Statistical Evaluation of Metaproteomics and 16S rRNA Amplicon Sequencing Techniques for Study of Gut Microbiota Establishment in Infants with Cystic Fibrosis.

Newborn screening for cystic fibrosis (CF) can identify affected but asymptomatic infants. The selection of omic technique for gut microbiota study is crucial due to both the small amount of feces available and the low microorganism load. Our aims were to compare the agreement between 16S rRNA amplicon sequencing and metaproteomics by a robust statistical analysis, including both presence and abundance of taxa, to describe the sequential establishment of the gut microbiota during the first year of life in a small size sample (8 infants and 28 fecal samples). The taxonomic assignations by the two techniques were similar, whereas certain discrepancies were observed in the abundance detection, mostly the lower predicted relative abundance of Bifidobacterium and the higher predicted relative abundance of certain Firmicutes and Proteobacteria by amplicon sequencing. During the first months of life, the CF gut microbiota is characterized by a significant enrichment of Ruminococcus gnavus, the expression of certain virulent bacterial traits, and the detection of human inflammation-related proteins. Metaproteomics provides information on composition and functionality, as well as data on host-microbiome interactions. Its strength is the identification and quantification of Actinobacteria and certain classes of Firmicutes, but alpha diversity indices are not comparable to those of amplicon sequencing. Both techniques detected an aberrant microbiota in our small cohort of infants with CF during their first year of life, dominated by the enrichment of R. gnavus within a human inflammatory environment. IMPORTANCE In recent years, some techniques have been incorporated for the study of microbial ecosystems, being 16S rRNA gene sequencing being the most widely used. Metaproteomics provides the advantage of identifying the interaction between microorganisms and human cells, but the available databases are less extensive as well as imprecise. Few studies compare the statistical differences between the two techniques to define the composition of an ecosystem. Our work shows that the two methods are comparable in terms of microorganism identification but provide different results in alpha diversity analysis. On the other hand, we have studied newborns with cystic fibrosis, for whom we have described the establishment of an intestinal ecosystem marked by the inflammatory response of the host and the enrichment of Ruminococcus gnavus.

Long-term docosahexaenoic acid (DHA) supplementation in cystic fibrosis patients: a randomized, multi-center, double-blind, placebo-controlled trial.

BACKGROUND: Cystic fibrosis (CF) patients have an alteration in fatty acid (FA) metabolism, associated with increased omega-6 and low omega-3 FA. Previous studies on supplementation with omega-3 FA in CF had contradictory results, and to date there is no evidence to recommend routine use of omega-3 supplements in CF patients. We hypothesized that long-term supplementation with docosahexaenoic acid (DHA) will have beneficial effects in these patients, by reducing pulmonary, systemic and intestinal inflammation. METHODS: This was a randomized, double-blind, parallel, placebo-controlled trial. CF patients (age >2 months) were randomized to receive a seaweed DHA oil solution (50 mg/Kg/day) or matching placebo for 48 weeks. Primary outcomes were pulmonary (interleukin [IL]-8), systemic (IL-8) and intestinal (calprotectin) inflammatory biomarkers. Secondary outcomes included other pulmonary (IL-1ß, IL-6, neutrophil elastase, lactate and calprotectin) and systemic (serum-IL-1ß, IL-6) inflammatory biomarkers, as well as clinical outcomes (FEV1, pulmonary exacerbations, antibiotic use, nutritional status and quality of life). RESULTS: Ninety six CF patients, 44 female, age 14.6±11.9 years (48 DHA and 48 placebo) were included. At trial completion, there were no differences in all primary outcomes [serum-IL-8 (p=0.909), respiratory-IL-8 (p=0.384) or fecal calprotectin (p=0.948)], all secondary inflammatory biomarkers, or in any of the clinical outcomes evaluated. There were few adverse events, with similar incidence in both study groups. CONCLUSION: In this study, long-term DHA supplementation in CF patients was safe, but did not offer any benefit on inflammatory biomarkers, or in clinical outcomes compared with placebo. (NCT01783613).

Effects of inhaled high-molecular weight hyaluronan in inflammatory airway disease.

Cystic fibrosis (CF) is a chronic inflammatory disease that is affecting thousands of patients worldwide. Adjuvant anti-inflammatory treatment is an important component of cystic fibrosis treatment, and has shown promise in preserving lung function and prolonging life expectancy. Inhaled high molecular weight hyaluronan (HMW-HA) is reported to improve tolerability of hypertonic saline and thus increase compliance, and has been approved in some European countries for use as an adjunct to hypertonic saline treatment in cystic fibrosis. However, there are theoretical concerns that HMW-HA breakdown products may be pro-inflammatory. In this clinical pilot study we show that sputum cytokines in CF patients receiving HMW-HA are not increased, and therefore HMW-HA does not appear to adversely affect inflammatory status in CF airways.

Síndrome de Donohue. Resistencia extrema a la insulina en el periodo neonatal / Donohue syndrome. Extreme insulin resistance in the neonatal period

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Tos crónica / Chronic Cough

La tos crónica (TC), o tos que perdura más de 8 semanas, ha merecido un interés creciente en los últimos años debido a los avances producidos que han motivado un cambio de visión respecto a la clásica tríada diagnóstica y terapéutica en vigor desde la década de los setenta. Unos resultados no óptimos en el tratamiento que alcanza los dos tercios de casos, junto a una nueva concepción de la TC como síndrome de hipersensibilidad con 2 polos, periférico y central, similares al dolor crónico, ocasionan que se contemple este problema tan frecuente en la práctica clínica de una nueva manera. Los receptores periféricos de la TC siguen teniendo vigencia bajo la tríada diagnóstica; sin embargo, tanto la convergencia de estímulos como la hipersensibilidad adquirida a nivel del sistema nervioso central son hechos que tienen una repercusión clave en el éxito del tratamiento

Chronic cough (CC), or cough lasting more than 8 weeks, has attracted increased attention in recent years following advances that have changed opinions on the prevailing diagnostic and therapeutic triad in place since the 1970s. Suboptimal treatment results in two thirds of all cases, together with a new notion of CC as a peripheral and central hypersensitivity syndrome similar to chronic pain, have changed the approach to this common complaint in routine clinical practice. The peripheral receptors involved in CC are still a part of the diagnostic triad. However, both convergence of stimuli and central nervous system hypersensitivity are key factors in treatment success

Chronic Cough.

Chronic cough (CC), or cough lasting more than 8 weeks, has attracted increased attention in recent years following advances that have changed opinions on the prevailing diagnostic and therapeutic triad in place since the 1970s. Suboptimal treatment results in two thirds of all cases, together with a new notion of CC as a peripheral and central hypersensitivity syndrome similar to chronic pain, have changed the approach to this common complaint in routine clinical practice. The peripheral receptors involved in CC are still a part of the diagnostic triad. However, both convergence of stimuli and central nervous system hypersensitivity are key factors in treatment success.

Pulmonary function in children with type 1 diabetes mellitus.

AIM: To assess lung function in children and adolescents with type 1 diabetes mellitus (T1DM). PATIENTS AND METHODS: We conducted a case-control study of 100 patients with T1DM [median age 13 (10.6-14.7), 44% men, 23% prepubertal, and all nonsmokers] and 77 controls. None had evidence of lung disease or any other comorbidity. We performed pulmonary function tests, including spirometry [forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1), and FEV1/FVC ratio], plethysmography [total lung capacity (TLC), residual volume (RV), RV/TLC ratio, and airway resistance (Raw)], and diffusing capacity of carbon monoxide in the lung (TLCO), alveolar volume (AV), and TLCO/AV ratio. The duration of diabetes, degree of metabolic control, insulin dose, and presence of diabetic complications were registered. The χ²-test and analysis of variance were used to compare categorical and quantitative variables, respectively. RESULTS: The duration of diabetes was 6.2±3.8 years with a median HbA1c of 7.08±0.4%. FEV1/FVC ratio was found to be significantly higher in patients with TIDM than in controls. Patients with diabetes also had a nonsignificant trend towards lower FVC, FEV1, Raw, and TLCO, and higher RV, TLC, and RV/TLC than controls. There were no differences in pulmonary function based on duration of disease or metabolic control. We found differences in pulmonary evaluation when pubertal stage was analyzed. CONCLUSIONS: The lung is functionally involved in children with T1DM. Pubertal development stage influences the evaluation of lung function.

Subcutaneous implant with etonogestrel (Implanon®) for catamenial exacerbations in a patient with cystic fibrosis: a case report.

BACKGROUND: Cystic Fibrosis (CF) is a genetic disease with equal prevalence across sexes. However, women present worse lung function with faster function decline, earlier onset of bacterial colonization, more frequent pulmonary exacerbations (PE), greater bronchial hyper-responsiveness, and higher mortality rates after puberty than men. The etiology of this gender disparity remains elusive but female hormones have been implicated in several studies. CASE PRESENTATION: A 20-year-old female with CF with severe recurrent PE, always related to the menstrual cycle since menarche, combined with lung function decline requiring multiple courses of intravenous antibiotics. We report the cessation of PE and recovery of pulmonary function following the insertion of a subcutaneous implant with 68 mg of etonogestrel (Implanon®, Organon Española S.A. Laboratories, Madrid, Spain). CONCLUSION: Our case report supports the key role of female hormones in the development of PE and in the decline of lung function in a woman with CF. When appropriate, hormonal manipulation through contraceptive methods should be considered as potential treatment.

Tos en el niño / Cough in Children

La tos en la infancia es un síntoma muy frecuente, y constituye uno de los motivos de consulta más comunes en la práctica pediátrica diaria. Las causas de tos en el niño son diferentes a las del adulto y se deben seguir las guías específicas de la edad pediátrica para su diagnóstico y tratamiento. En la mayoría de casos la causa son infecciones respiratorias banales que producen una «tos normal o esperada», pero todo niño con tos que persiste más allá de las 4 a 8 semanas se considera que tiene «tos crónica» y debe ser evaluado para descartar patologías específicas que abarcan todo el espectro de la neumología pediátrica. El tratamiento de la tos debe realizarse en función de la etiología. Con un abordaje adecuado se puede identificar la misma hasta en el 80% de los casos y el tratamiento será efectivo en el 90% de ellos. En algunos casos de «tos crónica inespecífica», tos en la que se ha descartado patología subyacente, se puede realizar un tratamiento empírico en función de las características de la tos. No hay evidencia científica que justifique el empleo de tratamientos sintomáticos que alivien la tos, como jarabes antitusivos, mucolíticos y/o antihistamínicos, ya que pueden tener efectos secundarios potencialmente graves, por lo que no se deben emplear

Cough during childhood is very common, and is one of the most frequent reasons for consultation in daily pediatric practice. The causes differ from those in adults, and specific pediatric guidelines should be followed for correct diagnosis and treatment. The most common cause of cough in children is viral infection producing "normal cough", but all children with persistent cough, i.e. a cough lasting more than 4-8 weeks or "chronic cough", must be carefully evaluated in other to rule out specific causes that may include the entire pediatric pulmonology spectrum. The treatment of cough should be based on the etiology. Around 80% of cases can be diagnosed using an optimal approach, and treatment will be effective in 90% of them. In some cases of "nonspecific chronic cough", in which no underlying condition can be found, empirical treatment based on the cough characteristics may be useful. There is no scientific evidence to justify the use of over-the-counter cough remedies (anti-tussives, mucolytics and/or antihistamines), as they could have potentially serious side effects, and thus should not be prescribed in children

Improvement of digestive health and reduction in proteobacterial populations in the gut microbiota of cystic fibrosis patients using a Lactobacillus reuteri probiotic preparation: a double blind prospective study.

BACKGROUND: Although scientific knowledge about the benefits of probiotic use in cystis fibrosis (CF) is scarce, their expectative is promising. The aim of this work was to analyze the effect of a Lactobacillus reuteri probiotic preparation versus placebo in CF patients. METHODS: A prospective, double blind, crossover and with placebo study was carried out in 30 CF patients from two Spanish hospitals. Patients were randomized in Group A (6 months of probiotic followed by 6 months of placebo) and Group B (6 months of placebo followed by 6 months of probiotic). GIQLI (gastrointestinal) and SF-12 (general) health tests were performed after probiotic and placebo intakes. Fat absorption coefficient, calprotectin, and inflammatory interleukin quantification were determined in fecal samples. Total fecal DNA was obtained and metagenomic 454-pyrosequencing was applied to analyze the microbiome composition. STATA v12 MP software was used for statistical analyses. RESULTS: Statistically significant improvement in the gastrointestinal health and decrease of the calprotectin levels were demonstrated in patients after probiotic exposure, in comparison with placebo. All CF subjects reported good tolerance to L. reuteri without secondary effects. Metagenomic analysis showed an important dysbiosis in CF gut microbiota associated with a high concentration of Proteobacteria. Probiotic intake was followed by a reduction in the total bacterial density, mostly due to a considerable reduction in the γ-Proteobacteria phylum; and an important increase of the microbial diversity with a higher representation of Firmicutes. CONCLUSIONS: Probiotics might ameliorate the dysbiosis of CF gut microbiota, characterized by a high density of Proteobacterial organisms. L. reuteri significantly decrease intestinal inflammation and increase digestive comfort.

Cough in children.

Cough during childhood is very common, and is one of the most frequent reasons for consultation in daily pediatric practice. The causes differ from those in adults, and specific pediatric guidelines should be followed for correct diagnosis and treatment. The most common cause of cough in children is viral infection producing "normal cough", but all children with persistent cough, i.e. a cough lasting more than 4-8weeks or "chronic cough", must be carefully evaluated in other to rule out specific causes that may include the entire pediatric pulmonology spectrum. The treatment of cough should be based on the etiology. Around 80% of cases can be diagnosed using an optimal approach, and treatment will be effective in 90% of them. In some cases of "nonspecific chronic cough", in which no underlying condition can be found, empirical treatment based on the cough characteristics may be useful. There is no scientific evidence to justify the use of over-the-counter cough remedies (anti-tussives, mucolytics and/or antihistamines), as they could have potentially serious side effects, and thus should not be prescribed in children.

Population structure and antimicrobial susceptibility of both nonpersistent and persistent Pseudomonas aeruginosa isolates recovered from cystic fibrosis patients.

Seventy-six Pseudomonas aeruginosa isolates recovered from chronically (n=18) and nonchronically (n=18) colonized cystic fibrosis (CF) patients (2002 to 2009) were grouped in separate polyclonal populations. International CF epidemic clones were not identified, but the high-risk clone ST274, also found circulating in Spanish hospitals, was present. Persistent isolates were more resistant to antibiotics than nonpersistent isolates.

Inhaled antibiotics for the treatment of chronic bronchopulmonary Pseudomonas aeruginosa infection in cystic fibrosis: systematic review of randomised controlled trials.

INTRODUCTION: Inhaled antibiotics are probably the safest and most effective therapy for Pseudomonas aeruginosa chronic lung infection in cystic fibrosis (CF) patients. AREAS COVERED: To summarise the available evidence, a systematic review of the three currently available inhaled antibiotics (aztreonam lysine (AZLI), colistin (COL) and tobramycin (TOB)) was performed. The three AZLI placebo-controlled studies showed that the improvements in FEV1 and mean sputum P. aeruginosa density were statistically significant better than with placebo. The two COL placebo-controlled studies involved few patients but showed that COL was better than placebo in terms of maintenance of some pulmonary function parameters. The tobramycin inhalation solution (TIS) and tobramycin inhalation powder studies showed that the efficacy of both formulations was similar but significantly better than placebo. In the comparative studies, TIS showed more efficacy than COL solution, colistin inhalation powder showed non-inferiority to TIS and AZLI was superior to TIS. EXPERT OPINION: Placebo-controlled and comparative clinical trials have shown that clinical evidence of inhaled antibiotics is very different. The choice of treatment for each individual CF patient must be based on the features of the drug (clinical evidence on efficacy and safety), the inhalation system and the patient characteristics. Development of new inhaled antibiotics will allow new end points of efficacy and therapy regimens to be assessed.

In vitro prevention of Pseudomonas aeruginosa early biofilm formation with antibiotics used in cystic fibrosis patients.

The ability of antibiotics used in bronchopulmonary infections in cystic fibrosis (CF) patients to prevent Pseudomonas aeruginosa early biofilm formation was studied using a biofilm microtitre assay with 57 non-mucoid P. aeruginosa isolates (44 first colonisers and 13 recovered during the initial intermittent colonisation stage) obtained from 35 CF patients. Minimum biofilm inhibitory concentrations (BICs) of levofloxacin, ciprofloxacin, imipenem, ceftazidime, tobramycin, colistin and azithromycin were determined by placing a peg lid with a formed biofilm onto microplates containing antibiotics. A modification of this protocol consisting of antibiotic challenge during biofilm formation was implemented in order to determine the biofilm prevention concentration (BPC), i.e. the minimum concentration able to prevent biofilm formation. The lowest BPCs were for fluoroquinolones, tobramycin and colistin and the highest for ceftazidime and imipenem. The former antibiotics had BPCs identical to or only slightly higher than their minimum inhibitory concentrations (MICs) determined by standard Clinical and Laboratory Standards Institute (CLSI) microdilution and were also active on formed biofilms as reflected by their low BIC values. In contrast, ceftazidime and imipenem were less effective for prevention of biofilm formation and on formed biofilms. In conclusion, the new BPC parameter determined in non-mucoid P. aeruginosa isolates recovered during early colonisation stages in CF patients supports early aggressive antimicrobial treatment guidelines in first P. aeruginosa-colonised CF patients.

MALDI-TOF MS improves routine identification of non-fermenting Gram negative isolates from cystic fibrosis patients.

Identification of non-fermenting Gram-negative bacteria (NFGNB) from cystic fibrosis (CF) patients is often limited. A collection of stored NFGNB isolates (n=182) recovered from CF patients over a 15 year period was examined. The routinely reported identification during this period was compared with that obtained by MALDI-TOF MS. Isolates giving discrepant identification at the genus level were further analyzed by 16S rDNA sequencing. The MALDI-TOF MS system identified 94% of the isolates, including Burkholderia cepacia and Pandoraea spp. isolates, the latter previously misidentified as other NFGNB by conventional microbiological methods. Lack of identification by MALDI-TOF MS was associated with the absence of entries in the database.

Assessment of the level of sedation in children after cardiac surgery.

BACKGROUND: There is no reference method for the evaluation of the level of sedation in children after cardiac surgery. The utility of the bispectral index and middle latency auditory evoked potentials has not been evaluated. METHODS: The bispectral index, middle latency auditory evoked potentials, Ramsay scale, and COMFORT scale were used for assessment of the level of sedation in critically ill children after cardiac surgery and other surgical procedures. The measurements with these four methods were recorded simultaneously once a day for five days. The level of sedation was categorized in two levels, moderate or deep, according to the values obtained from each method. Correlations and agreements among the methods and the best bispectral index and middle latency auditory evoked potential values that discriminated between the two levels of sedation were calculated. RESULTS: Thirty-two children after cardiac surgery were included in the study, together with eighteen children after other surgical procedures who formed the control group. In each group, the correlation and agreement between the four methods varied between moderate and good. In the cardiac surgery patients, when the level of sedation was determined by the Ramsay scale, the best values of bispectral index and middle latency auditory evoked potentials that discriminated between the two levels of sedation were 63.5 and 37.5, respectively, and these values predicted the level of sedation correctly in 84.4% of the patients with each method. CONCLUSIONS: Bispectral index and middle latency auditory evoked potentials could be useful to assess the level of sedation in children after cardiac surgery.

Analysis of bispectral index and middle latency auditory-evoked potentials parameters in critically ill children.

To study the parameters of the bispectral index and middle latency auditory-evoked potentials in critically ill children. We performed a prospective, observational study in 81 critically ill children categorized into two levels of sedation (moderate and deep) and three age groups (<1, 1-6, and >6 months). The parameters of the bispectral index and middle latency auditory-evoked potentials studied were the signal quality index, suppression ratio, total power, spectral edge frequency, electromyographic activity, and suppression ratio. Suppression rates were higher in children younger than 1 month than in older children and were also higher in deep sedation. Total power and spectral edge frequency increased with age. Electromyographic activity was higher in moderate sedation. Spectral edge frequency and suppression rates change with age and level of sedation. These variations are a consequence of the electroencephalographic structure at young ages and are secondary to the higher proportion of slow waves in deep states of sedation.

Unorthodox long-term aerosolized ampicillin use for methicillin-susceptible Staphylococcus aureus lung infection in a cystic fibrosis patient.

Staphylococcus aureus is a significant cause of pulmonary colonization in cystic fibrosis (CF) patients. The optimal strategy of therapy in chronically infected patients with this pathogen is not yet established. We report a successful long-term aerosolized ampicillin treatment of a 14-year-old girl with chronic symptomatic S. aureus lung infection.

Assessing sedation in critically ill children by bispectral index, auditory-evoked potentials and clinical scales.

OBJECTIVE: To evaluate the correlation and agreement between the bispectral index (BIS), middle latency auditory-evoked potential index (AEP index), Ramsay scale (RS) and COMFORT scale (CS) for evaluation of the level of sedation in critically ill children. DESIGN: Prospective observational study. SETTING: Pediatric critical care unit. PATIENTS: Seventy-seven critically ill children receiving sedation and mechanical ventilation. MEASUREMENTS AND RESULTS: Simultaneous recording of BIS, AEP index, RS and CS were performed once a day, for a maximum of 5 days. Two levels of sedation were categorized: light-moderate versus deep-very deep. Correlations between methods were determined using Spearman rank correlation test and the agreement using Cohen's Kappa test. The correlation and agreement between the four methods was moderate-to-good. Correlation was not found in paralyzed children. There was no correlation between the four methods and the heart rate or blood pressure, or with the type or dose of sedative medication. Receiver-operating characteristic (ROC) analysis revealed best discrimination between light-moderate and deep-very deep sedation at BIS and AEP index values of 63.5 and 33.5 when the level of sedation was classified by the RS, and at BIS and AEP index values of 67 and 37.5, respectively, when the level of sedation was classified by the CS. CONCLUSION: There is a moderate-to-good correlation and agreement of BIS and AEP index with the clinical scales in critically ill children without neuromuscular blockade. BIS and AEP index could be useful to evaluate the level of sedation in critically ill children with and without neuromuscular blockade.