal samples from 20 infants with CF and 45 healthy infants were collected regularly during the first 18 months of life and microbial composition was determined using 16S rRNA based sequencing. RESULTS: We observed significant differences in the overall microbiota composition between infants with CF and healthy infants (p<0.001). Akkermansia and Anaerostipes were significantly more abundant in control infants, whereas Streptococci and E. coli were significantly more abundant in infants with CF, also after correction for several clinical factors (p<0.05). Antibiotic use in infants with CF was associated with a lower alpha diversity, a reduced abundance of Bifidobacterium and Bacteroides, and a higher abundance of Enterococcus. CONCLUSION: Microbial development of the gut is different in infants with CF compared to healthy infants from the first months of life on, and further deviates over time, in part as a result of antibiotic treatment. The resulting dysbiosis may have significant functional consequences for the microbial ecosystem in CF patients.

Pharmacokinetics and safety of tobramycin nebulization with the I-neb and PARI-LC Plus in children with cystic fibrosis: A randomized, crossover study.

AIMS: We aimed to compare the pharmacokinetics (PK) and safety profile of tobramycin inhalation solution (TIS) using the I-neb device to the standard PARI-LC Plus nebulizer in children with cystic fibrosis. METHODS: A randomized, open-label, crossover study was performed. In 2 separate study visits, blood samples from 22 children were collected following TIS nebulization with I-neb (75 mg) and PARI-LC Plus (300 mg). Study visits were separated by 1 month, in which 1 of the study nebulizers was used twice daily. Tobramycin PK for both nebulizers was established using measured tobramycin concentrations and Bayesian PK modelling software. Hearing and renal function tests were performed to test for aminoglycoside associated toxicity. In addition to standard estimated glomerular filtration rate values, biomarkers for tubular injury (KIM-1 and NAG) were measured. Patient and nebulizer satisfaction were assessed. RESULTS: Inhalations were well tolerated and serum trough concentrations below the predefined toxic limit were reached with no significant differences in PK parameters between nebulizers. Results of audiometry and estimated glomerular filtration rate revealed no abnormalities. However, increased urinary NAG/creatinine ratios at visit 2 for both nebulizers suggest TIS-induced subclinical tubular kidney injury. Nebulization time was 50% shorter and patient satisfaction was significantly higher with the I-neb. CONCLUSIONS: Nebulization of 75 mg TIS with the I-neb in children with cystic fibrosis resulted in comparable systemic exposure to 300 mg TIS with the PARI-LC Plus and was well tolerated and preferred over the PARI-LC Plus. Long-term safety of TIS nebulization should be monitored clinically, especially regarding the effects on tubular kidney injury.

Cutaneotrichosporon (Cryptococcus) cyanovorans, a basidiomycetous yeast, isolated from the airways of cystic fibrosis patients.

Cystic fibrosis (CF) patients are colonized with a multitude of bacteria and fungi. From respiratory samples of two CF patients in our institute, a difficult to identify yeast was isolated repeatedly. This yeast was eventually identified as Cutaneotrichosporon (Cryptococcus) cyanovorans by internal transcribed spacer (ITS) and ribosomal large subunit (LSU) sequencing. C. cyanovorans is a basidiomycetous yeast originally reported as environmental isolate from South African soil and has not been described before as clinical isolate from CF patients.

Exercise oxidative skeletal muscle metabolism in adolescents with cystic fibrosis.

NEW FINDINGS: What is the central question of this study? Do intrinsic abnormalities in oxygenation and/or muscle oxidative metabolism contribute to exercise intolerance in adolescents with mild cystic fibrosis? What is the main finding and its importance? This study found no evidence that in adolescents with mild cystic fibrosis in a stable clinical state intrinsic abnormalities in skeletal muscle oxidative metabolism seem to play a clinical significant role. Based on these results, we concluded that there is no metabolic constraint to benefit from exercise training. Patients with cystic fibrosis (CF) are reported to have limited exercise capacity. There is no consensus about a possible abnormality in skeletal muscle oxidative metabolism in CF. Our aim was to test the hypothesis that abnormalities in oxygenation and/or muscle oxidative metabolism contribute to exercise intolerance in adolescents with mild CF. Ten adolescents with CF (12-18 years of age; forced expiratory volume in 1 s >80% of predicted; and resting oxygen saturation >94%) and 10 healthy age-matched control (HC) subjects were tested with supine cycle ergometry using near-infrared spectroscopy and (31)P magnetic resonance spectroscopy to study skeletal muscle oxygenation and oxidative metabolism during rest, exercise and recovery. No statistically significant (P > 0.1) differences in peak workload and peak oxygen uptake per kilogram lean body mass were found between CF and HC subjects. No differences were found between CF and HC subjects in bulk changes of quadriceps phosphocreatine (P = 0.550) and inorganic phosphate (P = 0.896) content and pH (P = 0.512) during symptom-limited exercise. Furthermore, we found statistically identical kinetics for phosphocreatine resynthesis during recovery for CF and HC subjects (P = 0.53). No statistically significant difference in peak exercise arbitrary units for total haemoglobin content was found between CF and HC subjects (P = 0.66). The results of this study provide evidence that in patients with mild CF and a stable clinical status (without signs of systemic inflammation and/or chronic Pseudomonas aeruginosa colonization), no intrinsic metabolic constraints and/or abnormalities in oxygenation and/or muscle oxidative metabolism contribute to exercise intolerance.

Physical activity assessment in cystic fibrosis: A position statement.

BACKGROUND: The aim of this position statement was to inform the choice of physical activity tools for use within CF research and clinical settings. METHODS: A systematic review of physical activity tools to explore evidence for reliability, validity, and responsiveness. Narrative answers to "four key questions" on motion sensors, questionnaires and diaries were drafted by the core writing team and then discussed at the Exercise Working Group in ECFS Lisbon 2013. RESULTS AND SUMMARY: Our current position is that activity monitors such as SenseWear or ActiGraph offer informed choices to facilitate a comprehensive assessment of physical activity, and should as a minimum report on dimensions of physical activity including energy expenditure, step count and time spent in different intensities and sedentary time. The DigiWalker pedometer offers an informed choice of a comparatively inexpensive method of obtaining some measurement of physical activity. The HAES represents an informed choice of questionnaire to assess physical activity. There is insufficient data to recommend the use of one diary over another. Future research should focus on providing additional evidence of clinimetric properties of these and new physical activity assessment tools, as well as further exploring the added value of physical activity assessment in CF.

Clinical evaluation of the Nanoduct sweat test system in the diagnosis of cystic fibrosis after newborn screening.

UNLABELLED: After a positive newborn screening test for cystic fibrosis (CF), a sweat test is performed to confirm the diagnosis. The success rate of the generally acknowledged methods (Macroduct/Gibson and Cooke) in newborns varies between 73 and 99%. The Nanoduct sweat test system is easier to perform and less sweat is needed. The main aim of this study was to measure the success rate of the Nanoduct compared to current approved sweat test methods in a newborn population. After informed consent of the parents, newborns with a positive screening test for CF were included. The Macroduct or Gibson and Cooke and Nanoduct were performed in all infants, during the same appointment. The chloride concentration was determined by standard coulorimetry; conductivity was measured directly and converted to a NaCl molarity. One hundred eight newborns were included: 17 with CF, 7 with cystic fibrosis transmembrane regulator (CFTR)-related metabolic syndrome (CRMS), and 84 healthy children. The success rate of the Nanoduct was 93% and for the Macroduct/Gibson and Cooke 79% (McNemar, p = 0.002). The Nanoduct detected the same CF patients as the Macroduct/Gibson and Cooke; one CF patient had an equivocal result for both tests, and no patients were missed. The area under the receiver operating characteristic curve for detection of CF with the Nanoduct was 0.999, with ideal cutoff levels of 91 and 66 mmol/l, comparable to former studies. CONCLUSION: The success rate of the Nanoduct to collect sufficient sweat in infants was higher compared to the Macroduct and Gibson and Cooke.

Estimating peak oxygen uptake in adolescents with cystic fibrosis.

OBJECTIVES: To predict peak oxygen uptake (VO2 peak) from the peak work rate (W peak) obtained during a cycle ergometry test using the Godfrey protocol in adolescents with cystic fibrosis (CF), and assess the accuracy of the model for prognostication clustering. METHODS: Out of our database of anthropometric, spirometric and maximal exercise data from adolescents with CF (N=363; 140 girls and 223 boys; age 14.77 ± 1.73 years; mean expiratory volume in 1 s (FEV1%pred) 86.82 ± 17.77%), a regression equation was developed to predict VO2 peak (mL/min). Afterwards, this prediction model was validated with cardiopulmonary exercise data from another 60 adolescents with CF (28 girls, 32 boys; mean age 14.6 ± 1.67 years; mean FEV1%pred 85.43 ± 20.01%). RESULTS: We developed a regression model VO2 peak (mL/min)=216.3-138.7 × sex (0=male; 1=female)+11.5 × W peak; R(2)=0.91; SE of the estimate (SEE) 172.57. A statistically significant difference (107 mL/min; p<0.001) was found between predicted VO2 peak and measured VO2 peak in the validation group. However, this difference was not clinically relevant because the difference was within the SEE of the model. Furthermore, we found high positive predictive and negative predictive values for the model for prognostication clustering (PPV 50-87% vs NPV 82-94%). CONCLUSIONS: In the absence of direct VO2 peak assessment it is possible to estimate VO2 peak in adolescents with CF using only a cycle ergometer. Furthermore, the regression model showed to be able to discriminate patients in different prognosis clusters based on exercise capacity.

An official American Thoracic Society workshop report: optimal lung function tests for monitoring cystic fibrosis, bronchopulmonary dysplasia, and recurrent wheezing in children less than 6 years of age.

Although pulmonary function testing plays a key role in the diagnosis and management of chronic pulmonary conditions in children under 6 years of age, objective physiologic assessment is limited in the clinical care of infants and children less than 6 years old, due to the challenges of measuring lung function in this age range. Ongoing research in lung function testing in infants, toddlers, and preschoolers has resulted in techniques that show promise as safe, feasible, and potentially clinically useful tests. Official American Thoracic Society workshops were convened in 2009 and 2010 to review six lung function tests based on a comprehensive review of the literature (infant raised-volume rapid thoracic compression and plethysmography, preschool spirometry, specific airway resistance, forced oscillation, the interrupter technique, and multiple-breath washout). In these proceedings, the current state of the art for each of these tests is reviewed as it applies to the clinical management of infants and children under 6 years of age with cystic fibrosis, bronchopulmonary dysplasia, and recurrent wheeze, using a standardized format that allows easy comparison between the measures. Although insufficient evidence exists to recommend incorporation of these tests into the routine diagnostic evaluation and clinical monitoring of infants and young children with cystic fibrosis, bronchopulmonary dysplasia, or recurrent wheeze, they may be valuable tools with which to address specific concerns, such as ongoing symptoms or monitoring response to treatment, and as outcome measures in clinical research studies.

Respiratory symptoms in a boy revealing Carney triad.

The association of gastrointestinal stromal cell tumor (GIST), paraganglioma, and pulmonary chondroma is known as the Carney triad, occurring predominantly in young adult females. We present the case of a 14-year-old male with respiratory symptoms resulting in the diagnosis Carney triad.