Abstracts from the 23rd Italian congress of Cystic Fibrosis and the 13th National congress of Cystic Fibrosis Italian Society

s from the 23rd Italian congress of Cystic Fibrosis and the 13th National congress of Cystic Fibrosis Italian Society Naples, Italy. 22-25 November 2017 Published: 16 January 2018 S1 Effects of diet on cystic fibrosis microbiome Annamaria Bevivino (annamaria.bevivino@enea.it) ENEA Casaccia Research Center, Sustainable Territorial and Production Systems Department, Rome, Italy Italian Journal of Pediatrics 2018, 44(Suppl 1):S1 The human body contains complex communities of microorganisms that play an important role in human health. In the last decade, next-generation sequencing (NGS) was employed to decipher the structure and composition of the human microbiome, defined as the totality of microbes, their genomes and interactions in a defined environment. Cystic Fibrosis (CF) disease affects the function of a number of organs, principally the lungs, but also the gastrointestinal tract [1]. An increasing number of studies have revealed a cross-talk between the two compartiments as exemplified by intestinal complications during respiratory disease and vice versa. Although so far mechanisms through which the lung could influence the gut environment are unclear, the existence of the gut–lung axis could open up new possibilities for therapeutic approaches to respiratory diseases. A growing body of evidence suggests that the gastrointestinal (GI) microbiome in people with CF is altered. These dysbioses contribute to disease manifestations in many organs, both within and beyond the GI tract [2]. In this lecture, I highlight how changes in the intestinal microenvironment, with a particular focus on the intestinal microbiota, impact upon respiratory disease. Together with reported benefits for respiratory exacerbations, such as a reduced rate of pulmonary exacerbations and upper respiratory tract infections in patients with CF, the administration of probiotcs may globally modify the GI microbiome, thus promoting bacterial communities that modulate host immune responses [3]. Observations from clinical trials of probiotics in CF patient revealed the probiotics can ameliorate the dysbiosis of CF, reducing the proteobacterial populations in the gut microbiota, as well as decreasing gut inflammation and the number of pulmonary exacerbations.


Background
Genetic analysis of cystic fibrosis transmembrane conductance regulator (CFTR) gene is fundamental for diagnosis, prognosis and personalized therapy of cystic fibrosis (CF). Despite CF is a monogenic disease, its genetics is not easy. Consequently, the genetic analysis of CFTR gene should take under account a lot of different situations that should be specifically handled.

Materials and methods
According to current definitions, the revision underway can be defined as a Consensus document. A group of 23 experts of SIFC composes the "working group for the revision of Consensus document about genetic analysis in CF". A list of the subjects to be treated was made by the working group. A first elaboration of each subject was done by small subgroups. The overall document was revised several times by the working group, harmonized and approved as a first draft. This draft is in submission for a first evaluation and revision to the board of SIFC and of the other scientific societies involved in the management of CF. After this revision, a further elaboration by the working group will be done, aimed to the inclusion of suggestions and to the production of a second draft to be finally approved by SIFC and other scientific societies.

Results
Three basic items of the Consensus document were identified: main text, risk tables and flowcharts. The first draft of the main text is ready. Risk tables and flowcharts will be added in the next step. The main text is composed of 6 preliminary sections: list of components of the working group; premise; abbreviations; definitions; abstract; introduction. Then, there are 7 general chapters: general description of the disease; genetic counselling; general strategies of mutational search (with appropriateness); mutational search in CF; mutational search in CFTR-related disorders; preimplantation mutational search; characterization of novel, rare and uncharacterized sequence variations. The next chapter describes specific strategies of mutational search and is divided into 13 sections each concerning mutational search applied to specific practical cases. The document finishes with the following 4 chapters: quality assurance; report; databases, website and software; references.

Conclusions
The need of documents assuring a homogenous behavior of geneticists and molecular biologists performing the CFTR genetic test is particularly felt. The last update of this type of document in Italy was done in 2005. The revised Consensus document will be an updated valuable practical guide, based on solid scientific evidences, for CF healthcare professionals. The main aim of clinical microbiology is the identification of bacterial, viral, fungal, and parasitic agents that cause human diseases, to enable an optimized clinical management of patients as well as to prevent infectious diseases transmission. In the last years the clinical microbiology has changed rapidly: in addition to culture-based methods that remain irreplaceable in routine clinical microbiology, news tools have been implemented, providing improved sampling and culture strategies and culture-independent methods. The inclusion of matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) as diagnostic tool has improved the accuracy of pathogen identification and shortened the time to achieve the final results. The technological advances in non-cultured methods have unveiled a much larger human-associated microbiota than was expected. The airway microbiome has been found to be much more diverse than previously thought based on bacterial culture, and relationship between that diversity and pulmonary function has been identified. This review summarizes the state of the art of culture methods and molecular techniques in clinical microbiology laboratory. The most advanced level of newly developed techniques includes disease-based sampling kits, new culture approaches, direct pathogen detection, point-of-care (POC) testing and clinical isolate identification with MALDI-TOF MS. Despite the advent of cutting edge molecular-based (real time PCR, 16S rRNA sequencing, next-generation sequencing) and protein-based microbial identification tools, there is an enormous need to continue culture-based testing to assess susceptibility to all antimicrobials and to identify pathogens with mutations that may escape detection by new technologies. Rapid detection and identification of infectious agents in clinical specimens are mandatory to implement appropriate therapeutic measures. For this purpose it is clear that combining culture based methods and molecular techniques can contribute to clinical management of airway infection, highlighting the importance of making these two methods complementary.

Background
Cystic fibrosis (CF) is a progressive genetic disease that affects multiple organ systems. Therapy is directed to maintain and optimize nutritional status and pulmonary function, as these are prognostic factors for maintaining good lung function during the pediatric-adolescent age and key factors in survival. For infants and young children, the aim is to achieve the 50th percentile of weight and length for a healthy same-age population up to age 2 years.CF diagnosis by means of neonatal screening, ever more precocious, offers the great opportunity to correct pancreatic insufficiency since its inception, thus enabling the correct therapy to achieve a good nutritional status for all children. However, as shown by studies in different CF population, this target is not achieved for all subjects. The purpose of this report is to present the growth data of children aged up to 24 months enrolled in the Italian Cystic Fibrosis Registry, in the years 2011-2014. Materials and methods Registry data of the period 2011-2014 were evaluated. Indicators used to assess growth and nutritional status were weight/length (W/L), and length per age. Data were analyzed by age (groups: 0-6 months, 7-12, 13-18, 19-24) and for each year of data collection. The indicators were standardized with the calculation of the z-score.

Results
The Table 1 presents the data (median z-score for length per age and percentage of infants with W/L z-score >50°centile) for the years 2011-2014. With regard to weight per length, the lowest median zscores are found each year in the 0-6m age group. Recovery takes place within the first 12 months of life in most patients: for all years of study, the median z-score has passed from a negative value in 0-6m group, to values around zero in the subsequent age groups, with progressive increase of children with parameters above 50°centile and progressive reduction of children below 10°centile. Conclusions Children with CF are born smaller than the average [1] and can already present at the time of diagnosis pancreatic insufficiency that has already impaired their growth. The six month figure therefore reflects the short period of diagnosis and the beginning of appropriate therapy. Over the period considered, at the age of 19-24 months, more than half of the infants have a W/L z-score above the 50°c entile.
The increasing interest in molecular biology applied to diagnostics is the result of the deep advances in scientific knowledge in genetics made possible by the introduction and the continuous development of applied technologies. The number of laboratories using molecular biology techniques is growing rapidly and the inevitable consequence of this "demographic explosion" is the need for standardization as well as a definition of the organization of laboratories. Guidelines recommend the participation of laboratories performing genetic testing in internal Quality Control processes, as well as in Quality Control Programs (CEQs). The importance of laboratory participation in CEQ programs is recognized internationally. Currently, quality control programs for the molecular analysis of the CFTR gene are available both at the Italian and the European level. In Italy the document of the State Region Conference of July 15, 2004 established that the Institute of National Public Health (Istituto Superiore di Sanità ISS) as the competent office to evaluate the technical and professional quality of the operators through external quality controls. Participation is open to both private and public laboratories. In 2016, ISS examined 62 laboratories, each having received four DNA aliquots together with clinical and technical information. The laboratories sent back through a computerized system raw data and reports, written according to their standard format. Raw data and references were then evaluated by experts and a performance rating assigned: sufficient or insufficient.
The evaluation was done following the general evaluation criteria on the CNMR website at http://www.iss.it/tege/index.php. An analogous scheme is proposed at European level by the European Cystic Fibrosis Thematic Network, which is a project approved under the 5th framework program of the European Union. This project is coordinated by the Human Genetic Center of the University of Leuven. Currently, CF Network collaborates with EuroGentest's European Molecular Genetics Quality (EMQN) in order to improve the harmonization of external quality evaluation systems in Europe. At the moment the CF network, in order to improve harmonization of External Quality Assessment (EQA) schemes within Europe, works in close collaboration with the EuroGentest Network of Excellence and the European Molecular Genetics Quality Network (EMQN). The European Network organizes an External Quality Assessment scheme for Cystic Fibrosis, for more than 200 laboratories worldwide, some performing diagnostic testing for cystic fibrosis, others working inside diagnostic kit companies. The aim of the CF EQA scheme is to evaluate the entire analytical process, from DNA sample receipt and genotyping up to the written report with the final interpretation of the data as it is normally being sent to the clinician who requested for the genetic test. Over the years, there has been a continuous improvement in performance both in Italy and in Europe, in accordance with the aims proposed by CEQ programs for educational and continuous improvement of quality.

O1
A gene targeting approach for cystic fibrosis Silvia Pierandrei 1,2  Background Homologous replacement can be used to modify specific gene sequences of chromosomal DNA in a process called "Small Fragment Homologous Replacement" (SFHR). A wild-type small DNA fragment (SDF) is used to correct the mutated genomic target. Cellular responses and molecular mechanisms involved in SFHR are only partially understood. Different DNA repair pathways and cell cycle checkpoints, as well as specific genes (as for example Trex1), appear to mediate the cellular response. This approach can be used as therapeutic CFTR correction aimed at restoring the wild-type CFTR sequence. Recent studies have shown the possibility to expand patient-derived epithelial cells from different human tissues, through the "Culture Reprogramming Condition" (CRC) methodology. The possibility to obtain with high efficiency large amounts of primary epithelial cells with stem-cell properties from CF patients with different mutations would represent a pivotal advancement for CF gene targeting.

Materials and methods
To optimize the efficiency of SFHR, we developed a reporter based assay system where the replacement frequency is quantified by cytofluorimetric analysis following restoration of a stably integrated mutated eGFP gene in the genome of SV-40 immortalized mouse embryonic fibroblasts (MEF

Materials and Methods
In order to explore the role of gap junctions (GJs) in this rescue, cocultures (hAMSC:CFBE, 1:5 ratio) were studied for the formation of GJs, before and after silencing them with siRNA directed against connexin 43 (Cx43), a major component of GJs. Under these experimental conditions, GJ-mediated intercellular communication was studied by the transfer of lucifer yellow, CFTR expression by confocal microscopy and Western blotting, while its activity by spectrofluorimetric measure of chloride efflux.

Results
Cx43 specific siRNA mediated down-regulation of Cx43 mRNA and protein, whereas a scrambled siRNA had no effect. Co-cultures could form functional GJs that were inhibited when the Cx43 protein expression was down-regulated. Transfection of co-cultures with siRNA against Cx43 resulted in the absence of CFTR expression on the apical membrane and reduction in the mature form of CFTR (band C), paralleled by the significant decrease in CFTR-dependent chloride channel activity.

Conclusions
These results indicate that GJs are involved in the correction of CFTR chloride channel activity upon the acquisition of an epithelial phenotype by hAMSCs in co-culture with CF cells. These results may indicate that undifferentiated hAMSCs may be administered to the CF lung where they would rescue CFTR expression and function by forming GJs. Further exploration of the usefulness of hAMSCs in the rescuing of CFTR-dependent basic defects involved in the pathogenesis of CF lung disease should be studied in primary airway epithelial cells obtained from CF patients and in "in vivo" models. Background Impaired glucose tolerance and cystic fibrosis related diabetes (CFRD) negatively affect respiratory function measured by FEV1. Insulin secretory defects are risk factors for CFRD and may be also related to early lung function abnormalities that cannot be detected by spirometry. Our aim was to assess the correlation between OGTT-derived indices and the Lung Clearance Index (LCI), a more sensitive marker of early lung abnormalities than FEV1.

Materials and methods
We performed a Multiple-Breath Washout Test in 61 CF patients with a mean age of 18 years (range: 10-30) who underwent an OGTT as part of their annual screening for CFRD. From OGTT, we retrieved data on 2h plasma glucose (as a marker of glucose tolerance) and 30 min plasma insulin (as a marker of early insulin response). We compared LCI values across strata of glucose tolerance and quartiles of 30 min plasma insulin after OGTT. Moreover, we fitted multiple linear regression models to take into account the potential confounding effect of sex and age.

Results
According to the OGTT, 7 patients (11.5%) had diabetes without fasting hyperglycemia and 11 patients (18%) showed impaired glucose tolerance. Background Scientific literature about pregnancies in women with a diagnosis of cystic fibrosis (CF) is still scattered. Family planning in this group of women is becoming commoner with health improvement and survival increase. The objective of the study is to assess the maternal health status of patients with cystic fibrosis through the analysis of maternal outcomes.

Materials and methods
We conducted a longitudinal analysis of functional outcomes in pregnant women over the period 2010-2015, using records from the Italian Cystic Fibrosis Registry (ICFR). Cases were matched by age and genotype with never pregnant control subjects (1:2). Characteristics at baseline were outlined in both comparison groups. Changes in lung function and nutritional outcome (BMI) were also assessed.

Results
A total of 84 pregnancies were identified in 81 women. Median maternal age was 31 years. Pancreatic insufficiency was reported in 50% of pregnant women. 55% presented a BMI <22, 44% had a moderate-severe respiratory function (FEV 1 % predicted <70%). Chronic P.aeruginosa was present in about half of cases. Evidence of difference in median age at CF diagnosis was found between pregnant and never pregnant women (P<0.05). A decrease in lung function was found statistically significant in pregnant women over the period 6-12 months before pregnancy and 12 months after delivery (P<0.5). No significant differences were found over the same study period between pregnant and the control group in nutritional status and lung function. Only one patient died 14 months after delivery.

Conclusions
This study showed a decline in lung function over the ante-post pregnancy period in patients with cystic fibrosis. However, further investigations are needed to improve knowledge about the long period impact of pregnancy on the cystic fibrosis disease. We would like to thank Italian CF Centers, which provide data to ICFR

Conclusions
The impairment of olfaction influences the quality of life in CF patients, decreasing appetite, aggravating nutritional problems and playing a significant role in social interactions. We found a significant frequency of smelling disorders in CF patients and a major impairment of odor threshold. Our data suggest that CF olfactory impairment can result from olfactory periphery dysfunction due to either conduction problems by CRS and impaired mucociliary clearance owing to thickened mucus. So therapies improving the mucociliary clearance might recover olfactory performance in CF patients with CRS. Moreover this olfactory test could represent a new clinical tool in the follow-up of CF patients.

Background
Atelectasis is a common complication in cystic fibrosis (CF) patients. Several patients do not respond to standardized therapies. As persistence of atelectasis is associated with poor outcome, bronchoscopy can represent an alternative tool for the treatment of this complication. Few data are available for its routine use. Our objective was to evaluate the efficacy of bronchoscopy as a therapeutic tool in CF pediatric patients with atelectasis/subatelectasis not responding to conventional therapies.

Materials and methods
Since 2013 10 CF patients with atelecatsis underwent one or more bronchoscopies. For each patient, microbiological and radiological data, respiratory function (FEV1% and MMEF%) were collected. Flexible Olympus Bronchoscopy was used. Broncho-alveolar-lavage was performed and mucolytics and antibiotics were instilled. In few occasions surfactant was employed. Following the procedure, patients underwent PEP-Mask respiratory physiotherapy/non-invasive ventilation in C-PAP mode; after 48-72h RX chest was performed. Subsequently, patients were regularly followed.

Results
Ten patients (4 males) were enrolled (mean age 9.6±4.7 years, 6 months-16 years); 9/10 had pancreatic insufficiency. Indication for 1st bronchoscopy was always the radiological finding of atelectasis/subatelectasis not responsive to conventional therapy. In 7/10, radiological examinations showed a single atelectasis (2/7 due to ABPA); in 3/10 multiple atelectasis. 7/10 and 3/10 underwent a single and repeated bronchoscopy (through a mean period of 6 months) respectively. 18 procedures were performed: dornase-alpha was always used and in 17/18 patients it was associated with 7% hypertonic solution and hyaluronic acid. In 15/18 procedures colistin (14/18) or tobramicin (1/18) was instilled in case of Pseudomonas aeruginosa infection, while in 2/18 vancomycin in presence of Staphylococcus aureus. In 2/18 cases, surfactant was used for the largest extension of the atelectasis area. In patients undergoing a single procedure, complete resolution of atelectasis was observed in 6/7, while in 1/7 resolution was partial. In 3/10 patients undergoing repeated bronchoscopy, complete resolution of atelectasis was observed in 1/3 after 3 procedures, in 2/3 X-ray showed a partial improvement. Our results show that the procedure is well tolerated and can lead to radiological resolution of atelectasis even after a single procedure. Clinical benefits were evident in terms of statistically significant improvement of spirometry parameters. A national longitudinal observational study could be useful to draw up a standardized protocol. Consent for publication: Written informed consent was obtained from the patient's parents. Despite new medical therapies that have delayed the progression of lung disease with the consequent improvement of life expectancy, lung transplantation is currently the only treatment for end-stage respiratory insufficiency in patients with cystic fibrosis (CF). In this study we analyzed retrospectively our center's experience since start of the transplantation program in 1996 with focus on survival analysis.

Materials and Methods
We reviewed CF transplant database and patient charts. Survival rates were calculated and compared between four era from 1996 to 2016. Categorical data were presented as number and percentage and comparisons done using the chi-square (χ 2 ) test or Fisher's exact test. Numeric data were presented as mean ± standard deviation (SD), for normally distributed data and comparisons done using the two sample independent t-test, while non-normal numeric data were presented as median (range).

Results
In a 20-year period, 243 patients with CF were listed for lung transplantation; 125 patients (62 males, 63 females) underwent transplantation and 85 patients died while waiting for donor organs. The mean and median age at transplantation was 27 ± 8 SD and 26 (range 9-52) years, respectively. FEV1 was 27.7 ± 6.3 % predicted, 115 patients (92 %) were pancreatic insufficient and 43 patients (34 %) had diabetes CFrelated. Overall 1-year survival after lung transplantation for CF was 71.2 %, 5-year survival was 56.9 %, 10-years survival was 46.8 %, 15year survival was 31.6 % and 20-years survival was 35%. Removing CF patients who died within the first three postoperative months, the mean and median survival after transplantation were 8 ± 5.6 SD years and 7.1 years (range 3 months -20 years), respectively. We found a trend for gender difference in survival rate (CF males 7 ± 6.2 SD years versus CF females 5.4 ± 5.6 SD years, p < 0.07).

Conclusions
Lung transplantation is a well-established life extending treatment for patients with CF. Survival after lung transplantation in our centre is good, although slightly lower than the international data. Further analysis is needed to identify the multifactorial mechanisms that might have contributed to reduce outcomes at our center. Background Allergic bronchopulmonary aspergillosis (ABPA) is a pulmonary disorder, which occurs in patients with chronic lung diseases such as Cystic Fibrosis (CF). ABPA results from hypersensitivity to Aspergillus antigens which determines airways inflammation, bronchiectasis and bronchospasm. Conventional therapy consists of the use of steroids combined with anti-mycotic drugs. In conventional treatment-resistant cases, it is accredited therapy with Omalizumab, a recombinant humanized murine monoclonal antibody that binds to free IgEs, impinging on the receptors present on mastocytes and basophils. This article describes our experience on the use of anti-IgE monoclonal antibody in three CF patients with ABPA. Case Reports I, A 17-year-old male CF patient was admitted to our hospital for respiratory distress, wheezing and persistent cough. His sputum cultures were chronically positive for S.aureus and recently positive for Aspergillus fumigatus (Af). He complained of persistent dry cough and his FEV1 has progressively reduced from 75% to 40% of predicted. Total IgE were 1324 UI/ml, specific IgE for Af levels were 23.9 kUA/l and IgG antibody to Af were positive, making possible the diagnosis of ABPA. Thus, we started treatment with Prednisone and Itraconazole. However, after several weeks his general conditions worsened. We decided to start treatment with Omalizumab according to standard protocols. Symptoms and FEV1 improved after the second administration. At week 8 of therapy, his FEV1 raised to 83% predicted. After three months IgG antibody to Af were negative and after one year total IgE were <363 UI/ml. II, 11-year-old male CF patient was admitted due to chronic respiratory failure.His airways were chronically colonized from for P.aeruginosa and in the previous two months from Af. Declining pulmonary function is associated with the Resting Energy Expenditure (R.E.E.) increase from 10 to 20%. Chronic lung disease exacerbations lead to an increased R.E.E. value, which returns to basal levels some weeks after resolution of inflammation. Attempting to balance the energy gap justifies precocious and aggressive nutritional intervention, which begins in the early years and continues throughout life. However, an increase in caloric intake is not sufficient to neutralize protein-calorie need resulting from R.E.E. value growth. Non-energy intake results in reduced respiratory muscle function and decreased exercise tolerance, causing a chronic and irreversible deterioration in patient status, until death.

Materials and methods
The aim of our study is to observe and analyze the evolution of Plasmatic AminoAcids in a sample of 34 CF patients, 17 men and 17 women, treated with appropriate low-carb, high-fat, high-calorie, high-glucose diet, tailored to anthropometric values, age and gender, as well as recommended by the latest guidelines, and to assess a possible correlation with the patient's clinical phenotype nutritional state.

Results
Aminoacidogram showed that: 16/34 patients (47%) had significantly reduced Plasmatic Amino Acid levels; when considering patients with severe malnutrition, 63% presented an altered Amino Acid profile, although 30% of those with good nutritional status also had lowered levels (Table 4). These results suggest the presence of a metabolic disorder, which does not depend solely on nutritional status.

Conclusions
In conclusion, the amino acid profile seems to be influenced by different factors and somehow identifies a "metabolic disorder" that characterizes CF. Furthermore it does not appear to be outweighed only through highcalorie diet. Future studies and larger clinical samples will be needed. Consent for publication: The patients gave the consent to publish clinical data.  on the pathway from the mutated genotype to residual functional protein and, finally, to the clinical phenotype. In turn, this knowledge will improve diagnostic and prognostic capabilities, as well as therapeutic strategies. In particular, the occurrence of complex alleles could explain a part of the clinical variability found in CF, since sequence variations in cis on the same allele often affect transcript levels and/or the residual functionality of the CFTR protein. The presence of unidentified complex allele in a group of patients with the same apparent mutations, but different clinical manifestations, could explain the different severity of their conditions.

Materials and methods
In order to collect the currently available information on CFTR complex alleles, a thorough bibliographic research has been conducted. Overall, 130 papers were evaluated and all the information was collected in a summary table including the list of CFTR complex alleles currently known.

Results
About one hundred complex alleles were listed. For each complex allele one or more bibliographic sources, the occurrence in CF or CFTR-RD patients and, whenever possible, the differential effect at clinical and cellular level (compared to the effect of single variations), as well as any confirmatory studies, were reported.

Conclusions
Only for a small part of the CFTR complex alleles already known the mechanism of action and the pathogenic role has been clarified. Functional studies are mandatory to understand the role of the identified complex alleles in the different clinical manifestations of disease. The knowledge of the pathogenic effect of the complex alleles may improve diagnosis and prognosis of the disease and is expected to be crucial both to define the individual outcome and to guide the choice of the most suitable therapeutic approach for each patient.

P3
Haplotypes possibly modulating CFTR The correlation between sequence variations of CFTR (Cystic Fibrosis Transmembrane conductance Regulator) gene (genotype) and clinical manifestations (phenotype) of Cystic Fibrosis (CF) is still difficult to understand completely. CFTR gene produces a primary transcript of 6132 bases; a complex transcriptional regulation occurs on the entire locus of the gene, with a dynamic not yet completely clear. In order to study the possible mechanisms involved in the modulation of these processes, we have conceived a study that analyzes selected intragenic haplotypes, possibly modulating CFTR expression, in different populations of CF and CF-like patients.

Materials and Methods
From a previous work, we selected eight markers (six SNPs and two polymorphic traits), that we analyzed through DNA sequencing of the CFTR gene in the following seven different populations: men with idiopatic seminal hyperviscosity (ISHV) and with congenital bilateral absence of vas deferens (CBAVD), patients with CFTR related disorders (CFTR-RD), CF patients with or without pancreatic sufficiency (CF-PS and CF-PI) as target populations; general population (GC) and normospermic men (NC) as controls. We obtained RNA samples from nasal brushing of selected patients, previously genotyped. RNA was analyzed by reverse transcriptase PCR assays to study a possible induction of anomalous splicing based on the single variations composing the haplotype.

Results
Data analysis highlighted statistically significant differences in frequencies of selected haplotypes (frequency >5% in at least one population) between populations with different clinical manifestations. Our study shows that some haplotypes are more frequent in target populations compared to controls; in particular, one haplotype marks 63% of FC-PI, 33% of FC-PS, 36% of CFTR-RD, 20% of CBAVD, 13% of ISHV, 3% of GC and 6% of NC alleles. No anomalous splicing was produced by the individual sequence variations. Studies concerning the modulation of the CFTR mRNA levels by the presence of selected haplotypes in populations under investigation are still running.

Conclusions
Our results confirm that the simultaneous presence in cis of multiple variations composing particular haplotypes is characteristic of some CF and CF-like populations in respect to controls. We hypothesize that the presence of a specific haplotype, rather than single variations, can affect the processing of the primary transcript of the CFTR, reducing its level. Moreover, haplotypes may be markers of mutated alleles that may be associated with different clinical manifestations. The use of haplotypes can be the basis of broad-spectrum screening programs, with a considerable saving of costs and reduction of investigation times. The most frequent complication was liver disease (11.9%), whereas diabetes was rare (2.9%). These data are similar in subjects whose genotype shows the presence of the RF mutation in heterozygosis with F508del or with another different mutation. Subjects with more frequent (N > 30) genotypes (double heterozygosis of F508del with 3849 +10kbC→T, 2789+5G→A and D1152H, respectively) showed similar clinical features, but lung disease is more severe in subjects F508del/ 3849+10kbC→T. F508del homozygous patients showed younger age at diagnosis, meconium ileus in 12.2% of cases, and worse results as regards nutrition, lung disease and prevalence of complications.

Conclusions
Patients with RF of CFTR protein are numerous in Italy and have a milder phenotype than F508del homozygous. Lung disease is present, although it can be delayed in onset in most of these patients, but it may became severe in some.

Background
The poly-T tract, located at the junction of intron 8 (IVS-8) and exon 9, is well known. It influences transcription, and thereby reduces the amount of normal CFTR protein. The number of T residues present, 5, 7 or 9, affects the splicing efficiency of exon 9. If the T5 allele is present, a proportion of CFTR transcripts will lack exon 9, which produces a nonfunctional protein and variable CF symptoms. The TG repeats, 5' of the poly-T, also influence splicing of exon 9, and when present on the same allele as a 5T repeat, the longer the TG-repeats, the higher the proportion of CFTR transcripts that will lack exon 9. In this report we describe the finding of a new (TG)13T6 allele.

Materials and methods
In this study, we described a pregnant 36 years old woman, who has come to our attention for CF screening. The analysis has been performed by means of Next Generation Sequencing with Multiplicom MASTR™ Dx (Multiplex Amplification of Specific Targets for Resequencing) using MiSeq platform. Data analysis has been carried out by Sophia Data Driven Medicine (Sophia DDM), Sophia Genetics®. In addition, we performed a nasal brushing of the patient. RNA was extracted, reverse transcribed and amplified using standard methods. RT-PCR results were analyzed by a semiquantitative densitometric assay.

Results
The genomic analysis did not reveal any known pathogenic variant, but it showed an atypical poly-T/TG repeats genotype.

Background
The CFTR gene encodes for a chlorine-transporting protein essential for the correct hydration of epithelium in many organs and tissues. The malfunction of CFTR protein causes Cystic Fibrosis, a genetic disease with an incidence of about 1 out of 3000 births. To date, more than 2000 CFTR gene mutations are known and many of them alter the correct splicing of its mRNA. The qualitative evaluation of the effect of a splicing genetic variant is a simple procedure that by an RT-PCR followed by an electrophoretic separation can reveal the alternative splicing products. Conversely, making a quantitative and accurate evaluation of splicing products, in order to evaluate the percentage of proper residual splicing, requires complicated and expensive procedures.

Materials and methods
We evaluated the use of digital droplet PCR (ddPCR) with EVAGreen technology to quantify the amount of exon skipping due to splicing mutations. In particular, we used total RNA extracted from nasal cells brushed directly from subjects bearing different intron 9 polyT variants, including the 5T-12TG allele. The total RNA was retro-transcribed and amplified by ddPCR using a single primer pair to amplify both PCR products, 305 bp (correct splicing) and 122 bp (Exon 10 skipped).

Results
Using a single primer pair it was possible to determine different percentage of exon 10 skipping depending on the poly T tract size, as reported in literature. In particular, we obtained about 50% and 95% correct splicing from 5T/7T and 7T/7T subjects.

Conclusions
These data, even if preliminary, strongly suggest that it is possible to accurately quantify the percentage of correct residual splicing with a simple pair of primers, without the use of special probes. This methodology could be helpful especially in the analysis of composite heterozygous, in which splicing mutations are associated with other severe mutations and it is necessary to know the exact contribution of the splicing variant to the phenotype. Mutations of CFTR gene, responsible of Cystic Fibrosis (CF), cause abnormal chloride transport in the airways, pancreas, intestine, and vas deferens, leading to progressive lung and pancreatic dysfunction, elevated sweat electrolyte levels and male infertility, respectively. Chronic pancreatitis (CP) is a complication of CF due to prematurely activated trypsin within the pancreas that plays a pivotal role in triggering the activation cascade of pancreatic digestive zymogens. We studied several genes belonging to different pancreatic pathways to evaluate if mutations in such genes may represent risk factors for CP also in CF patients.

Materials and methods
We enrolled: 48 patients affected by CF and pancreatitis, recruited through a multicentric study; 35 patients with CF without symptoms nor history of pancreatitis; 80 unrelated healthy controls. We designed a panel of 8 genes involved in the intrapancreatic activation of trypsin PRSS1, PRSS2, SPINK1, CTRC, CASR, CFTR, CTSB and KRT8 and 24 additional genes classified into four groups according to the activity of the encoded protein in the "Pancreatic Secretion Pathway" (PSP). Targeted resequencing was performed using a TruSeq Custom Amplicon Low Input technology (Illumina) with the MiSeq platform. All identified variants were analyzed with bioinformatic softwares evaluating the impact of the change in amino-acidic structure on protein functionality. The variants predicted as "damaging" by at least three of these softwares were validated by standard Sanger sequencing. The study obtained the approval of local ethical committees.

Results
We found 14 patients (29.16%) with mutations in genes involved in the intra-pancreatic activation of trypsin in the group of CF patients with CP, while mutations in such genes were found in 2/35 (5.7%) patients with CF without experience of CP and in 3/80 (3.8%) healthy subjects (chi square, p<0.001). Thus, we found mutations in 12 genes of 11 patients for the PSP in the cohort of patients with CF and CP. Finally, 14/48 patients (29.2%) showed one or more mutations in the genes involved in the intra-pancreatic activation of trypsin and 6 patients had also mutations in the genes involved in PSP. Of the remaining 34 patients, 5 presented mutations in at least one gene of the other pancreatic pathways of the panel.

Conclusions
This study suggests that the trans-heterozygous association between CFTR mutations and the genes involved in the pathway of pancreatic enzyme activation may be a risk factor and predispose to the development of pancreatitis in patients with CF. Background Transient nutritional disorders are common pathological conditions during childhood, especially when children progress throughout critical steps of their development. About 25% of children with a normal physical and mental development can indeed be affected by nutritional disorders. This percentage increases to 35% in children with developmental defects. Temporal continuity and the tendency to persist might determine the evolution of the difficulties in a real food disorder [1,2]. Cystic fibrosis (CF) diagnosis can be a significant risk factor in the development of the primary relationship between caregivers and children, favouring anxiety and eating difficulties.

Materials and Methods
A qualitative research on nutritional issues and behavioural during meals was performed by the psychologist and the dietitian in CF pancreatic insufficient patients (aged 0 to 18 years). Clinical charts were reviewed and BMI centiles were recorded. In 0-2 years group weight/length centiles were collected. The study obtained the approval of local ethical committee.

Results
Seventy seven patients were selected for this study, their ages being: 0-2 years old: 8 patients; 3-5 years: 14 patients; 6-10 years old: 21 patients; 11-18 years old: 34 patients. 12% of parents reported inadequate behaviours during the main meals or described the necessity of using non canonical methods to feed their children (i.e., distraction, forced feeding); moreover 38% of parents described an anxious status correlated to their children' weight gain and referred to the moment of the weight check during follow up clinical visits.In Table 6 nutritional status and frequencies of eating disorders are showed in the different age groups.

Conclusions
The data analysed in our Centre showed that nutritional disorders are common pathological conditions among CF patients and they need to be considered carefully. Symptoms detected during the early childhood do not physiologically resolved and they have the tendency to persist during puberty and adolescence. Eating difficulty does not concern only patients with pathological BMI or at risk of malnutrition; we observed children with a normal BMI who are affected by mild or serious nutritional disorders and parents seriously worried about their child's growth, although the presence of a normal BMI. The high incidence of nutritional disorders among paediatric patients affected by cystic fibrosis highlights the importance of this topic. The medical staff with the help of the psychologist and the dietitian should carefully discuss possible strategies to avoid the onset of these conditions since the time of first communication with parents after CF diagnosis and during follow up. Background Nutrition is a main feature of the management of patients affected by Cystic Fibrosis (CF) and their nutritional status is directly related to pulmonary function and survival. Several studies suggest that a diet implemented with omega 3 fatty acid, such as Docosahexaenoic Acid (DHA), shows some anti-inflammatory effects, attenuates pulmonary and gastrointestinal symptoms, reduces the disease progression. Materials and methods DHA basic has been introduced as a nutritional supplement into the diet of 26 CF patients (12 males and 14 females) with an average age of 11,5 years affected by loss of weight and poor growth. All patients have been followed-up for 6 months monitoring weight, BMI, FEV1 and pulmonary exacerbations.

Results
We found an average weight gain of 3,5 kg in 22 patients, whereas 4 patients did not show any weight modification. Spirometry has been performed on 12 patients, 6 of whom had an improved FEV1 with an average increase of 7%, 6 did not presented any change. Only 6 cases of pulmonary exacerbations, requiring hospitalization, have been observed. Conclusions Diet integration with DHA basic can be certainly considered as a part of the management of CF patients, because of the improvement of their nutritional and respiratory status. Our study strenghtens the evidences yet published about the anti-inflammatory effects of DHA, demonstrating both an improvement of auxological parameters and pulmonary symptoms, and a decrease of exacerbation episodes and subsequently of the use of antibiotics therapy. Consent for publication: The patients gave the consent to publish clinical data.

Materials and methods
In our study we enrolled 54 CF children (23 females and 31 males; 0-12 years old, mean age 6.4 years). Periodic nasal and pharyngeal swabs were performed in every enrolled patient for 2 consecutive years every 3 months, during routine outpatient visits. For every Mc+ patient, we collected data regarding presence of nasal symptoms (rynitis, polyposis, adenoidal hypertrophy), development of relapse and relapse timing. In 19 of the 35 Mc+ patients, we performed eradication antibiotic therapy with oral amoxicillin/clavulanate course for 15 days.

Conclusions
Our study can allow us to postulate that: 1) Mc is isolated more frequently in nasal swab than in pharyngeal swabs; 2) Nasal Mc+ and nasal symptoms resulted to be associated with a more frequent and earlier relapse; 3) Oral amoxicillin/clavulanate eradication therapy for Mc is not useful to prevent microbiological and clinical relapse in CF children.
Further study on larger populations will be crucial in order to better define Mc incidence and its ideal management in CF children. Background Chronic Pseudomonas aeruginosa (PA) lung infections are the major cause of morbidity and mortality in cystic fibrosis (CF) patients. During the initial phase of infection, patients can be treated with early eradication therapy to prevent or delay chronic PA lung infections. Following eradication, lung re-infection can occur with bacteria with the identical genotype. This may be due to re-colonization from the patient's paranasal sinuses. Although the CFTR defect equally affects respiratory cells in the upper airways (UAW) and lower airways (LAW), the microbiological assessment of UAW is not included in the standard of care. The aims of the study were to evaluate, using non-invasive methods, the microbiological status of UAW of CF patients not chronically infected with PA.

Materials and methods
During the period 2014-2017, 47 patients not chronically infected by PA, according to the Leeds' definition, were evaluated. We simultaneously sampled the LAW by expectorated sputum or deep throat swab and the UAW by nasal lavage, using the Mainz method. Genotyping of PA isolated from UAW and LAW were also performed by BOX-PCR in order to assess if the UAW may represent a re-infection source.

Results
A total of 64 nasal lavages and concomitant LAW specimens were analyzed from 47 patients (median age 13 years, range 2-48). During the study period 14 out of 64 (22%) nasal lavage and 32 (50%) LAW were found positive for PA. Positive samples were found simultaneously from upper and lower airways in 10 (15.6%) out 64 specimens and they carried identical PA genotypes in the two compartments. In 4 patients PA was cultured from the UAW after successful eradication therapy. PA isolates from UAW showed high susceptibility to the majority of tested antibiotics. No resistance to colistin, meropenem or ceftazidime was found. Only 2 PA isolate showed a mucoid phenotype.

Conclusions
The presence of PA isolates in the nasal lavage suggests that the UAW can play a role in the acquisition and/or persistence of these bacteria, even after eradication therapy, this may be due to the fact that the UAW is undertreated in CF. Since the PA strains isolated from the UAW had low antibiotic resistance, the efficacy of antibiotic treatment of the paranasal sinuses should be evaluated. In summary, upper airway involvement requires prospective investigation and an interdisciplinary consensus on diagnosis and therapy. Background Recent microbiome studies suggest that the airways of individuals with cystic fibrosis (CF) are colonized by a complex microbiota, including strict anaerobes that traditionally inhabit the oral cavity. Prevotella species are consistently one of the most prevalent members of the CF microbiome, however few studies with cultural methods have investigated the antibiotic resistance of these specie in CF. Exposure to antibiotics used routinely in the treatment of CF pulmonary infection may also increase the resistance to antimicrobials even in anaerobic strains. Therefore in this study, we determined the abundance and antimicrobial susceptibility of Prevotella isolates in a cohort of CF patients at Giannina Gaslini Institute.

Materials and methods
Anaerobic and aerobic culture techniques were used to culture sputum samples from 60 (43 adult and 17 pediatric) CF patients. Sputum (20μl) were plated onto Brucella Blood Agar with Hemin and Vitamin K1 (BD) and Schaedler Kanamycin-Vancomycin Agar with 5% Sheep Blood (BD). The plates were incubated under anaerobic conditions at 37°C for 5-7 days then were examined and unique isolates were enumerated and subcultured. The specie identification were performed in duplicate by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) using a Vitek MS Spectrometer (bioMérieux). To assess the susceptibility of the obligate anaerobe isolates to antibiotics, was used the Thermo Scientific™Sensititre™ Anaerobe MIC Plate. Interpretative criteria for susceptibility to antibiotics were in accordance with Eucast.

Conclusions
Our study confirms an abundance of Prevotella in lower airway of CF patients. Regarding the antimicrobial susceptibility, previous studies on isolates from CF patients described a high resistance to amoxicillin and macrolides. Our results demonstrate that metronidazole, meropenem/imipenem and piperacillin/tazobactam are likely to be most effective against Prevotella. Further research into anaerobic pathogens, in particular Prevotella, will lead to improve treatments to reduce the severity of CF lung disease.

P14
The challenge of macroscopic sputum quality assessments with a colour grading system in Cystic Fibrosis Background Lung disease in CF is characterized by airway inflammation, persistent cough with production and stagnation of thick mucus. Sputum samples are a standard part of the clinical assessment of CF patients and are routinely collected for microbial surveillance and respiratory physiotherapy evaluation. Macroscopic sputum assessment, which consists primarily on visual examination of volume, colour, apparent viscosity and patientperceived thickness, is a subjective measurement with a potentially high variability. The aim of the study is to explore the feasibility and agreement of a 5-point colour scale for macroscopic evaluation of sputum colour.

Materials and methods
Clinically stable subjects with CF were recruited during their scheduled follow-up or the day before discharge from hospitalization. After performing a medical examination and spirometry patients were asked to withhold their usual bronchodilator therapy and start their usual airway clearance technique during which a sputum sample was collected by a respiratory physiotherapist (RPT) in a wide mouthed capped plastic container. The sample was assessed with a 5-point colour scale (from 0=lighter to 5=darker) by the patient, two RPTs and two residency doctors (RDs), which were blind to the patient identity, within ten minutes. Samples containing more than minimal salivary contamination were not evaluated. Sign test was used to test for consistent differences between patients and health professionals and within RPTs and RDs. Statistical significance was accepted at p<0.05. Sample characteristics are presented as mean (Table 7) unless specified.

Results
Nineteen subjects were recruited and a single sputum was collected per each. Six patients were excluded for excessive salivary contamination of the sample. Sputa from 13 subjects (7 females) aged 29(11) years with a FEV 1 of 48,2(20,1%) pred. were assessed. Median colour point was the same within patients, RPTs and RDs (score=4). Sign test revealed agreement in colour assessment between patients and RPTs, patients and RDs, RPTs and RDs. Patients self-assessment scores were lower than the median of RPTs and RDs in 5(38%) subjects.

Conclusions
Although sputum properties and their changes are not correlated with frequency of infection, use of antibiotics, and quality of life, a sputum colour scale could be an objective useful tool to standardize the macroscopic description of sputum on medical record and progress notes among different health professionals. The study is still ongoing to evaluate agreement on a larger sample size along with a feasibility comparison with a 9-point colour scale.

Background
In the last few years, the range of lung infections due to emerging and atypical microorganisms has been widen by the increased availability of molecular diagnostic procedures. Pulmonary nocardiosis is a well-recognized cause of disease in patients with risk factors such as immunosuppression, malignancies, and severe lung disease. However, only sporadically, Nocardia spp have been considered pathogenic in CF. In this report we describe the identification by whole genome sequencing (WGS), of N. otitidiscaviarum as the cause of chronic pulmonary disease in BV, a 14 years-old CF girl.

Materials and methods
An observational retrospective analysis was carried out from 2010 to 2016 among adult CF patients. Respiratory function indexes (FEV 1 %pred, FVC, FEV 1 /FVC) and nutritional status (BMI) were collected within three months from first NTM isolation and un to one year from the start of treatment. The prevalences of the slowgrowing Mycobacterium avium complex (MAC) and the rapidgrowing Mycobacterium abscessus complex (MABSC) species were calculated. Simple isolations and non-tuberculous mycobacteria pulmonary disease (NTM-PD) were differentiated according to ATS/IDSA 2007 Statement. The efficacy of antimycobacterial therapy was assessed through radiographic imaging, changes in pulmonary function and sputum conversions at 3, 6 and 12 months from isolation (NTM) or from the onset of therapy (NTM-PD).

Results
One hundred fifty five CF adults were screened for NTM between 2010 to 2016. Among them, 23 patients (10 males), aged 30.5 (18-56) years with a FEV1 % pred. of 70.2 were found to have at least one positive sputum culture for NTM (prevalence of 15%); 41% of NTM positivity is due to MABSC and 38% to MAC. The overall prevalence for NTM-PD was 5% (8 patients) which were treated for NTM-PD with anti-mycobacterial multidrug treatment.
There is a statistically significant difference on lung function and BMI between patients with NTM and NTM-PD. Improvements in lung function and nutritional status after treatment haven't reached the statistical significance within the NTM-PD group. The average duration of therapy was 407 (68-609) days with an adverse effects incidence of 55%. Two patients interrupted treatment for serious side effects. In NTM-PD group 78% of patients underwent chronic therapy with azithromycin versus 35% of patients with NTM infection and 38% of NTM-PD patients had more than two pulmonary exacerbations the year before NTM isolation against 29%.

Conclusions
Prevalence of NTM-positive cultures in our sample is consistent with the literature. The radiological progression of the disease in NTM-PD despite anti-mycobacterial treatment and the considerable drug toxicity in CF adult patients highlights the need for a close monitoring of this population. Background Methicillin-resistant Staphylococcus aureus (MRSA) represents one of the most important new microrganisms that interest patients with cystic fibrosis (CF); it also represents an increasing clinical problem with limited, not always effective, therapeutic options. Because chronic infection with MRSA can be associated with accelerated decline in lung function, its eradication is attempted in most CF centers today. We present two case reports in which chronic infection with MRSA shows two different evolutions. Case reports DM, male 38 years old, with a complete form of CF, chronic infection with MRSA and Pseudomonas Aeruginosa, CFRD, chronic kidney failure, severe osteoporosis, showed since the first MRSA colonization a decline in lung function, with frequent need of hospitalizations, he also developed multi-drug resistance and many adverse reactions to drug. The patient also showed a decline in life quality through the compilation of CFQ-R questionnaire. The patient had always showed a successful adherence to antibiotics therapy and an intensive chest physiotherapy, with great limitations because of multiple adverse reactions to antibiotics with severe symptoms, so it was not always simple to treat lungs exacerbations and we treated him with antibiotics tested in sputum antibiogram. In the last year he was successfully admitted to the hospital for a lung transplant. FC,16 years old, with a complete form of CF, chronic infection with MRSA and also Pseudomonas Aeruginosa, showed since the first colonization from MRSA a decline in lung function, many drug resistance including also resistance to Linezolid, presents many lungs exacerbations, with a great need to hospitalization (10 hospitalization in the last 12 months ). It was not always simple to treat lung exacerbations and we treated him with antibiotics tested in sputum antibiogram. The second patient had not always an optimal adherence to all therapies concerning CF, first of all chest physiotherapy and use of inhaled antibiotics. The patient showed a deep decline of life quality through the compilation of CFQ-R questionnaire because of recurrent hospitalization and lungs exacerbations. Both the patients showed the same clinical and bacteriological situation instead of the presence in the first patient of multiple comorbidity such as CFRD, chronic kideys failure and osteoporosis.

Conclusion
We want to underline the importance of a correct adherence and compliance to all the therapies concerning CF. This behaviour lead to an improvement of clinical conditions, but also to an improvement of quality of life. Consent for publication: The patients gave the consent to publish clinical data. In this study, we performed a time-resolved analysis of functional and taxonomic feature of lung microbiota in order to identify the key microbiome signatures associated with the increased risk of adverse outcomes and understand how microorganisms evolve in response to changes in clinical conditions along time.

Materials and methods
Twenty-one subjects with CF were enrolled in this study and followed over a 15-month period. Sputum samples were taken every three months during clinic visits. Patients were monitored during and after exacerbation. Library construction and metagenomic sequencing were performed following standard pipelines in Illumina Hiseq 2000 platform.

Results
An extraordinary resilience of the main CF pathogens to antibiotic treatment was detected. Hierarchical clustering based on microbial strain-level profiling of marker genes detected from metagenomics samples produced one cluster for each patient, containing all time points including those sampled through one or more exacerbation events. This effect was more evident for the main CF pathogens detected such as Pseudomonas aeruginosa and Staphylococcus aureus, but also for species than can be considered as emerging CF pathogens, such as Rothia mucillaginosa and Prevotella melaninogenica. Taxonomy distribution inferred from metagenomics data was quite heterogeneous both across patients and within time points of the same patient with some species that were not detectable especially during recovery, probably due to the antimicrobial treatment that might have drastically reduced the abundance of pathogen species below the revelation threshold.

Conclusions
Our results revealed that the airway colonization is highly selective and that a single strain, which started it, continues to survive and thrive in the lung of the patient. The possibility to analyze the microbiome dynamics in CF airways will permit to discover novel biomarkers involved in the pulmonary disease dynamics and can give us a set of tools to unlock the potential of microbiome-based personalized medicine in major disease areas including CF. Background Cystic Fibrosis (CF) is a potential source of distress and there is growing recognition that the child's care must focus on medical therapies and on the psychosocial well-being of patients and their families. We will describe a case report of two siblings that highlights the importance of integrate psychosocial care and standard medical practice. The case underlines the importance of a multidisciplinary approach to achieving global patient well-being.

Materials and Methods
Marco and Francesca come to our medical attention in 2014 for a "second opinion" after being taken care of at another service. They showed pancreatic insufficiency, Pseudomonas Aeruginosa colonization and frequent pulmonary exacerbations. Marco presented: FEV 1 = 70%; food allergy (milk, egg, gluten and nuts) and a severe malnutrition (weight 26 Kg, height 133 cm, BMI = 14.7) that required the placement of a Percutaneous Endoscopy Gastrostomy (PEG). Francesca presented: FEV 1 = 66%; (weight 36 Kg, height 143 cm, BMI = 17.6). They showed psychosocial problems (anxiety and depression symptoms, parental conflict after separation, poor knowledge of disease and poor adherence to treatment). The two brothers were followed up through a multidisciplinary approach that envisaged: a) medical care (hospitalization and day hospital); b) educational sessions about the use and maintenance of the devices, the management of therapy and proper nutrition; c) individual and family psychological support; d) multidisciplinary and systematic meetings of the medical team (doctors, nurses, psychologists, physiotherapists, social workers and dieticians) in order to identify common strategies. Background Patient Engagement should be considered as a key priority to innovate healthcare services delivery in the context of Cystic Fibrosis (CF), a chronic severe disease. The objectives of this study have been to identify pivotal variables associated to a greater Patient Engagement in patients with CF and to investigate the management of the disease. Materials and methods This is a cross-sectional study involving a sample of 60 patients affected by CF. The survey featured 4 validated scales. Particularly the following measures were used to indagate the level of: Patient Engagement (PHE-S), Patient Activation (PAM), Self Efficacy (SE), Positive and Negative Affect States (PANAS). Data were analysed performing Bivariate Correlations.

Results
We have conducted the survey in a sample composed by 29 males and 31 females, mean age 26,5 (±10,55); mean BMI 20,76 (±2,67); also 37,7% (n=23) featured exacerbations in the last year and 53,3% (n=32) takes from 5 to 15 drugs. We have measured the level of Patient Engagement in this sample and 20% (n=12) is in the Arousal phase: they are starting to act; 60% (n=36) in the Adhesion phase: they have learned behavioural skills to act in a proactive manner towards the disease. The last 20% (n=12) is in the Eudaimonic project phase: they have fully accepted the disease and they are good stakeholder for a positive engagement. We have measured also the Patient Activation and 50% of the sample (n=30) results to be in a phase of full activation: the patients have realised most of the needed behavioural changes. The last interesting outcome observed is a low adherence to pharmacological therapies in 55% (n=33) of the sample. Furthermore we have analysed the correlations between Patient Engagement and the aforementioned variables (Table 8).

Conclusions
The study highlights the correlations among Patient Engagement and some important variables: Patient Activation, Self Efficacy and Negative Affect States. These factors are essential to promote a positive engagement. In closing, we have to notice that the low adherence to therapies suggests to improve an effective communication between patient and clinician.  .7 years) followed at our Centre were screened by the PHQ-9 (Patient Health Questionnaire-9) and GAD-7 (Generalized Anxiety Disorders -7 item) to investigate depression and anxiety scores, respectively. The CFQ-R questionnaire was also submitted. Patients agreed to personal data treatment.

Results
The PHQ-9 score was in the normal range (0-4) in 62 patients (54.9%), between 5 and 9 (mild depression) in 33 patients (29.2%), and > 10, suggesting a moderate-severe depression in 18 patients (15.9%). Considering the GAD-7 questionnaire, we found a normal score in 63 patients (55.8%), a score suggesting a mild anxiety in 33 patients (29.2%) and a score suggesting a moderate-severe anxiety in 17 patients (15.0%). We found a negative correlation between anxiety score and all domains of CFQ-R, except for physical and respiratory domains and between depression score and all domains of CFQ-R, except for respiratory domain.

Conclusions
According to the CFF and ECFS consensus statements about a third of our population should receive a supportive intervention and psychoeducation, and about 15% of our adults should have a psychological and/or psychopharmacological intervention. When we used the HADS scale in a different group of CF subjects, we found a similar prevalence of elevated, > 10, anxiety (17.2 vs 15.4%) but a lower prevalence of elevated, >10, depression (6.6 vs 15.9%) in comparison with the data obtained in this study. We wonder if the proposed modality of screening and treatment for anxiety and depression is also applicable in Italy. Two aspects are critical: i) there is a very small number of psychologists, who works full-time in the Italian CF Centers; ii) the psychological intervention is more directed to improve the coping of patient-family with the disease during specific life times (diagnosis, growth and nutrition, adolescence, self-care and independence, worsening of disease and decisions on organ transplantation, end of life issues).

P23
Cystic fibrosis is a known disease? We present an observational study made to know how cystic fibrosis (CF) is known by two samples: nurses population and society.

Materials and Methods
Every sample was investigated using a questionnaire. It consists of 18 multiple choise questions, submitted to two samples: the "Nurses" sample, consisting of 50 nurses belonging to different wards not concerning CF and the "Society" sample, including 50 subjects who don't study or work in the healthcare field. Questions was aimed to investigate the CF knowledges about incidence, symptoms, diagnosis, treatment and life expectancy and to understand if the samples know the existence of the CF Center and which healthcare professionals are involved in the patients care.

Results
The results suggest that: 18% of nurses surveyed and 68% of subjects belonging to the "Society" sample don't know CF; 72% of nurses interviewed and 90% of subjects belonging to the "Society" sample don't know people with CF; 2% of nurses and 48% of the "Society" sample say that CF is not a genetic disorder; 70% of subjects in both samples think that CF is a very rare disease; 28% of subjects belonging to the "Society" claim that patients with CF are contagious; 22% of surveyed nurses and 35% of the "Society" sample don't know the organs involved, symptoms, diagnosis and treatments; very often CF is being exchanged for a neurodegenerative/neuromuscular disease; 64% of the two sample don't recognize that life expectancy is greatly improved by the advancement of scientific research, the implementation of new treatments, and the improvement of care processes. Almost 58% of the "Society" sample dont' recognize the existence of the CF Center, which is dedicated to the care and assistance .14% of nurses interviewed and 22% of subjects belonging to the "society" sample doesn't know that there is a CF Center also in Palermo; 14% of nurses surveyed and 36% of subjects belonging to the "Society" sample are not interested in learning more about CF.

Conclusions
The results obtained show that this disease it's not so well-known among two sample due to poor media involvement and also probably due to poor university and post-based training on. These results must make us reflect on the role that healthcare professionals have in educating society about the knowledge of this pathology. It's necessary to increase the awareness of the scientific world and not to give the right attention to CF. Background Rigorous systematic reviews have found that in developed countries, adherence to long-term therapies among patients with chronic disease is only 50% [1]. Cystic fibrosis (CF) is a genetic disease that affects several organs, including the respiratory, digestive and reproductive systems, but above all is a "time-consuming" disease. CF requires complex medical management throughout the patient's life. The therapeutic load becomes increasingly intense with the course of the years and with the worsening of the disease and it is known that adherence to therapies dramatically decreases since adolescence, if not before. Adherence between children and adults with CF varies from 38% for chest physiotherapy to 50% for inhalation therapies and dietary supplements [2] and 68% for the new drug Kalydeco© [3].
Talking about adherence to therapy during an outpatient visit is not always easy and it is not always done. Riekert et al provided a questionnaire to US CF Foundation accredited CF centers, showing that only 8% of respondents measure adherence, and only 64% discuss about adherence during the outpatient followup [4]. The most common used strategy to increase adherence of CF patients is disease education.

Materials and methods
We analyzed the disease awareness of 79 patients older than 8 years followed at the Regional Support Center for CF in Brescia. For each age group (8-11, 12-17,>18) an electronic questionnaire (Google Forms©) was created. Questionnaires were subdivided into thematic sections, each provided with several multiple choice or short answer questions. The topics covered are as follow: knowledge of the disease and self-management, knowledge about health, infection control, chest physiotherapy, lifestyle, psychosocial well-being, sexual education, professional/educational plan and social care.

Results
Results are reported in Table 9.

Conclusions
Below are the key points of educational intervention based on the outcome of our patients' needs: -Patient education starts at the time of diagnosis and must be integrated into each stage of the evolution of the disease. -All members of the multidisciplinary team must contribute to patient education. -Instructing the patient to self-management of the disease, adapting the information and therapy according to his/her needs. -Developing therapeutic plans in collaboration with all members of the team and patients. -Encourage adherence to the therapeutic regimen.

Background
Drug reaction with eosinophilia and systemic symptom (DRESS) syndrome is a rare life-threatening hypersensitivity reaction with an estimated incidence between 1:1000 and 1:10000 drug exposures occurring 2-6 weeks after a causative therapy. DRESS syndrome is characterized by skin eruption, fever, elevated liver enzymes and leukocytosis with eosinophilia. Renal, pulmonary, cardiologic and neurologic manifestations may be also associated. This condition may be fatal if unrecognised, especially in patients with hepatic failure. Diagnosis may be difficult, particularly in paediatric and cystic fibrosis (CF) patients in which this condition is rarely described. Case report A 4-year-old girl with CF admitted to our hospital for a pulmonary exacerbation was treated with piperacillin-tazobactam (150 mg/kg/die) and tobramycin (10 mg/kg). After 14 days she developed fever (up to 40°C) and a diffuse maculopapular erythematous rash. Generalized polyadenomegaly and hepatomegaly were also detected. Laboratory investigations revealed a marked increase in C-reactive protein, lactate dehydrogenase, aspartate and alanine aminotransferse and a significant alteration in coagulation tests. On the 18 th of hospitalization hypereosinophilia was also observed (Table 10). Autoimmune, infective and hematologic detection tests were negative. Due to the clinical features the diagnosis of DRESS syndrome was performed and the ongoing antibiotic treatment discontinued. Without others therapies, clinical resolution was progressively achieved. Conclusions CF patients have an increased incidence of adverse drug reactions due to the heavy therapeutic burden they are exposed in order to prevent progressive lung damage. To our knowledge, this is the first report of DRESS in a paediatric patient with CF. Due to its highly variable clinical presentation, DRESS may be difficult to diagnose as it can mimic infections, autoimmune diseases, hematologic and lymphocytic disorders. Our case highlights the importance to be aware of this condition as the removal of the offending drug is the main management step for the resolution of the clinical and laboratory features. Consent for publication: The authors confirm written informed consent was obtained from the patient's parents.

P26
The support of the thoracic ultrasound in the reexpansion techniques of a lung parenchymal atelectasic area: case report Matteo Giuliari 1    To report the clinical course of a preterm infant with diagnosis of cystic fibrosis. Case report A female infant was born via cesarean section for podalic presentation secondary to premature onset of labor, weighing 1140 g at 28 weeks and 3 days of gestational age, with Apgar scores of 3 and 7. Her mother had attended many prenatal consultations with negative serology for vertical infections and normal obstetrical ultrasound. Immediately after birth, the infant was referred to the neonatal critical care unit due to early respiratory distress, abdominal distension and frequent regurgitations. Serial abdominal radiographs revealed air fluid levels.
Initially she was stabilized with nasogastric and rectal decompression. In the tenth day of life the patient underwent laparotomy surgery for intestinal occlusion/ meconium ileus. Ileal resection and two ileostomies were performed, followed by reconstruction of the bowel transit at 17 days of life. She had late neonatal sepsis during the hospital stay, requiring prolonged antibiotic therapy. She was intermittently ventilator dependent, secondary to multiple surgical procedures and sepsis. Two transfusions of red blood cells were performed. Parenteral nutrition was gradually transitioned to infant formula and pancreatic enzyme supplements with satisfactory weight gain. On the basis of her complicated clinical course, Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) mutation analysis had been performed using the Cystic Fibrosis Genotyping Assay (Celera-Abbott). Mutation analysis showed homozigosity for the common CFTR mutation [delta]F508. The sweat chloride test was carried out with a positive result (89 mEq/L). At three months her chest X-ray revealed left pulmonary hyperinflation and right pulmonary consolidation compatible with pneumonic disease. After culture, and based on the microorganism isolated from the upper respiratory tract secretion (Klebsiella pneumoniae and Escherichia coli), antibiotic therapy was prescribed. After prolonged hospitalization, she was discharged home, but after few days she was hospitalized again for bronchiolitis. Laboratory tests for respiratory syncytial virus was positive. During hospitalization, due to progressive worsening of the respiratory pattern, noninvasive ventilation (HFNC) was applied for seven days. She was discharged home on room air and she has not been hospitalized anymore. Ongoing she receives continuous care from paediatric cystic fibrosis centre, has a good nutritional status taking infant formula with pancreatic enzymes supplementation and performs PEP-mask physiotherapy with benefit.

Conclusion
This case report highlights the importance of advanced neonatal care with cystic fibrosis therapy, and in particular the joint management of two care units and of appropriate treatment of bronchiolitis in CF patient. Consent for publication: Patient's parents signed consent for the publication of patient's clinical data.

P29
Efficacy and safety of Kalydeco Ivacaftor is a CFTR potentiator that corrects chloride transport in most CFTR class III mutations affecting channel gating, some class IV mutations exhibiting abnormal gating or conductance defect and several missense mutations associated with defects in protein processing or function. Ivacaftor is globally approved for class-III and R117H CFTR mutations and, in the United States, for further 28 mutations (23 missense mutations and 5 splicingaffecting mutations) resulting in partially functioning CFTR protein. The 3272-26A→G variant is a class V mutation. Phenotype associated to this variant is variable, with either mild and severe lung disease described; pancreatic sufficiency (PS) is frequent. The 3849+10kbC→T variant is also a class V mutation. Phenotype of patients having the 3849+10kbC→T variant is highly variable, with lung disease delayed in onset in most of these patients, but then become severe in some. The majority of patients have PS, borderline or normal sweat chloride (SCL) values and sometimes male fertility. We describe the effectiveness and safety of Ivacaftor in two women with CF with the CFTR genotype 3272-26A→G /E585X and 3849+10kbC→T /F508del, respectively, with severe lung disease.

Materials and methods
Ivacaftor 150 mg bid was started after a request of compassionate use was approved by the local Ethics Committee (EC). Before starting the therapy with Ivacaftor, a basal assessment was performed (lung function, 6-minute walking test (6MWT), CFQ-R questionnaire, sweat test, biochemical tests, microbiology test, ophthalmologic evaluation). Data about lung function, 6MWT, antibiotic therapies were retrieved by the local database. Subjects were admitted as outpatients once a month.

Results
Main results are summarized in Table 11. The administration of Ivacaftor resulted in improvement of lung function, 6MWT, CFQ-R scores and a decrease of antibiotic therapy in both patients. Sweat chloride decreased only in the patient with 3849 +10kbC→T/F508del genotype, whereas nutrition and sputum microbiology were unchanged. No safety concerns were registered for both patients.

Conclusions
These cases expand our knowledge about potential benefits of Ivacaftor for CFTR mutations with RF, including certain mutations affecting splicing. The clinical and functional improvement in subjects with severe lung damage suggests that Ivacaftor could alter the natural history of the disease, when an adequate target is present, as a CFTR with altered gating function or defective conductance or a quantitative partial defect of CFTR, as in presence of mutations affecting splicing.

Consent for publication:
The patients gave the consent to publish clinical data. Background Cystic fibrosis (CF) is a life-limiting disease that is caused by defective or deficient CF transmembrane conductance regulator (CFTR) protein activity. Phe508del is the most common CFTR mutation with high rates of premature death. It is a multisystem disease that is characterized by pancreatic insufficiency and chronic airway infections associated with loss of lung function, repeated pulmonary exacerbations. Lumacaftor is a CFTR corrector that has been shown in vitro to correct Phe508del CFTR misprocessing and increase the amount of cell surface-localized protein. Ivacaftor is an approved CFTR potentiator that increases the open probability of CFTR channels. The TRAFFIC and TRANSPORT trials were two phase 3, multinational, randomized, double-blind, placebo-controlled, parallel-group studies in which Lumacaftor was orally administered combined with Ivacaftor.

Materials and methods
Between March 2016 and September 2017 at the Regional Reference Center of Palermo for the CF, a total of 7 patients in critical need (female:5, male:2) aged 17-44 years (mean 26,6) with CF, who were homozygous for the Phe508del, were enrolled and assigned to receive Lumacaftor/Ivacaftor. At first we evaluated auxometry (weight, hight and BMI), respiratory function (%FEV 1 and %FVC), ECG, liver, pancreatic and renal function, sweat test, respiratory infections and CFQR. Patients agreed to personal data treatment.

Results
Significant improvements in the percentage of predicted FEV1 were seen, the respiratory function got better in term of FEV1 an FVC in this 18 month of observation: FEV1 + 11,4 % after 3 months (T3), + 12,2% after 6 (T 6 ), +9,8 one year later (T 12 ) and + 6,8 at the end of the observation (T 18 ). Also the FVC increased of 10,4 %, 13%, 9,3% and 9,5% respectively after 3, 6, 12 and 18 months (T 3 , T 6 , T 12 , T 18 ). We observed the improvement of the mean-BMI: +0,16 percentage points after six months (T 6 ) and 0,21% at the end (T 18 ) (Table 12). We recorded nine cases of pulmonary exacerbations and any side effects at the end, except for a patient who suffered of a subopacity of the crystalline. The sweat test results were not reliables. The quality of life was investigated by the Cystic Fibrosis Questionnaire-Revised (CFQ-R) respiratory symptom scale, that revealed an improved tolerance to effort and a decreased night bronchorrea.

Conclusions
The discovery of this therapeutic strategies gave new prospective of life to the patients who carried a Phe508del mutation in CFTR. Our data demonstrated, according to the international literature, that combination therapy of Lumacaftor/Ivacaftor improves the respiratory function and the quality of life of these patients.
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