Abstracts from the 24th Congress of the Italian Society of Cystic Fibrosis and the 14th National Congress of Cystic Fibrosis Italian Society

s from the 24th Congress of the Italian Society of Cystic Fibrosis and the 14th National Congress of Cystic Fibrosis Italian Society Salerno, Italy. 8 -10 November 2018 Published: 16 April 2019 CFTR/CELLULAR STRESS A1 Myriocin potential as a phenotype-modifying therapeutical in Cystic Fibrosis Natalia Cirilli, Alessandra Mingione, Emeranziana Ottaviano, Fabiola Bonezzi, Michele dei Cas, Matteo Barcella, Silvia Tedesco, Tatiana Armeni, Anna Caretti, Rita Paroni, Elisa Borghi, Paola Signorelli Cystic Fibrosis Care Centre, Mother-Child Department, United Hospitals, Ancona, Italy; Italian Research Cystic Fibrosis Foundation, Verona, Italy; Biochemistry and Molecular Biology Laboratory, Health Sciences Department, University of Milan, Milan, Italy; Microbiology Laboratory, Health Sciences Department, University of Milan, Milan, Italy; Clinical Biochemistry and Mass Spectrometry Laboratory, Biochemistry and Molecular Biology Laboratory, Health Sciences Department, University of Milan, Milan, Italy; Dipartimento Scienze Cliniche, SpecialisticheDI.S.C.O., Università Politecnica delle Marche, Ancona, Italy Correspondence: Natalia Cirilli (natalia.cirilli@ospedaliriuniti.marche.it) Italian Journal of Pediatrics 2019, 45(Suppl 1):A1 Background Cystic Fibrosis (CF) is caused by a variety of mutations of the CFTR ion channel, with deletion of phenylalanine 508 (F508del) representing approximately 70% of patients. This mutation induces a proteinopathy, characterized by aggregates of mutated and unfolded proteins, hyper-inflammation, impaired trafficking and altered metabolism at cellular level. CFTR dysfunction has primarily a devastating effect on lungs, causing chronic inflammation and recurrent unresolved infections. CF patients develop severe comorbidities: male infertility, biliary cirrhosis, osteopenia/osteoporosis, renal failure, diabetes (50% of adult patients), malabsorption and increased synthesis of cholesterol and its accumulation in liver but also in other tissues, dyslipidemia with increased plasma triglycerides, pancreas fibrolipomatosis, hepatic lipogenesis and steatosis. Lung chronic inflammation and infection have been extensively associated to the lipotoxin ceramide, core component of membrane lipids. such as sphingomyelins and glycosphingolipids. We previously demonstrated that the sphingolipid synthesis inhibitor Myriocin is able to reduce inflammation and to ameliorate defense response against bacterial and fungal infection. Materials and methods 43 CF patients with selected genotypes were enrolled. Monocytes from peripheral blood samples were extracted and also clinical data were collected. Results We here demonstrate the mode of action of this molecule. First, we show in IB3 cell line, that Myriocin is an effective inducer of autophagy which is defective in CF proteinopathy and can sustain pathogen © The Author(s). 2019 Open Access This artic International License (http://creativecommons reproduction in any medium, provided you g the Creative Commons license, and indicate if (http://creativecommons.org/publicdomain/ze clearance (xenophagy). Second, we demonstrated that Myriocin activates key transcriptional factors, TFEB, FOXO1a and PPARgamma involved in autophagy induction, mitochondria genesis and activity, energy production and lipid mobilization and consume. We proved that Myriocin significantly increases the transcription of downstream genes regulating fatty acids entry in mitochondria (CTP1a and 1b; FATP) and their oxidation (ACAD L). Next, we showed that Myriocin drammatically reduces pathological accumulation of lipid unorganized deposits in IB3 cells. By Mass spectrometry we observed that inhibition of sphingolipid synthesis causes a reduced content of non sphingoid-base containing lipids, such as glycerol lipids and cholesterols. We then proved that Myriocin is able to change the transcriptional profile of IB3 treated cells, by gene sequencing analysis, enhancing the transcription of genes involved in lipid transport and consume and energy metabolism that resulted partially downregulated in CF. Finally, Myriocin treatment of peripheral blood monocytes from CF patients, infected with A. fumigatus conidia, significantly increases their pathogen killing ability. Conclusions Myriocin is a potent modifier of pathological gene expression profile in CF and a potential therapy for CF related infection. A2 miR-125b/NRF2/CFTR circuitry: a pilot study on oxidative stress in cystic fibrosis Maria Pelullo, Francesca Megiorni, Giuseppe Cimino , Antonio Pizzuti, Serena Quattrucci, Claudio Talora, Samantha Cialfi Department of Molecular Medicine, Sapienza University of Rome, Rome, Italy; Department of Pediatrics, Sapienza University of Rome, Rome, Italy; Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy Correspondence: Samantha Cialfi (samantha.cialfi@uniroma1.it) Italian Journal of Pediatrics 2019, 45(Suppl 1):A2 Background In physiopathology of Cystic Fibrosis (CF), oxidative stress implications were recognized and widely accepted. The CFTR defects in ionic transport disrupt the intracellular redox balance causing CF classical pathologic hallmarks. Therefore, oxidative stress together with detoxification genes and miRNA aberrant expression could be associated with clinical outcome. Materials and methods Patients (n=8) with CF diagnosis with the same mutation (F508del), but various clinical status, compared to healthy individuals. After nasal brushing, we extracted total RNA of patients for consecutive expression analysis of CFTR, NRF2 and its targets as well as miR-125b, by quantitative real time PCR (qPCR) using TaqMan chemistry. Written informed consent was obtained by each participant to the study. le is distributed under the terms of the Creative Commons Attribution 4.0 .org/licenses/by/4.0/), which permits unrestricted use, distribution, and ive appropriate credit to the original author(s) and the source, provide a link to changes were made. The Creative Commons Public Domain Dedication waiver ro/1.0/) applies to the data made available in this article, unless otherwise stated. Italian Journal of Pediatrics 2019, 45(Suppl 1):43 Page 2 of 26 Results In this pilot study, we analyzed the existence of a correlation among CFTR, miR-125b, NRF2 and oxidative stress genes in a representative number of CF patients. In CF patients with chronic P. aeruginosa (PA) lung infection, the mRNA levels of the NRF2 gene were significantly down-expressed and showed a direct correlation with CFTR gene expression. Interestingly, the NRF2 downmodulation was accompanied by an induced expression of miR-125b. On the contrary, PA negative CF patients showed NRF2 expression levels more similar to those of healthy individuals, with a reduction of miR-125b, even if CFTR levels remained downmodulated. These data are in line with the expression of the oxidative stress target genes NQO1 and GST-T1. Moreover, we found that PA positive patients with a FEV1>60% showed the expression of HMO1, another NRF2 target gene, higher than those with a lower FEV1. Conclusions The evidence of a CFTR, NRF2 and miR-125b impaired network as oxidative stress response in CF patients, prompt us to hypothesize that these molecular mechanisms could explain the wide CF phenotypic variability as an additional control level over the CFTR gene mutations. Furthermore, this study may allow the discovery of potential therapeutic targets in order to improve CF patient’s quality of life by screening the expression of these oxidative stress factors as prognostic markers.


Background
Cystic Fibrosis (CF) is caused by a variety of mutations of the CFTR ion channel, with deletion of phenylalanine 508 (F508del) representing approximately 70% of patients. This mutation induces a proteinopathy, characterized by aggregates of mutated and unfolded proteins, hyper-inflammation, impaired trafficking and altered metabolism at cellular level. CFTR dysfunction has primarily a devastating effect on lungs, causing chronic inflammation and recurrent unresolved infections. CF patients develop severe comorbidities: male infertility, biliary cirrhosis, osteopenia/osteoporosis, renal failure, diabetes (50% of adult patients), malabsorption and increased synthesis of cholesterol and its accumulation in liver but also in other tissues, dyslipidemia with increased plasma triglycerides, pancreas fibrolipomatosis, hepatic lipogenesis and steatosis. Lung chronic inflammation and infection have been extensively associated to the lipotoxin ceramide, core component of membrane lipids. such as sphingomyelins and glycosphingolipids. We previously demonstrated that the sphingolipid synthesis inhibitor Myriocin is able to reduce inflammation and to ameliorate defense response against bacterial and fungal infection.

Materials and methods
43 CF patients with selected genotypes were enrolled. Monocytes from peripheral blood samples were extracted and also clinical data were collected.

Results
We here demonstrate the mode of action of this molecule. First, we show in IB3 cell line, that Myriocin is an effective inducer of autophagy which is defective in CF proteinopathy and can sustain pathogen clearance (xenophagy). Second, we demonstrated that Myriocin activates key transcriptional factors, TFEB, FOXO1a and PPARgamma involved in autophagy induction, mitochondria genesis and activity, energy production and lipid mobilization and consume. We proved that Myriocin significantly increases the transcription of downstream genes regulating fatty acids entry in mitochondria (CTP1a and 1b; FATP) and their oxidation (ACAD L). Next, we showed that Myriocin drammatically reduces pathological accumulation of lipid unorganized deposits in IB3 cells. By Mass spectrometry we observed that inhibition of sphingolipid synthesis causes a reduced content of non sphingoid-base containing lipids, such as glycerol lipids and cholesterols. We then proved that Myriocin is able to change the transcriptional profile of IB3 treated cells, by gene sequencing analysis, enhancing the transcription of genes involved in lipid transport and consume and energy metabolism that resulted partially downregulated in CF. Finally, Myriocin treatment of peripheral blood monocytes from CF patients, infected with A. fumigatus conidia, significantly increases their pathogen killing ability.

Conclusions
Myriocin is a potent modifier of pathological gene expression profile in CF and a potential therapy for CF related infection.

Background
Intestinal Current Measurements (ICM) have been utilized for several decades for measuring ex vivo CFTR function as support for difficult diagnosis and for detection of effects of CFTR modulators in preclinical studies, as well as for monitoring therapies of patients affected by cystic fibrosis (CF). Transport of both chloride and bicarbonate is mediated by CFTR, a transmembrane channel permeable to anions. Defective bicarbonate transport plays a role in CF and CFTR related disorders leading to pancreas and lung damage. Selective defect of bicarbonate transport was previously reported for a group of CFTR variants in patients with pancreatitis and no lung disease (LaRusch et al., PLOS Genetics 2014; Park et al., Gastroenterology 2010). We set up ICM in appropriate conditions for detecting selective CFTR mediated/regulated bicarbonate transport defect.

Materials and methods
We performed ICM in rectal biopsies of 8 non CF subjects, 8 CF patients and 8 patients with unknown diagnosis referring to the Cystic Fibrosis Center of Verona for CFTR function measurements. ICM were performed according to both the procedure of European Cystic Fibrosis Society ICM Standard Operating Procedure and variations using Clfree or HCO 3 free medium and carbonic anhydrase/ transport inhibitors in non CF controls vs. wide spectrum of CFTR mutations including CFTR bicarbonate defective mutants.

Results
Normal tracings for both anions were observed in non CF controls while abnormal tracings in terms of Cl and bicarbonate transport were obtained in all CF patients except one with Q1463H/R553X/ D806G CFTR mutation with pancreatitis with pancreas divisum and lung infection (Staphylococcus aureus methicillin resistant). In subjects with inconclusive diagnosis in the presence of disseminated bronchiectasis we found normal tracings for both bicarbonate and Cl except in two patients with selective defect of bicarbonate transport in the presence of I807M+/-, R668C+/-, CFTR genotypes and Cl sweat values of 18 and 23 mmol/L and 28 and 68mmol/L, respectively (sweat test by Gibson and Cooke performed twice). None had clinical history of pancreatitis in addition to lung disease, evaluation is in progress.

Conclusions
Selective defect of bicarbonate transport might be responsible for clinical phenotypes milder than classical CF, including not only pancreas, but also lung disease. It can be detected by bioassays such as ICM that is very relevant for understanding functional impact of CFTR variants and their response to CFTR modulators. Informed consent was obtained from all subjects. Study supported by: Italian Cystic Fibrosis Research Foundation grant#7/2016, Lega Italiana FC-Associazione Veneta Onlus.

Background
Although the median age of survival is increased in Cystic Fibrosis (CF) patients, females continue to have a lower median age of survival compared with males. Several lines of evidence indicate that estrogens play a relevant role in the pathophysiology of CF. In vitro studies have shown that 17β-estradiol inhibits airway surface liquid production, and in vivo studies in CF animals have demonstrated that 17β-estradiol enhances inflammation, increases mucus production and favors the conversion from the non-mucoid to the mucoid form of P. aeruginosa. The aim of this study was to investigate the effects of 17β-estradiol and GB-1877 on calcium-dependent chloride channel (CaCC) currents in human bronchial epithelial cells carrying the F508del-CFTR mutation both in homozygosis and in heterozygosis.

Materials and methods
Perforated patch clamp experiments were performed on single cells using two immortalized cell lines, CFBE (F508del/F508del) and IB3-1 (F508del/W1282X). Gramicidin (10 or 20 μM) was added to the electrode solution to reach the whole cell configuration. Electrical stimulation protocol consisted in square voltage steps from -80 to +80 mV with 20 mV interval and 800 msec duration.

Results
The presence of 17β-estradiol significantly reduced the CaCC currents, both in basal conditions and in the presence of ATP (100 μM).
The addition of GB-1877 (10 μM) completely restored the currents abolished by 17β-estradiol, in basal conditions and after stimulation with ATP in both CFBE and IB3-1 cells. GB-1877 had a strong, direct action on membrane current density, which significantly increased more than 4-fold in both cases. The membrane current stimulation produced by GB-1877 was further enhanced by the addition of ATP. CFBE cells incubated for 24 hours with 3 μM VX-809 (a CFTR corrector) and then acutely stimulated with VX-770 (a CFTR potentiator) in the presence of forskolin, showed an increase of chloride currents

Background
In the last few years, the field of therapeutic gene editing is rapidly developing with the emergence of CRISPR/Cas9 system. This developing technology represents a new opportunity for the treatment of many genetic diseases, including Cystic Fibrosis. However, despite the great potential of this technique, the low frequency of genome editing events, along with the off-target events generation, remains a limitation in the clinical application of this powerful method. In an attempt to develop methods to improve the efficiency of genome editing events, we set up a protocol for quantifying these editing events using the droplet digital PCR (ddPCR) technology.

Materials and methods
For this purpose, the ddPCR has already been performed using labeled probe (i.e. fam, hex or vic) technology. Our method, based on EVAGreen technology, uses two primer pairs: one primer pair amplifies a reference sequence distant from the Cas9 cleavage site (reference assay); and a second primer pair include the Cas9 cleavage site (Editing assay). When a reporter expression cassette, included in the HDR construct, is inserted in the Cas9 cleavage site, the amplification of the editing assay is blocked as the amplicon length far exceeds 1000 bp, resulting in a reduction of positive droplets. At this point, comparing the positive droplets from the two assays (reference and editing one) is possible to calculate the percentage of edited allele. The quantification of the genome editing events, using the ddPCR assay, was performed on gDNA of 293T cells transfected with two constructs: 1) one home-made HDR construct (bearing a reporter cassette expressing the puromycin and the yellow fluorescent protein between the homology arm region including the CFTR exon 11, 2) one construct expressing the Cas9 enzyme and the gRNA targeting the genomic sequence of F508del mutation.

Results
The combination of the reference and editing assay resulted in a fine quantification of gene editing events. Indeed, we were able to accurately quantify the edited alleles in 293T cells transiently transfected and after puromycin selection.

Conclusion
Finally, we demonstrated that the Droplet Digital PCR assay, based on EvaGreen technology, represent an easy and cost effective assay to evaluate the genome editing efficiency, showing in our case, a genome editing efficiency up to 90% of alleles in puromycin selected cells.
We thank Ministero della Salute (Rome, Italy) L.548/93 for the regional research funding quote 2013-2015

A6
The role of functional studies in the diagnosis and treatment of cystic fibrosis: comparing the case of the G970D and G970R Background More than 2000 CFTR mutations have been identified so far, but only few of them are clearly defined as CF-causing based on functional studies. We present a case of a rare mutation, the G970D, that has been shown using transfected cDNA in HEK293 cells to be sensitive to ivacaftor. However, a similar missense mutant, G970R in the same codon, was found to be sensitive to potentiators in vitro but not in vivo due to splicing alteration. Thus, we used several basic research methodologies to evaluate if this patient was eligible for treatment with ivacaftor. Materials and Methods 1) nasal epithelial cells (HNEC) were collected from patients to evaluate the effect of mutations on splicing by RT-PCR assay, 2) HNEC were expanded and polarized for evaluation of CFTR function by ussing chamber system 3) the use of a minigene system was used to confirm in vitro the splicing pattern.

Results
Firstly, we used in silico tool to predict the fisio-pathological effect of mutations, confirming that the G970R completely abolishes the canonical 5' splice donor site of exon 17 resulting in a likely retention of intron 17. On the contrary, the G970D predicted not to affect splicing. This prediction was confirmed by RT-PCR analysis of mRNA extracted from HNEC cells and from in vitro minigene assay. Finally, the functional behavior of CFTR, from HNEC bearing the G970D, was evaluated by short-circuit recordings. The cells responded to cAMP agonist with an increase in trans-epithelial current and this current was nearly doubled by stimulation with ivacaftor. Moreover, in cells that were also incubated with VX-809 (1 μM) for 24 hours, CFTR function was significantly enhanced, with a proportional increase of both cAMP-and potentiator-dependent responses.

Conclusion
Our results show that the G970R actually disrupts the RNA splicing thus leading to a severely altered CFTR protein. This event explains the lack of success of treatment with potentiator. In contrast, the G970D does not alter RNA splicing. The resulting G970D mutation affects the gating and trafficking of the CFTR channel that can be targeted with VX-770 and VX-809. These results represent the evidence needed to justify the treatment of the patient with these drugs. Finally, our study is an interesting example of the precision medicine approach by emphasizing the role of appropriate in vitro studies, in this case focused on RNA analysis, to fully characterize the effects of rare CFTR mutations. We thank Telethon Foundation (TMLGCBX16TT) and Ministero della Salute (Rome, Italy) L.548/93 for the regional research funding quote 2008-2015.

Background
Cystic Fibrosis (CF) is the most common genetic disease with an incidence of 1 case per 2500-3000 births. More than 1800 mutations are responsible of a heterogeneous symptoms cortege in relation to the number of organs and systems involved. The classical form is characterized by multi-organ involvement like the exocrine glands. Outside the classical forms are those in which the manifestations of the disease mainly affect a single organ and are generically indicated as "atypical". In comparison to classical genotypes for CF there are polymorphic variants that if in association with classical mutations are those considered responsible for atypical phenotypes. TG12-13-5T represent polymorphisms whose isolated presence is not relevant in a subject's genotype. Its association with classical mutations responsible for CF, leads to reduced production of cystic fibrosis transmembrane conductance regulator protein (CFTR).

Cases report
We describe the cases of five adult, male patients who came to our observation for genetic counseling for infertility. All patients are carriers of the TG12-13 5T polymorphism associated with classical mutations responsible for CF. Mean age was 34.6 years. All patients underwent functional and instrumental respiratory evaluation, pancreatic and hepatic function, sweat testing, ENT evaluation, genetic study for CF. In the cases described, the presence of the TG12 13-5T polymorphism associated with classical mutation responsible for CF correlates with phenotypic pictures characterized by infertility, normal respiratory function, with FEV 1 medium 109% and isolated bronchiectasis, pancreatic and hepatic function were normal, minimal alteration of the liver structure, average pathological sweat test with an average chloride concentration equal to 64.8 mEq/l, only one patient with polyposis and pansinusitis.

Conclusion
The R31C mutation, according to the literature data, is defined as "Non CF causing", therefore without giving symptoms of disease if not associated with "serious" mutations. Our case is a variant R31C associated with a "mild" mutation (TG12-5T) that presents a clinical suggestive of CF. Can we exclude therefore that this mutation, even associated with non-serious mutations, cannot develop a CFTR-RD picture with mild symptoms or, in males, a possible sterility secondary to agenesis of deferent ducts? What happens in children diagnosed as CFSPID? Many will remain asymptomatic, 10-20% instead will develop symptoms suggestive of CF ( for example PA infection), but the trend is still under study. Parents gave consent to patient data publication.

Results
In both patients, the genetic characterization confirmed the presence of F508del mutation. The analysis of the exon 9 and surrounding intronic regions of CFTR showed an extra amplicon larger (of about 300 nucleotides) than the wild-type. The DNA sequence revealed, in both patients, the presence of an insertion of part of intron 9 in intron 8 of the CFTR gene, within the (TG)m repeat, with a poly-T stretch. The molecular lesion resulted to be the same in both patients, with only a small difference in the number of T in the poly-T stretch. The functional characterization at RNA level revealed a near complete anomalous splicing, without exon 9, from the mutated allele of both patients. Consequently, the mutated allele is expected not to contribute to the formation of functional CFTR protein.

Conclusions
This alteration has not been previously described in the literature. Its molecular and functional features are compatible with the definition of new CF-causing mutation of the CFTR gene. This allowed the completion of the genetic characterization of both patients. The fact that the only difference in the two mutated alleles is the small change in the number of T within the poly-T stretch allows speculating about the molecular mechanism of divergence from a common ancestral allele. We declare to have the informed consent statement for the processing and use of personal data.

A10
Understanding the network of the regulation of PON2 Background 3OxoC12 acylhomoserine lactone (3OxoC12) is the master regulator of Quorum Sensing (QS) regulating the expression of several bacterial virulence. Paraoxonase2 (PON2) has lactonase and antioxidant activities that are inhibited by 3OxoC12. PON2, expressed in all tissues, appears involved in many areas of defence of the organisms, including bacterial infections, inflammation, oxidative stress, and innate immune systems. PON2 polymorphic sites (148A/G and 311 S/C) have been previously described to be related to cardiac problems and diabetes type 2. The hydrolysis of 3OxoC12 can be a rational approach to the cure of CF. We focused on the comprehension of the transcriptional and the posttranslational regulation of PON2 expression.

Materials and methods
We produced in E.coli an engineered version of PON2, with activities matching those described for the native protein, used to generate mutants to understand its structural and biochemical details. We set up qRT-PCRs to highlight the presence of the most abundant mRNA isoforms and to genotype for the two most common PON2 SNP coding variants p.Ala148Gly and p.Ser311Cys. We investigated the regulation of expression of PON2 mRNA, by checking an hypothesis of control via an "mRNA operon", through the silencing of target genes.

Results
We focused to identify the post translational modification and we showed a 3OxoC12-dependent ubiquitination (UBQ) (LYS 168) of PON2 nearby a polymorphic site, able to influence the lactonase activity. Recently a second UBQ site, nearby a second polymorphic in position 311, has been identified by us; then we have produced mutants of the two PON2 polymorphic sites (148A/G and 311 S/C) to study the activities and relationships with PTMs. Studying the mechanism of control of PON2 regulation, we identified a RNA binding protein and a E3 ubiquitin ligase that, when silenced, is able to increase the expression of the PON2 gene. Moreover we genotyped for the SNP 311 several cell lines identifying normal, heterozygotes and also mutant homozygotes.

Conclusions
Considering the impact of this gene on the defence against grambacterial, its involvement in the inflammation, the indication that the polymorphisms could dictate the severity of disease -as already demonstrated for other pathology-it could be useful to analyze CF patients to understand if the gene can have a role of modifier in relation to the presence of SNPs. Background Cystic fibrosis (CF) is the most common genetically determined, lifelimiting disorder in populations of European. The genetic origin of CF is the mutations in the CF transmembrane conductance regulator (CFTR).CFTR mutations may be classified into 6 different categories based on the mechanisms that are affected. Class I result from mutations leading to absent CFTR production. Class II, including Phe508del, are caused by defective CFTR processing. Class III mutations result in expression of CFTR, the channel gating is defective and results in impaired chloride transport function. Conductance defects are seen in class IV mutation. Class IV result in a milder phenotype. Finally, class VI mutations are characterized by a functional but unstable CFTR [1]. Ivacaftor is a potentiator that augmented chloride transport and increased airway surface liquid height and cilia beat frequency in airway epithelial cells expressing a CFTR gating mutation (III Class) [2]. Case report C.N. female, 5 years old. Born at the end of gestational age, birth weight 3600 g. Diagnosis by screening and familiarity with FC. Sweat test: 98 mEq/L Cl -. Genetics: F508del/G1244E. Intermittent colonization by Pseudomonas aeruginosa and Staphylococcus aureus. Growth curve under 25 0 centile from the first months of life. She has only 6 months of follow-up. She assumes Ivacaftor at a dose of 75 mg every 12h with good compliance. From the beginning of the therapy program, an improvement of the auxometric parameters and of the score of the CFQ-R respiratory domain was observed; she did not present respiratory exacerbations and showed a good tolerance to the drug, in fact no adverse events were reported. There was not enough compliance to perform the spirometry, necessary for the assessment of respiratory function Conclusion Kalydeco is an example of innovative therapeutic strategy for carriers of a CFTR channel gating mutation. The further development of such approaches offers great promise for future therapeutic strategies in CF [3]. Parents gave consent to patient data publication.

Background
Ivacaftor (IVA) is a CFTR potentiator approved in Italy in Cystic Fibrosis (CF) patients carrying gating mutations and R117H mutation. In these subjects, IVA was shown to increase percent predicted forced expiratory volume in the 1st second (ppFEV 1 ) and BMI and decreased sweat chloride [1,2]. In the last years, the use of IVA is under evaluation for CFTR mutations resulting in residual functioning protein, due to the consistent improve of the chloride transport in vitro and the FEV 1 in vivo [3,4]. The 2789+5G->A is an alternative splicing mutation of class V associated with reduced synthesis of CFTR protein [5]. We report the use of IVA in four CF adult patients with at least one 2789+5G>A CFTR mutation.

Materials and methods
We retrospectively evaluated all CF patients in follow-up at CF Centre of Florence, Italy, carrying at least one 2789+5G->A. We enrolled only patients aged 12 years and older, with a median ppFEV 1 in the last year ≤ 40% to receive IVA, 150 mg every 12 hours. For each of these, in the year before IVA-starting and on IVA, we evaluated the following characteristics: median ppFEV 1 , best FEV 1 , BMI, Cystic Fibrosis Questionnaire-Revised (CFQ-R) and potential adverse events.

Conclusions
Ivacaftor treatment seems to have a potential benefit in CF patients carrying at least one 2789+5G>A CFTR mutation improving lung function, BMI and CFQ-R. Our results, although derived from few patients, support the necessity of further studies to confirm the benefit.

A13
Long-term effectiveness of ivacaftor in patients with Cystic Fibrosis carrying CFTR mutations with residual function and severe lung disease Donatello Salvatore 1 , Cesare Braggion 2 , Barbara Messore 3  Ivacaftor is a CFTR potentiator approved for class-III and R117H CFTR mutations and, in the US, for other 28 mutations with "residual function" (RF) of CFTR. RF mutations are usually associated with lung disease that can be delayed in onset and slower in progression, but it may become severe in the adult age.

Conclusions
The high heterogeneity of our casuistry related to respiratory disease and functional capacity underlines the need for a personalized approach to assess the responsiveness to these new drugs. To date data were not available for the entire sample, nevertheless we speculate that a close monitoring of adherence to respiratory physiotherapy should be mandatory and 6MWT distance could be an inaccurate outcome for Iv-Lu responsiveness without the contribution of other more reliable outcomes of maximal exercise performance.

A15
Effects of Lumacaftor/Ivacaftor on physical activity and exercise tolerance in three adults with Cystic The combination of the cystic fibrosis transmembrane conductance regulator (CFTR) corrector lumacaftor with the potentiator ivacaftor has been approved for the treatment of patients with cystic fibrosis (CF) homozygous for the Phe508del CFTR mutation. The major benefits of lumacaftor-ivacaftor therapy may reside in the reduction of pulmonary exacerbations and in a lower annual loss of lung function. However, there are no reports detailing the effect of lumacaftor-ivacaftor on daily physical activity (PA) and exercise tolerance.

Materials and methods
We performed incremental cardiopulmonary exercise testing (CPET) and we assessed PA pre-and post 2 years initiation of lumacaftorivacaftor in three adults with CF (CFTR genotype F508del/F508del) and chronic airflow obstruction. CPET-related measurements included oxygen uptake (V'O 2 ), carbon dioxide production (V'CO 2 ), ventilatory profile, heart rate (HR) and oxygen pulse (V'O 2 /HR) throughout exercise and at lactic threshold (LT) and peak. LT measures represent submaximal exercise related data. PA was assessed using the accelerometer Sense Wear Pro3 Armband.

Conclusions
Daily physical activity levels and exercise tolerance improved after two years of LUM/IVA therapy, despite no patient education or lifestyle advice. All patients provided informed written consent for this case report. Ivacaftor is a cystic fibrosis transmembrane conductance regulator (CFTR) potentiator, approved for patients with CF with gating and residual function CFTR mutations. We report the results of a singlecenter observational study investigating its effects in CF patients with gating mutations.

Materials and methods
Patients with gating mutations were recruited to an open-label study evaluating Ivacaftor. Primary outcomes included: lung function, sweat chloride, sputum microbiology, number of pulmonary exacerbations and weight gain. Results 7 subjects (6 females) were enrolled and completed 24 months follow-up on Ivacaftor; mean age at enrollment was 29.3 years (range 12 -45 years) with 1 subject <18. Two subjects were excluded from the analysis because of very low adherence to the treatment, documented by large variations of sweat Cl and missed collect of Ivacaftor at the pharmacy. Another patient was excluded because not able to perform the lung function tests, having very severe lung disease with Burkholderia cepacia infection. The remaining 4 participants experienced significant improvements in ppFEV 1 (mean absolute increase of 18% at 1 month that was sustained along the follow up period (24 months); sweat chloride decreased from a mean value of 101 mmol/l at baseline to 39 mmol/L at 24 months. Mean absolute BMI increased of 2.8% after 1 month of treatment until 10.8 % at 12 and 24 months. Pulmonary exacerbations were absent and no subject needed hospitalization during the follow up. Microbiology of sputum was unchanged. No safety concern was reported.

Conclusions
Patients with gating mutations experienced improved lung function and nutritional status, and a very pronounced decrease of pulmonary exacerbations. This study supports ongoing use of Ivacaftor for patients with these mutations. Despite the promising nature of Ivacaftor, our data suggest that adherence rates can be suboptimal.
Monitoring and motivational interventions are important also for this treatment. Background Cystic fibrosis (CF) is a life-limiting disease that is caused by defective or deficient cystic fibrosis transmembrane conductance regulator (CFTR) protein activity. Phe508del is the most common CFTR mutation. CF affects approximately 70,000 people around the globe including more than 30,000 in Europe. CF is a multisystem disease affecting organs where CFTR is expressed (airways, gastrointestinal and the reproductive tract). Lumacaftor is a CFTR corrector that has been shown to correct Phe508del CFTR misprocessing and increase the amount of cell surface-localized protein. Ivacaftor is CFTR potentiator that increases the open probability of CFTR channels. Case report M. P., male 12 years old. Diagnosis of CF in complete form with meconium ileus. Sweat test: 96 mEq/L Cl -. Genetics: F508del/F508del. Intermittent colonization by Pseudomonas aeruginosa from the first months of life. Poor nutritional situation with slow weight gain. In 2015, diagnosis of GH deficiency, starting from January 2016 GH treatment. In August 2017, in consideration of the "critical need"-status, he started taking the pediatric dosage of Lumacaftor/Ivacaftor as it was part of the compassionate use program. For the first week he took Lumacaftor/Ivacaftor 100/125mg every 12h preceded by short acting bronchodilator. From the second week began full pediatric dosage 200/250mg every 12h. At the age of 12 years of age he continued with the same dosage until the time when the marketing of the association Lumacaftor/Ivacaftor was made available for patients over 12 years of age. During one year of observation the patient presented an improvement of FEV1 equal to 14% of the starting value, and of the FVC equal to 30%; the 6MWT performance showed an increase in the distance traveled of 295 meters measured at the T12. The assessment of the nutritional status of the child showed a slight increase in weight and height, despite the concomitant use of GH treatment, with a consequent increase in BMI of +0.6. During the treatment, no respiratory exacerbations were observed compared to at least 4 episodes that occurred during the previous 12 months. By submitting the patient to the CFQ-R quality of life questionnaire, the score for respiratory symptoms improved significantly from 50 to 83. The sweat test also showed a reduction in chloride concentration. The only parameter that has not undergone changes has been the microbiological data because the patient has maintained colonization by Pseudomonas aeruginosa.

Conclusion
The use of this therapeutic strategies gave new prospective of life to the patients who carried a Phe508del mutation in CFTR. Parents gave consent to patient data publication.

Background
Waiting for all available new modulators could be approved for patients with Cystic Fibrosis (CF) who carry class I and /or class II mutations not homozygous for F508del mutation alternative therapies could be evaluated. A previous phase-2 clinical trial, showed that oral cysteamine (CYS), already approved for the treatment of cystinosis, combined with the kinase-inhibiting epigallocatechin-gallate (EGCG, a safe over-the-counter approved nutraceutical), could be effective in terms of clinical response and functional rescue of CFTR [1]. On the basis of these results we used as off-label therapy this combined treatment for two selected patients. Case report 1 Matteo (genotype: F508del/2183AA>G) is a 7 years boy who presented chronic lung disease, bronchiectasis, liver steatosis, kidney tubular dysfunction and pancreatic insufficiency. One year treatment with CYS (30 mg/Kg qds) plus ECGC (270 mg qd) was completed.
During one year follow-up he improved his lung function: FEV1% increased from 92 to 113%, while MMEF% from 64 to 78%. The sweat chloride decreased from 117 mmol/l at baseline to 55 mmol/l after 12 months. No pulmonary exacerbation was registered compared to one exacerbation in the previous year. The patient reports an improvement of daily exercise capacity. Case report 2 Ivana (genotype: N1303K/L1065P) is an 18 years girl affected by a severe lung disease, atelectasis, chronic pansinusitis, liver disease treated with UDCA, frequent respiratory exacerbations requiring hospitalization, multidrug allergy, CF related diabetes and pancreatic insufficiency. She assumed the combined therapy with CYS plus EGCG through 2 years. During two years follow-up she improved lung function: FEV1% increased from 68.7 to 79.9%, while MMEF% from 22.3 to 32.6%. The sweat chloride decreased from 113 mmol/l at baseline to 55 mmol/l after 24 months. The transmembrane conductance flow (measured by SPQ on nasal brushing sample) gained 50% of wild type after 2 years of treatment. Pulmonary exacerbations reduced in severity with a reduction in the number of hospitalizations of 50% (from 6 to 3) during 2 years follow-up.

Conclusion
Our experience confirms previous data of efficacy and safety of the association CYS plus EGCG in a selected group of patients. In order to confirm the efficacy of CYS plus EGCG combination in subjects with CF carrying two minimal residual function CFTR mutations, a Phase III randomized-controlled multicentre study has to be encouraged. Patient's parents gave consent for the publication of clinical data. Background Gastroesophageal reflux (GER) is common in patients with cystic fibrosis (CF) and is often regarded as playing a role in the pathogenesis of CF lung disease. Individuals with CF have many predisposing factors to the development of GER, with a reported prevalence ranging from 35 to 81%. Both the acid and nonacid components of GER may have an effect on lung disease. The lung clearance index (LCI) is a measure of ventilation distribution obtained from the multiple-breath washout (MBW) test. As the MBW test uses relaxed tidal breathing without the requirement of any maximal effort, it is an attractive test for infants and pre-school-aged children. LCI is abnormal in a significant proportion of pre-school children with cystic fibrosis (CF), and correlates with the presence and extent of structural lung disease detected via chest computed tomography (CT) and lower respiratory tract inflammation and infection.

Materials and methods
We enrolled a cohort of 14 patients with CF both children and adults who were investigated with 24-h pH-metry. All patients were free from proton pump inhibitors (PPI) therapy for 1 month and in a stable phase of the disease. At the same time (one day before or after the exam) we performed the study of Lung Clearance Index (LCI) with Multiple Breath Washout (MBW) technique.

Results
We found the presence of relevant episodes of acid gastroesophageal reflux in 10 (71%) patients (6 adults and 4 children Case report 1 Ilary, 7 years, comes to our observation for acute slow-resolution pancreatitis. Second-born from normal course of pregnancy, emission of meconium occurred within the first 24 hours of life. Normal statureweight growth. During the hospitalization a positive result of the sweat test was carried out and consequently the genetic study was performed with a compound heterozygosis of the CFTR gene (F508del / T338I mutation); the latter is associated with a slightly expressed phenotype, especially with regard to the pancreas secretory function. Case report 2 Giulia, 11 years old, anything relevant in remote medical history, regular weight-weighted growth. She arrives in emergency area for abdominal pain in the epigastric site radiated to the back, with increasing intensity, not responsive to antalgic therapy. At the blood chemistry tests, high values of pancreatic enzymes are found. Excluding infectious and malformative causes of acute pancreatitis, in the suspicion of cystic fibrosis (CF), a sweat test resulted positive , and genetic testing showed mutation in heterozygosity of G542X and 711 + 3A> G intron 5. The latter mutation intervenes on the splicing of CFTR and allows the synthesis of a partially functional protein, or a reduced portion of normally functioning CFTR. In both cases other exams such as broncho-aspirate culture, chymotrypsin and faecal elastase were in normal and it was undertaken fasting, infusion support and subsequently therapy with pancreatic extracts and ursodeoxycholic acid, with gradual normalization of enzyme levels.

Conclusion
Pancreatitis is a relatively rare complication affecting 1-2% of CF patients and may be an initial isolated manifestation of the disease and therefore be the first indicator for the diagnosis of CF. This complication occurs in cases with a functioning pancreas, so in order to manifest inflammation, it is necessary that the organ is still able to secrete enzymes. Approximately 10% of CF patients maintain good pancreatic function until adulthood or for life: they have at least one mutation of the CFTR gene defines as mild. In some of these mild forms, the pancreas causes partial stasis of secretion in the pancreatic canaliculi, inducing inflammation with important abdominal pain and marked increase in pancreatic enzymes in the serum. Follow-up is important for understanding the possible onset of pancreatic insufficiency. In the presence of pancreatitis history, without triggering causes, both in a child and in an adult, it is advisable to investigate for CF. Parents gave consent to patient data publication.

Background
Cystic Fibrosis (CF) is an autosomal recessive genetic disease. CFassociated Liver Disease (CFLD) is a common complication and represents the third cause of death in 2.5% of patients with CF. In clinical practice liver biopsy is not routinely applied to evaluate the severity of liver disease in CF, as it is a painful and invasive procedure. Since much more studies support the use of liver ultrasound to correctly assess the degree of hepatic involvement, we aimed to study a group of children diagnosed as having CF to evaluate the correlation between liver stiffness assessed by ARFI elastograghy and conventional US qualitative examination.

Material and methods
Data were collected from 34 consecutively enrolled CF patients including age, gender, antropometric measures, CFTR mutations. Liver stiffness derived from acoustically generated tissue shear wave propagation (Shear-wave elastography-SWE) and Ultra Sound (US) parameters of liver pathology such as hepatomegaly, variation in echogenicity, fibrosis, signs of portal hypertension were also registered in all patients in a bladder manner.

Results
Preliminary results include data from 34 CF patients (17 Males; mean age 8.9 years, range 3.3-15.6). All of them had pancreatic insufficiency and carried minimal residual function mutations on both alleles. 10/34 (29.41%) (8 Males; mean age 12.3 range 7.9-15.0) patients showed hepatic stiffness over the cut-off value (range 6.4-15.0) compared to normal value < 6.3 kPa. In 8/10 patients liver stiffness alteration was concordant with US signs of hepatic abnormalities (steatosis, fibrosis), while 2/10 didn't show any other sign of liver pathology. In 24/34 patients with liver stiffness in the normal range 6 patients showed US qualitative signs of mild liver abnormalities as steatosis and one with fibrosis.

Conclusions
According to previous data our preliminary results show a correlation between conventional US and increased values of liver stiffness, examined by Shear-Wave elastograghy [1] in order to detect CFLD. More data are needed to identify liver stiffness as a diagnostic tool of liver disease with advantage of real-time imaging and lower cost and to correlate it to clinical characteristics. Malnutrition is a complication of Cystic Fibrosis (CF) and has been recognized as a negative prognostic factor. Therefore malnutrition has to be corrected and a step wise approach has been recommended including enteral nutrition (EN) via percutaneously placed gastrostomy (PEG) tube [1]. The aim of this study was to investigate the efficacy of EN via PEG to improve nutritional status, respiratory function and incidence of complications.

Materials and methods
A retrospective cohort study on CF patients who underwent PEG between 2000 and 2016 was performed at our Center. Sixteen patients were included in the study with a median age of 13 years, 94% with pancreatic insufficiency. The anthropometric data were recorded 6 months prior PEG placement, at the time of placement and 6 months after. Patients were classified according to CF Foundation (CFF) nutritional criteria. Malnutrition was considered to be present in patients with BMI <10th percentile up to 19 years and <18.5kg/m² over 20 years. In addition, in 15 parents of 13 patients, the level of parental stress was investigated through a self-administered questionnaire; results of PEG-group (n=4) were compared to a group of control subjects with a CF child but not needing EN.

Results
Malnutrition was present in almost all patients who underwent PEG, with the exception of 3 children in whom PEG was necessary due to total aversion to oral feeding. After 6 months of EN median values of the Z-score of W/L or BMI increases by 1 standard deviation. At the start of EN, 13 patients (81.3%) were malnourished; 6 months after the malnourished subjects decreased to 31.3% (N=5). Complications (mechanical, gastroenterological, metabolic) occurred in 50% of patients and were solved; only in one case NE was stopped. The mean percentile value of the Total Stress levels falls within the norm in both groups. Only one mother reported a value in the range of clinical interest due to the coexistence of other problems in addition to CF.

Conclusions
Patients needing PEG constitute a small minority in CF population; from our experience it emerges that EN via PEG is safe and helps to achieve an adequate weight when usual interventions fail. Patients with PEG must be evaluated from a nutritional point of view at every clinical check and supported in the management of this device. Background optimizing growth from early childhood in Cystic Fibrosis (CF) is critical, as lung function and survival correlate strongly with nutritional status. Guidelines recommend a persistent optimal nutritional status. The objective of this preliminary study is to evaluate nutritional status in a cohort of children with CF in the first 2 years of life, in follow-up at Cystic Fibrosis Centre of Naples Materials and methods Data from 12 children (5 Males) affected by CF, born between 2015 and 2016, were retrospectively evaluated. CF was diagnosed in 10/12 by neonatal screening and in 2/10 for suggestive symptoms but with negative newborn neonatal screening. 9/12 were pancreatic insufficient (PI). We evaluated data about nutritional status (body weight, height and P/A ratio with the relative percentile) according to CDC growth curves, feeding modalities, prescriptions of nutritional supplements, clinical outcome and vitamin D levels. Furthermore, we investigated mothers anxiety, evaluated by STAI -Y questionnaire, in relation to nutritional status of enrolled children.

Results
Results of anthropometric parameters are summarized in Table 1. 4 patients at a high risk of malnutrition improved through personalized nutritional intervention at 2 years of age. From the analysis of feeding modalities, 4/12 children were breastfed, 5/12 were formulafed exclusively and 3/12 received mixed feeding. The dietary intake for children was about 120% (range 100%-130%) estimated average requirement. Insufficient children received PERT at a dosage increasing from 2500 lipase/g fat units at the diagnosis to 3300 lipase /g fat units at 24 months of follow-up in order to correct malabsorption. In particular, steatocrit values normalized on average from 34% (range 12%-40%) to 2.32% ( 1% -6.5%) All enrolled children received salt supplementation. Despite vitamin supplementation in all children since diagnosis only one with severe pancreatic insufficiency showed hypovitaminosis D.
Beyond their nutritional status preliminary data concerning 8 mothers showed a level of anxiety (trait: 35-63 state: 33-72) measured by STAI -Y questionnaire with no correlation to nutritional status.

Conclusions
Early individualized nutritional intervention is useful to ensure adequate growth and correct nutritional status in CF. More data are needed to correlate nutritional status and mother anxiety that could impact on rescue of malnutrition. Backgroung Identification of pulmonary exacerbation (PE) in CF patients is important for proper treatment. Serological markers as C-reactive protein (CRP) and leukocyte (WBC) counts are not very specific and not included in the Fuchs criteria for PE diagnosis. Serum amyloid A (SAA) proteins are a family of apolipoproteins expressed or secreted by liver during the acute phase of inflammation. SAA genes are regulated by the proinflammatory cytokines (IL-1, IL-6, TNF-α) and induced rapidly under inflammatory conditions like exposure to bacterial LPS. SAA is also involved in amyloidogenesis and tumor pathogenesis. At low concentrations (10-100ng/ml), as in early inflammation, SAA induces chemokines or matrix degrading enzymes and functions as a chemoattractant. When an infectious or inflammatory stimulus persists, the liver continues to produce SAA that becomes a direct bacterial opsonin or interferes with viral infection of host cells. AA amyloidosis is a rare, but severe complication of CF with predominant renal involvement. Aim of the study is to evaluate the usefulness of SAA as a marker of inflammation and a diagnostic criterion for PE.

Conclusions
Literature is still discussing the definition of PE. New and more sensitive biochemical markers of early inflammation are necessary to diagnose PE. SAA may represent a more sensitive biochemical marker than CRP and WBC for PE diagnosis and might be included, after an adequate validation study, in the definition criteria for PE. Cenocepacia, is associated with a more rapid decline in lung function and considered a relative or absolute contraindication to lung transplantation in almost all transplant centers for a markedly adverse impact on survival. Bcc infections are particularly difficult to treat due to their intrinsic multidrug resistance to many antibiotics, including aminoglycosides, cephalosporins, polymyxins and traditional antipseudomonal β-lactams. One of the most common resistance mechanism in Burkholderia species is the production of β-lactamases. Non beta-lactamase-mediated resistance, like efflux pumps and reduced outer membrane permeability plays also a role in the decreased susceptibility of BCC species towards beta lactam antibiotics. Avibactam is a novel β -lactamase inhibitor that restores the susceptibility to ceftazidime by preventing enzymatic hydrolysis. The combination of ceftazidime/avibactam (CEF-AVI) represents an important new therapeutic option for Bcc, as shows the highest in vitro susceptibility and may be a potential salvage therapy for Bcc infections when the other treatment options have failed. The main pathogenic germs are Staphylococcus aureus (Sa), Pseudomonas aeruginosa (Pa). In CF a targeted and aggressive therapy of chronic Pa infections is one of the mainly strategy to allow the improvement of the prognosis. The new combination of a β-lactam and a β-lactamase inhibitor such as Ceftolozane-Tazobactam shows a rapidly bactericidal action, a linear kinetics, it spreads well in the lung and acts on numerous mechanisms of resistance of Pa. Case report Female aged 16, weighing 51 Kg, suffering from CF in a complete form, waiting for lung transplants, is hospitalized for fever and dyspnoea. EGA and blood exams showed a septic state. She arrived with Levofloxacin, Tobramycin, Meropenem antibiotic iv treatment. Cultural examinations were performed and started antibiotic therapy with Ceftolozane/Tazobactam and Phosphomycin. From the fifth day of therapy the clinical conditions gradually improve and also the blood tests showed a reduction in CRP. Therapy is continued through 20 days. In the meantime, a subsequent sputum culture examination isolated 2 strains of Pseudomonas (mucoid and wrinkled) with a sensitivity similar to that of the entrance except for a resumption of sensitivity to Piperacillin-Tazobactam. So we decided to modify the therapy from ceftalozane to Piperacillin/Tazobactam and Colistina iv. After 7 days the clinical conditions worsened, with a reappearance of fever and increase in inflammation indices. The last sputum culture test documented the reappearance of resistance to Piperacillin-Tazobactam, therefore we decided to stop the therapy and to resume the association Phosphomycin+ Ceftolozano/Tazobactam. There was a progressive improvement of the clinical conditions.

Conclusion
The patient came to our observation in a highly compromised general state, that is, with an exacerbation of her chronic Pa MDR infection. We started a combination therapy with the aim of improving the clinical outcome i waiting for the lung transplant. The choice fell on Ceftolozano-Tazobactam due to its strong antipseudomonas action, used with good clinical evidence also in rescue therapies. We did not observe any side effects and the hepato-renal and medullary parameters remained in the normal range.
Parents gave authorization to patient data publication. In this study we evaluate the performance and the safety of a NWs solution, with or without surfactant, to reduce symptoms and bacterial load.
All subjects underwent the Sniffin'Sticks to evaluate the olfactory performance.

Results
Twelve patients completed 4 months of treatment: 6 patients performed the treatment with Naridek and 6 patients with physiological solution.
Due to the small sample size, the scores were added together to have an overall indication of the treatment (Table 1) The bacterial colonization in NWs shows no statistically significant difference. However, in 2 patients, we detected a reduction of the bacterial load, while there was no difference in the saline-treated group.
Conclusions Considering our small sample we can only draw some great deal to think about: treatment with NWs allows an improvement of the ENT symptoms and is well tolerated by patients. These data are confirmed by the ENT signs score and by the reduction of the SNAQ11 score in both treatment arms; the solution with surfactant (Naridek) significantly improves the ENT signs and decreases the nasal endoscopy and the SNAQ11 scores; no benefit was detected at the evaluation of olfactory performance.
In conclusion, even if further confirmations are necessary on broader cases, it seems to emerge as significant the role of surfactant in the therapeutic advantage of NWs.

Background
Burkholderia gladioli is an opportunistic Gram-negative, not belonging to Burkholderia cepacia complex (BCC The emergence of multidrug-resistant Pseudomonas aeruginosa (MDR-PA) strains is an important and growing issue in the care of cystic fibrosis (CF) patients. Ceftazidime-avibactam and ceftolozane/tazobactam are novel antimicrobials combining a third-generation cephalosporin with β-lactamase inhibitor the use of which are restricted to some clinical cases, including CF patients infected with MDR-PA. In this study, we assessed the ceftazidime-avibactam and ceftolozane/tazobactam susceptibility of MDR-PA isolated from adults with CF. Materials and methods MDR-PA (strains resistant to all antibiotics in two or more classes of antipseudomonal agents), were isolated from CF patients in our center in the last year. The antimicrobial susceptibility for all the isolates were performed by using the micro broth dilution method following EUCAST guidelines.

Conclusions
Ceftazidime-avibactam and ceftolozane/tazobactam are novel antimicrobial drugs targeting difficult to treat Gram-negative organisms. The data showed that these antimicrobials were highly active in vitro against CF isolates of Pseudomonas aeruginosa, including isolates that exhibit resistance to multiple drug classes. This study demonstrates excellent susceptibility profiles specially for ceftazidime-avibactam against MDR Pseudomonas aeruginosa strains collected from the sputum of individuals with CF.  Background Management of paranasal sinuses complications in Cystic Fibrosis (CF) is a challenge for the otolaryngologist. Paranasal sinuses system represents a P. aeruginosa reservoir affecting lower airways system. Early diagnosis and follow-up of ENT complications of CF, like mucoceles and polyposis, are important to treat them. A clinical-symptoms, radiological score for treatment, stratification and evaluation of CF patients has been developed.

Materials and methods
Between May 2015 and May 2018, 52 patients (27 males, 25 females; mean age 15.90 years, range 6-30 years) attending our ENT-CF clinic were evaluated for paranasal sinuses complications with maxillofacial CT; cone-beam CT (CBCT) investigation was used for pediatric patients. Radiological results were described qualitatively and quantitatively using Lund-Mackay score. CT and CBCT report with the radiological score, endoscopic Meltzer's Score and SNAQ-11 questionnaire for clinical ENT symptom were used for the assessment of a CF sinus score (CFSS), with a maximum score of 112 points. ENT treatment was chosen depending on CFSS results.

Conclusions
Our CFSS results confirmed that CF patients present an early clinical objectivity of nasal polyposis with pluri-sinonasal disease, asymptomatic in most of the cases. CBCT has demonstrated to be a suitable radiological investigation and follow-up for CF patients, due to its reduced radiation exposition. Future objectives of CFSS is to identify CF patients who can benefit the most from surgery and to become a mean for comparison among ENT specialists treating CF. Went to our referred center complaining dyspnea at rest, worsening of airway clearance and the need for more oxygen support during the day.

Background
We presented the case of a 27-yr-old man with Cystic Fibrosis (CF) due to a DF508 homozygotic mutation. The patient is colonized with Pseudomonas aeruginosa, Aspergillus fumigatus and Mycobaterium ascessus that were treated in the past with no evidence of reactivation, and an allergic bronchopulmonary aspergillus (ABPA) was detected. He is currently treated with a CFTR corrector (lumacaftor) and a CFTR potentiator (ivacaftor). Patient was treated with low oxygen flow (LOF) support continuously throughout the day. Before the admission, the health perception was assessed according to CFQ-R Teen/Adult Italian Version 2.0. The patient went to our referred center complaining dyspnea at rest, worsening of airway clearance and the need for more oxygen support during the day. Materials and methods Patient was admitted with high respiratory rate (35 breaths/min). His first gas assessment showed hypoxemia (pO2 55.8 mmHg) and low pO2/FiO2 ratio (1.36). Chest X-ray was suggestive for a new opacity. Then, high flow nasal therapy (HFNC) (AIRVO 2 Fisher and Paykel) was administered to the patient (34°C, 50 L/min and FiO2 42%). Patient continued high flow nasal cannula therapy almost continuously for seven days and for a further four nights. We prescribed HFNC domiciliary at night time.

Results
Patient reported great comfort and showed high tolerance to this device and setting. Respiratory rate decreased shortly after the start of high oxygen flow therapy. Gas assessment at the end of treatment with HFNC revealed increased level of pO2 and pO2/FiO2 (pO2 85.9 mmHg, PO2/FiO2 2.45). The patient acknowledged an improvement in cleaning the secretions. At the time of discharge, the patient pointed out the great benefits of the HFNC treatment and his general health perception increased according to CFQ-R Teen/Adult Italian Version 2.0.

Conclusions
In patients with CF the clearance of airway secretions remains one of the most common issues. It is well known that HFNC delivers warm and humidified gas and thus improves mucociliary function by maintaining clearance to a greater degree than other methods for delivering oxygen. Due to the capacity of the device to reach higher and relatively fixed FiO2 than LOF, hypoxemia improves throughout the treatment. Furthermore, HFNC generates positive end-expiratory pressure which reduces the work of breathing and respiratory rate. The low burden of the device allows the patient to continue the treatment also at home with potential benefits in terms of health perception, cleaning secretions and respiratory physiotherapy. The patient gave the consent to publish clinical data. In Cystic fibrosis (CF) lung disease is characterized by progressive airflow obstruction, due to mucus plugging and airway inflammation, causing destruction of the lung parenchyma secondary to bronchiectasis. These alterations result clinically in an increase of the work of breathing, leading to alveolar hypoventilation predominantly during sleep, exercise and acute respiratory exacerbations. In clinical practice, nocturnal cardiorespiratory polygraphy (PG) role is growing and may represent an important diagnostic test compared with conventional pulmonary function test as spirometry and lung clearance index (LCI). Furthermore, signs of lung function decline, marked principally by impaired gas exchange and/or increased work of breathing during sleep, may be represent powerful and validated criteria to start preventive nocturnal noninvasive ventilation (NPPV) in CF patients. The purpose of this study was to identify respiratory patterns over the spectrum of disease severity in patient with CF. The overall hypothesis for the current study is that patients with CF demonstrate gas exchange abnormalities and increased respiratory loads during sleep, reported and recognized by conventional cardiorespiratory PG and also, using non-invasive ventilation as preventing respiratory failure in contrast with conventional practice in clinical studies.

Materials and methods
Analysis of nocturnal breathing patterns and gas exchange on PG was performed in patients with CF (30) patients with different clinical characteristics. They performed also spirometry and LCI with multiple-breath washout (MBW) test.

Results
Children with CF demonstrated lower oxyhemoglobin saturation (95% ± 1.6%), higher respiratory rate (20.5 ± 6.9 breaths per minute). The Apnea Hypopnea index differ between age, particularly in preschool age (5.5 ± 2.7 events per hour,). Ventilation distribution outcomes (LCI,) was significantly higher (>7) in patients with advanced CF. 5 patients with serious nocturnal hypoxemia and/or increasing respiratory loading started nocturnal noninvasive Bi-level ventilation. After 6 months, control polygraphy showed an improvement of respiratory pattern. Conclusions NPPV may be represent a potential treatment in CF to prevent lung function decline, in contrast with conventional use in acute respiratory failure, or as a bridge to pulmonary transplant. Using Bi-level PPV, upper airway obstruction and/or work of breathing induced by intrinsic positive pressure of end expiration (PEEP) are prevented by expiratory positive airway pressure (EPAP) and thus pression support (PS) can be triggered easily by the patient. In conclusion, different ventilatory modes have been used in patients with CF, but bi-level positive pressure ventilation is used by the majority of the patients for the comfort and the security regarding to the inspiratory pressures. Background Extracorporeal life supports (ELS) are devices that enable direct oxygenation or CO 2 extraction from the blood. In recent years their use has increased among CF patients with end-stage respiratory condition and failure as a bridge for lung transplantation (LTx). Nevertheless, extracorporeal membrane oxygenation ECMO support (ECMO) is considered when a patient presents a rapid deterioration of a chronic lung disease and only when it has been already included on a LTx waiting list or an evaluation process has already started. Case Report A 25 years old woman with CF, CF-related diabetes (CFRD), CF liver disease (CFLD) with portal hypertension and a history of moderate but unstable lung disease, chronic Pseudomonas Aeruginosa colonization and allergic bronchopulmonary Aspergillosis (ABPA) presented with fever (38°C), persistent cough, excessive stagnation of thick mucus and dyspnea. Chest X-ray showed diffuse bilateral parenchymal thickening, several bronchiectasis with mucoid impaction, presence of voluminous sub-pleural bubbles. Intravenous antibiotics and corticosteroids were started along with intensive regimen of respiratory physiotherapy for airway clearance. During the first hours her status deteriorated, so that she needed to be transferred to the intensive care unit (ICU) where she required intubation and invasive mechanical ventilation (IMV) with assisted pressure support mode. After 4 hours, despite IMV at maximal protective pressure and maximal antibiotic coverage, arterial blood gas (ABG) analysis showed a persistent unresponsive hypoxaemia, severe respiratory acidosis and severe hypercapnia (pH 7.18, pCO 2 87 cm H 2 O), so veno-venous ECMO was initiated. After 40 hours, ABG analysis improved, so she was successfully extubated and placed on continuous NIV. During ECMO period, mucolytic therapy was doubled and two sessions per day of airway clearance were performed by respiratory physiotherapists. Ventilation parameters improved, so she has been "bridged" from NIV to heated humidified high-flow nasal cannula therapy. ECMO de-cannulation occurred on day 10 and after 48 hours she was discharged from ICU on spontaneous breathing with 5 lt/min oxygen supplementation. Respiratory physiotherapy was continued, reassessed and optimized along with a training program. She was discharged home on after 10 days without oxygen supplementation.

Conclusion
Considering clinical history and the stage of pulmonary disease, this patient wasn't considered yet for LTx. ECMO is an important support to severe CF exacerbations unresponsive to conventional treatments in patients that are not yet candidates to LTx. ECMO "bridge-to-recover" use is emerging in CF and could extend native lungs function in order to take time for waiting list evaluation and optimize allocation. The patients gave the consent to publish clinical data. , secondary outcomes were 6 minutes walked distance, dyspnoea (measured with a Borg scale before and after the 6MWT) and muscular fatigue perception measured in the same way. A comparative analysis was conducted to test the significance of differences within same group and between groups. SPSS version 24.0 was used for data entry and statistical analysis. Univariate comparisons were made using Student's t-test. Alpha was set at 0.05.

Results
Participants of both groups had improvement in all outcomes, presumably due to the overall treatment received. The study shows no statistically significant difference between groups for the main outcome while the 6 minutes walked distance (from 566.1 ± 47.0 m to 604.7 + 43.8 m; p=0.001), dyspnoea (from 6.5 ± 1.5 to 3.7 + 1.3; p=0.004) and muscular fatigue perception (from 3.8 ± 1.5 to 1.7 + 0.7; p=0.001) have improved in the trampoline group in a statistically meaningful way. The retrospective design of the study did not allow to know patients' preference between jumping and cycling.

Conclusions
Jumping on a trampoline could be a valid training modality for people with CF, but more research is needed to better understand its efficacy, safety and patients preference compared with other training modality.

A44
Manual therapy for musculoskeletal pain in cystic fibrosis: review of the Six-year-old girl admitted in the Cystic Fibrosis Centre, "Policlinico Umberto I" of Rome, for a pulmonary exacerbation colonized with Pseudomonas Aeruginosa. She had a CT thorax: various bronchiectasis filled with mucus in lingular area, in superior right lobe and superior left lobe. During hospitalization she received antibiotic therapy, corticosteroids, dornase alpha and, in the first days, daily respiratory physiotherapy with pep-mask. Before the admission, the patient used to have dance classes, four times a week and jumped on a trampoline every day for 45 minutes. The patient's adherence was good -her family actively participated in treatment plan -but her attitude towards the cure, physiotherapy in particular, was critical, because the girl perceived the therapeutic burden as very high.

Materials and methods
In an attempt to increase efficacy and the girl appreciation for treatment, during the 14 days hospitalization she received the prescribed treatment plan together with a new physiotherapy modality using at the same time the "High frequency chest wall oscillation" (Respin 11 Bronchial Clearance System®, Respin-USA Inc. Clarksburg (MD) USA) and the "Intrapulmonary percussive ventilation" (IPV® Percussionaire Corp. (ID) USA), twice a day, each session lasting 30 minutes. The program used for Respin 11® system was "Minnesota kids". We used three modalities for IPV®, each one lasting 9 minutes: middle flow rate, high frequency; middle flow rate, middle frequency; middle flow rate, low frequency. During the one minute break between the modalities, the patient performed assisted coughing. Furthermore, she had a 40 minutes playful physical activity session.

Results
At discharge, there was an improvement of the patient's clinical status: less rattlings, good lung air penetration. Although we couldn't use any validated evaluation scale, there are several evidences that she liked this new physiotherapy treatment: the girl's teacher showed us some draws of her having physiotherapy, her parents reported she was happy during the treatment so the perceived treatment burden decreased.

Conclusions
Medical treatments improve patients' clinical status, but submitting them to a heavy treatment burden. Hence, it is important to find a way to make the treatment more appreciated. In this patient, a nonconventional tool has proved to be successful: the girl changed her attitude to positive, by showing happiness during physiotherapy. This could be an important aspect we could deepen to increase adherence in pediatric patient subjected to heavy treatment burden. The patient gave the consent to publish clinical data.

Materials and methods
The platform Insight Online (IoL) used by respiratory physiotherapists at the CF Centre of Milan was initially screened to detect patients actively on I-neb. Adherence, inhalation and nebulization time, and cleaning data were reviewed and analysed.

Conclusions
IoL gives a unique opportunity to monitor patients' adherence, the performance of the aerosol device and can provides information that could have been missed if using self-reporting. On average, the majority of patients showed good results, however there is a great variability when using this device. Therefore, it may be necessary to provide an EI to train patients at inhaling. It may be also useful to make the training software available at home to improve the inhalation technique. Background Standardized exercise is part of the regular assessment of patients with CF [1]. It is used for the evaluation of physical limitations, to document exercise-associated symptoms or to screen for possible adverse effects of exercise [2]. Exercise is an important therapeutic modality for individuals with CF, with higher physical activity levels being associated with greater aerobic fitness [3], pulmonary function, glycemic control [4], and bone mineral density. The Godfrey protocol has been used for the clinical assessment of adverse reactions to exercise in CF. The aim of this study is to evaluate exercise tolerance in paediatric patients regularly followed-up at Milan CF centre.

Materials and methods
After reviewing the literature and the feasibility of different exercise testing at our centre, we adopted the Godfrey cycle protocol since November 2014 as routinary assessment of exercise. Informed consent and test operator procedure were then realized. After March 2018, following the donation of a paediatric cycloergometer, we have been testing the paediatric population below 120 cm-height as well.

Conclusion
These data suggest that in the pediatric population exercise testing offers an opportunity of detecting significant abnormalities. It would be important to perform cardio-pulmonary exercise testing in patients with abnormal response.

Background
Sweat chloride is the gold standard test for the diagnosis of cystic fibrosis (CF). Internal and External quality controls are mandatory to assess precision and accuracy of test results and are recommended to improve the performance of the laboratory.

Materials and methods
In 2014 the National Centre for Rare Diseases (CNMR) of the Istituto Superiore di Sanità (ISS) established the Italian pilot external quality assessment scheme for CF sweat test (IEQA-ST). In 2015 this activity was recognized as a third party service carried out by the ISS. The program is performed once a year and participation is voluntary. Private and public laboratories, performing sweat test, are invited to participate. The IEQA-ST consists of a coordination board (managing the working plan) and of an evaluation board (assessing the performance of each laboratory) composed by experts from CNMR-ISS and Scientific Societies (SIFC, SIMMESN and SIBiOC). An IEQA-ST webbased utility is used to facilitate communication and data sharing among all parties. Each round consisted of the following items: 1) meeting between the boards to ameliorate items and criteria to be adopted in the upcoming round; 2) agreement by participants on the general rules and the assessment criteria; 3) delivery of 3 sweat like samples (including mock identification data, clinical and technical information; 4) collection of technical, institutional and analytical results (including report) from participants; 5) assessment of results; 6) a decisional meeting between the boards; 7) delivery of the final report to all labs; 8) annual meetings. The latest item represents the qualifying event of this plan. In 2016 the poor performance criteria was also adopted.

Results
The number of labs involved in this activity increased (from 10 to 16); analytical errors decreased by about 45% from 2016 ahead Main feedbacks from labs: to maintain the annual meeting; to improve the content of the final report; to improve the communicating process.

Conclusions
The EQA-ST scheme is continuously evolving in accordance with national and international sweat test recommendations and feedbacks from participants. In this respect, laboratories opinion on the communication process, the technical support and the discussion about the assessment criteria are the driving force to improve the quality of the scheme.

Background
Cystic fibrosis (CF) is the most common recessive genetic disorder in Caucasians. The suspicion of disease comes from the detection of typical symptoms, familiar history and newborn screening (NBS The increased implementation of Cystic Fibrosis (CF) newborn screening (NBS) had led to identification of infants with a positive NBS test but inconclusive diagnostic testing, classified as "CF screen positive, inconclusive diagnosis" (CFSPID) in Europe [1][2]. We retrospectively evaluated the prevalence and clinical outcome of CFSPID patients by two NBS programs in the period 2011-2016 at CF Centre of Florence, Italy.

Materials and methods
We retrospectively evaluated CFSPID and CF patients diagnosed by CF NBS in the years 2011-2016 and followed until 31.12.2017. In this period a pilot project was aimed to assess the diagnostic impact of DNA analysis on the NBS algorithm (immunoreactive trypsin (IRT)meconium lactase -IRT2). IRT was considered elevated for values above the 99 th centile laboratory cut-off. The CFSPID definition was according Munck A et al 1 . All CFSPID patients repeated SC ever 6 months and performed extended CFTR gene analysis (detection rate 98%). During follow up we reclassified children as: CF diagnosis, healthy carrier or healthy in presence respectively of at least two pathological SC or two normal SC for age and 1 or 0 CF-causing mutation. We kept the CFSPID definition when SC was persistently in borderline range or in presence of two CFTR mutations, at least one of which has varying clinical consequence (https://www.cftr2.org/).

Conclusions
In six years CF: CFSPID ratio modified from 0.64:1 to 2.85:1 and 10% of CFSPID progressed to CF. Genetic analysis improved positive predictive value and identified a higher number of CFSPID infants progressing in CF.

Background
The lung transplant's experience is a complex situation where a series of factors interact each others. A good physical condition in the posttransplantation period is not always a guarantee of positive emotional perception. The progressive resumption of life, sometimes are not experienced in a good emotional state, as if the patient could not take his "new time". We report a clinical case of transplant cystic fibrosis patient where the psychological sessions showed this apparent contradiction.

Materials and methods
During the follow-up post-transplantation, we evaluated a patient, currently of 35 years old, with psychological assessment, for a period of six years (2012-2018). To assess depression and anxiety, at first evaluation in 2014, we administered respectively Hospital Anxiety Depression Scale (HADS) and State Trait Anxiety Inventory (STAI), and subsequently, as indicate by the Mental Health International Committee, we administered Patient Health Questionnaire (PHQ-9) and General Anxiety Disorders (GAD-7). Furthermore, during every evaluation, we administered Cystic Fibrosis Questionnaire-Revised (CFQ-R).

Results
During this period the general physical condition of the patient, the lung function and the nutritional state did not show critical aspects. The patient didn't' live any negative personal events. The score of depression, anxiety and quality of life after lung transplant are described in Table 1.

Conclusions
The results of psychological evaluation show higher levels of depression and lower score in QoL than the physical condition could be suggest. This case report underlines that a post-transplant, although without clinical complication, is not always an opportunity for the patients to re-discover all the aspects of the "new life". This patient, like other similar in our experience, seems not to be able to enjoy his "time" of life. As well as, when the disease was disabling, they do not live their time even after transplantation. These patients are looking for a more intensive life and are worry about death. They have the perception of having lost "time" irremediably, due to cystic fibrosis.
Knowing the average life expectancy in the post-transplant, they live like in a "turned hourglass", where the transplant is the moment when the countdown started. We should ask ourselves if we are underestimating some deep aspects of their personal experience, consequently limiting the support we can give them. For this reason it is not only necessary do the psychological screening, but also, every time, the clinical interview. In this way we can really know the patient's needs. Patient gave the consent for publication.

Background
Cystic fibrosis-related diabetes (CFRD) patients suffer from accelerated rates of pulmonary decline [1], also before the onset of overt CFRD [2][3]. This study aimed to determine if in a young CF population early alteration of glucose metabolism correlates with lung function or microbiological respiratory status.

Materials and methods
Clinical (BMI and cBMI), functional (FEV 1 and FVC % predicted) and microbiological (presence of chronic Pseudomonas infection) data were collected from CF patients (>10yrs) who underwent a screening oral glucose tolerance test (OGTT) as part of their routine clinical care since September, 2016 to August, 2018. All data were collected at the time of the last OGTT test. Statistical analysis was performed using T-test to compare means, linear and multiple linear regression (Stata/SE 12.0) after normalization for age. A P-value <0.05 was considered significant in all tests.

Conclusions
Fasting blood glucose is not a helpful screening to diagnose early glucose metabolism alteration in CF patients. CF patients with an impaired glucose metabolism (IGT + CFRD) show a statistically significant lower FEV 1 and FVC percent predicted. Chronic Pseudomonas colonization was higher in IGT and CFRD patients, however not statistical significant. Our data confirm the need of implementation of annual OGTT test in CF patients >10 yrs to identify early alteration in glucose metabolism, thus prompt nutritional advices and therapeutic measures might be implemented.

Background
Cystic Fibrosis is a systemic pathology with associated loss of salts with sweat. Pseudo-Bartter syndrome (PBS) is an uncommon cause of metabolic alkalosis that has been described as a presenting feature of CF, as well as a complication in those with known disease. It is accompanied by chronic salt depletion and sometimes failure to thrive without severe dehydration. PBS is characterized by hyponatremic, hypochloremic metabolic alkalosis that mimics Bartter syndrome, but with no pathology in the renal tubules.

Case report
We present the case of a 1-year-old male with Eastern Europe origins, affected by CF in homozygous for the F508del. The patient comes to our attention very frequently due to repeated dehydration attacks during the whole year, despite the treatment with sodium chloride substitution (2-4 mmol/kg/day), recurrent vomiting and failure to thrive requiring 4 admissions to our ward without real improvement between hospitalizations.

Conclusion
The poor response to therapy and the extremely early onset of such manifestations have made us suspect that the patient may not have the CF-related PBS but the true Bartter syndrome.

Background
Cystic fibrosis (CF) is a multi-organ disease with various manifestations of various organs, but mainly concerning the pulmonary area. Also, pancreatic failure with consequent malnutrition, correlation with diabetes mellitus, obstructive azoospermia and the typical infertility in males, bone involvement with the appearance of osteoporosis have been widely demonstrated as cause of morbidity in CF. Furthermore, vascular complications have not been clearly discussed in clinical trials. The aim of this study is to demonstrate the involvement of the peripheral microcirculation in patients affected by CF, a little debated and in depth aspect in the literature.

Materials and methods
We analyzed 15 patients (age between 10 and 38 years, 6 males and 9 females). The patients underwent a capillaroscopic examination, a noninvasive and easily repeatable instrumental technique that in vivo and in real time study the morphological and functional characteristics of the microcirculation.

Results
The capillaroscopic examination showed morphological abnormalities of the peripheral microcirculation in 13 patients out of 15. 5 patients out of 13 have a reduced capillary density; 9 patients out of 13 have a tortuosity of the loops between 20 and 50%. Other highlighted alterations are t dilatation of the sub papillary plexus in 5 patients out of 13 and a moderate angiotectonic disorder in 10 patients out of 13 (Table 1).

Conclusions
The analysis carried out on the sample of patients examined, highlights a correlation between CF and t microcirculation alterations. This aspect, unlike other places of involvement, is not much discussed in the literature, but could underlines a close link with systemic microcirculation disorder. Therefore, we conclude that these alterations founded in CF may be linked with systemic disease and represent a cause of morbidity.

Materials and methods
In a retrospective observational study, we selected all subjects with diagnosis of cystic fibrosis with nasal polyposis who underwent a FESS in our center in Rovereto hospital. We cultured samples from lower airways (sputum) before and after surgery and samples from paranasal sinuses at surgery date. We compared: 1. the detection of multiresistant bacteria (PA, SM, SEM and MRSA) in lower airways and in paranasal sinuses. 2. the multiresistant pathogens in sputum before and after surgery.

Results
The study included 11 subjects. We detected PA in 8 subjects, SM in 4 subjects and SEM in 2 sunjects, no MRSA was detected.
In the first analysis we observed different pathogens in lower airways and in sinuses samples in 8 out of 11 subjects: we detected SM (4 subjects) and SEM (2 subjects) in samples from lower airways only. In 2 subjects PA was found in lower airway only and in other 2 subjects in paranasal sinuses only. In the second analysis we observed that in 3 subjects we could not detect multiresistant pathogens before surgery, two of them became positive after surgery.8 subjects were positive before surgery, 4 of them became negative after FESS. The paired t test showed a decrease of 27% (95CI -71/+16 %, p-value 0.17) of probability in detection multiresistant bacteria after FESS.

Conclusions
We observed a discrepancy in bacterial detection using samples from lower airways and paranasal sinuses. Moreover, our study shows a negative trend in detection of multiresistant bacteria in sputum after surgery. This might suggest a positive effect of FESS in controlling pathogen colonization in cystic fibrosis.
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