A. Biffi

Triazole resistance in Aspergillus fumigatus isolates from patients with cystic fibrosis in Italy.

BACKGROUND Aspergillus fumigatus is frequently recovered from respiratory secretions of cystic fibrosis (CF) patients. Azole resistance has been increasingly reported. OBJECTIVES To assess the prevalence of azole resistance in A. fumigatus isolates from patients followed by two CF centers of northern Italy. METHODS 423 isolates (220 patients) were screened for azole resistance. Resistance was confirmed with the EUCAST method and cyp51A gene sequencing. Microsatellite genotyping was performed and results were compared with those of environmental resistant isolates. RESULTS No resistance was detected in one center, while 8.2% of the patients of the other center harbored resistant isolates. The TR34/L98H alteration in the cyp51A gene, present in seven cases, resulted associated with poor in-vitro activity of all tested azoles. CONCLUSIONS The environmental origin of the resistance seems to be probable since azole resistance was found also in naïve patients and an identical microsatellite genotype in clinical and environmental isolates was observed.

Effects of treatment in the levels of circulating cytokines and growth factors in cystic fibrosis and dialyzed patients by multi-analytical determination with a biochip array platform.

Chronic inflammatory diseases need non-invasive sensitive, reliable and predictive clinical biomarkers for diagnosis and monitoring therapy. Since inflammation is a complex phenomenon, simultaneous evaluation of different analytes in the same sample may help in defining this complexity and in developing specific anti-inflammatory intervention strategies. In this study, we used a biochip array system capable of measuring 12 cytokines and growth factors (IL-2, IL-4, IL-6, IL-8, IL-10, IL-1 α, IL-1 β, IFN-γ, TNF-α, MCP-1, VEGF, and EGF) in three groups: 97 control subjects; 24 cystic fibrosis (CF) patients before and during the antibiotic treatment (6 and 15days) for acute pulmonary exacerbation as well as 15days after the withdrawal of therapy; 22 children and young adults on chronic hemodialysis (HD) at the beginning and at the end of a standard HD session. CF patients in acute exacerbation displayed higher IL-2, IL-6, VEGF and MCP-1 levels than the control subjects. IL-6 significantly decreased during therapy (P<0.01) but not 15days after the withdrawal of therapy. IL-8 and EGF levels were significantly lower after 15days from the interruption of therapy (P<0.05 and P<0.01 respectively). Regression analysis showed that IL-4 and IL-6 correlated with the amelioration of the respiratory function during therapy. Patients on HD displayed higher IL-6 but lower IL-2, IL-4, IL-8, IFN-γ and EGF levels than control subjects. Serum levels of IL-8, IL-10 and IFN-γ were significantly higher at the end of the HD session (P<0.05 for all three). A biochip array allowed to define a pattern of cytokines/growth factors associated with an acute exacerbation in CF patients and IL-4 and IL-6 as predictors of response to therapy. In younger HD patients, we identified a biomarker pattern which is different from that of older patients. Finally, further studies are warranted to examine the role of these biomarkers in the pathogenesis of complications in HD patients.

290 Oral DHA supplementation in children with cystic fibrosis: a randomized placebo-controlled study

290 Oral DHA supplementation in children with cystic fibrosis: a randomized placebo-controlled study G. Alicandro1, R. Gagliardini2, B. Santini3, P. Rise’4, A. Biffi1, A.S. Tirelli1, R.M. Tiso1, L. Valmarana1, N. Cirilli2, C. Colombo1. 1Fond. IRCCS Ca’ Granda Osp. Maggiore Policlinico, CF Center, Universita degli studi di Milano, Milan, Italy; 2CF Center, U.O. Medicina Pediatrica, A.O.U. Ospedali Riuniti “G. Salesi”, Ancona, Italy; 3CF Center, Centro Pediatrico Pneumologia Pediatrica Ospedale Regina Margherita, Torino, Italy; 4Universita degli studi di Milano, Dip. Di Scienze Farmacologiche, Milano, Italy

Prevalence, geographic risk factor, and development of a standardized protocol for fungal isolation in cystic fibrosis: Results from the international prospective study “MFIP”

Abstract Background Fungi are increasingly recognized for their potential role in contributing to pulmonary damage in Cystic Fibrosis (CF). We therefore designed a prospective international study aimed at (i) determining the prevalence of fungi isolated from sputum samples collected from a large CF population, (ii) comparing the performance of different media used for fungal culture, and (iii) proposing a standardized protocol suitable for CF routine microbiology. Methods An international, consensually designed prospective study was set up ( https://www.ecfs.eu/special-projects/mucofong-international-project ). All centers worked according to the same protocol approved by Lille Ethical Committee. CF sputa were inoculated onto eight semi-selective media incubated at 37 °C or 25 °C–30 °C for 15 days, and inspected twice weekly for fungal growth. Results A total of 469 sputa were collected from patients at 18 European and one Australian CF centers. Positive cultures for fungal growth were significantly associated with patient ages. Aspergillus fumigatus was the most frequently isolated mold. We identified a growing European North-to-South gradient of Scedosporium prevalence, while yeasts, Aspergillus section Fumigati, Cladosporium and Penicillium were significantly more prevalent in the Northern regions. Conclusions According to the CHi-squared Automatic Interaction Detector method, we propose a consensual protocol based on two media (YPDA or Sabouraud medium, and B(+) medium) to detect the main opportunistic molds in CF context; the use of an additional medium being recommended according to the patients clinical status. This standardized protocol allows us to have an accurate overview of the respiratory mycobiome on the culturomic side in CF.

86 Molecular typing of rapidly growing mycobacteria (RGM) in patients with cystic fibrosis (CF)

Objectives To perform molecular typing of clinical strains of RGM isolated from the lower respiratory tract of CF patients with persistent, transient and intermittent infections. Secondary, to assess whether chronic infections are sustained by the same bacterial species, over time. Methods Twenty-three patients, out of the 793 followed at the Regional Reference Centre for Cystic Fibrosis of Milan, have a persistent RGM infection, and 19 show an intermittent infection. From October 2013 to October 2014, 65 strains were isolated and typed by gene sequencing of the rpo B gene. The gene sequences obtained were compared to those already present in the GenBank database to discriminate subspecies of RGM. Results Molecular typing of 46 strains (at two different follow-up times: T1, T2), isolated from 23 patients with chronic RGM colonization, reveals that chronic infection are sustained, in 100% of cases, by MAC (61% M. abscessus sensu stricto, 26% M. bolletii and 13% M. massiliense ), and species identified at T1 is the same identified at T2. In 19 patients, the intermittent infection is sustained by different species of RGM: 37% M. chelonae, 26% M. abscessus sensu stricto, 26% M. bolletii, 5.5% M. fortuitum and 5.5% M. massiliense. Conclusion The chronic infections are sustained by MAC, with a higher prevalence of M. abscessus sensu stricto, and that species acquired at T1 persists over time. Intermittent infections are sustained by M. chelonae, M. fortuitum and MAC. Further correlation studies between the clinical course of the pulmonary disease and the presence of MAC different subspecies are recommended in order to create appropriate therapeutic schemes.

89 Molecular characterization of strains of methicillin-resistant Staphylococcus aureus (MRSA) in patients with cystic fibrosis (CF)

Objectives To evaluate the molecular profile of MRSA strains isolated from the low respiratory tract of CF patients chronically colonized, in order to investigate its origin (HA-MRSA or CA-MRSA) and to examine, in a subgroup of patients, the acquisition of strains with a different molecular profile in the course of time. Methods Forty-four patients (9%) out of the 793 followed at the CF Centre of Milan suffer from MRSA chronic infection. Seventy-eight MRSA strains isolated from these patients have been characterized by: Multiplex PCR ( mec A gene and the SCC mec -type), spa -typing and a specific PCR for Panton Valentine (PVL) gene. Thirty-four out of 44 patients have been monitored over time (T1 = 2012; T2=2014) in order to investigate the molecular profile of MRSA strains isolated after the first infection. Results All 78 MRSA strains have the mecA gene; 76% present SCC mec type IV (CA-MRSA), 21% belong to spa-type t032 (UK-EMRSA-15) and 20% to spa-type t008. Among 4 strains with spa-type t008 and SCC mec type IVa (USA-300), only two show the PVL gene. The remaining 24% present SCC mec type I (HA-MRSA). The molecolar profile of the strains isolated in T2 was the same of the one isolated at T1 in 29/34 patient while 5/34 had different profile. Conclusion The molecular characterization of MRSA strains isolated at Milan CF Centre has shown a high prevalence of CA-MRSA (SCC mec type IV). The distribution of spa-type shows to be similar to that described in other European countries. The different MRSA molecular profiles suggest to carry out genotyping analyses as well, in order to highlight the presence of clones in our CF population.

ePS02.1 Anti-Aspergillus fumigatus antibodies in cystic fibrosis (CF) patients: A comparison of three methods

Objectives The aim of the study is to compare data from an ELISA ( A. fumigatus – Bordier Affinity Products SA, CH) and a Western Blot kit (WB Aspergillus IgG – LDBio Diagnostics, France) with double diffusion (DD) method in agar gel (Ouchterlony). Methods 91 sera from CF pediatric and adult patients followed at our hospital were tested retrospectively. DD, a semi-quantitative method, was performed using somatic and metabolic extracts of A. fumigatus (BioRad and FSK, Microgen Bioproducts Ltd). The interpretation is based on the absence/presence and number of precipitation arcs. ELISA and WB were performed according to manufacturers instructions. Conclusion The 91 sera were classified, according to the DD results, in negative (29), positive (49) and uncertain (13). Six out of the 29 negative sera were positive by ELISA, two out of the 6 were also positive to the WB, both belonging to patients with positive cultures. Notably, one of the two patients was affected by allergic bronchopulmonary aspergillosis. Among the 49 DD positive sera, 3 belonged to patients with positive culture but with stable clinical conditions and were negative for ELISA and WB. Statistical analysis of the data (excluding the doubtful cases) suggests that the distribution of inconsistent outcomes between the ELISA vs DD is compatible with the hypothesis of random variability (p = 0.508). The greatest number of positive results by ELISA compared to WB is not compatible with the hypothesis of random variability (p = 0.022). These assessments suggest that the replacement of DD with the ELISA test does not involve a deviation between the results obtained and that ELISA test confirms its validity as screening test.

56 Colonization by Rasamsonia argillacea in cystic fibrosis patients: A two-year retrospective study

Objective Rasamsonia is a new genus which comprises both thermotolerant Talaromyces and thermophilic Geosmithia species. Rasamsonia argillacea (RA) has recently been reported as the cause of invasive mycosis in human chronic granulomatous disease and also as airway colonizer in patients with cystic fibrosis (CF). The aim of this work is to follow patients colonized by emerging fungus Rasamsonia and study their clinical characteristics and conditions. Methods Over a two years period (2013–2014), seven out of the 798 CF patients, followed at Milan centre, showed at least one positive culture for Rasamsonia . RA was identified macroscopically and microscopically by lactic blue stain. Molecular biology analysis on RA strains will be carried out. Results No correlation between RA colonization and patient sex (4F, 3M) was found. Six patients carried at least one copy of mutated allele DF508. Five patients received antimycotic oral therapy (4 itraconazole and 1 voriconazole) before RA recovery. Two out of the patients (both treated with itraconazole) were intermittently colonized with RA. One of patients was also chronically colonized with Scedosporium apiospermum. Pulmonary function (FEV1) remained substantially unvaried. Conclusion All patients were chronically colonized also by various bacteria species: probably the presence of bacteria and the resulting inflammatory response can predispose CF airways to RA acquisition. Further studies are needed to asses the possible association between genotype of patients and colonization by RA, whether antimycotic administration could be a risk factor for RA acquisition and to investigate the RA impact on pulmonary function.

138 Genetic fingerprinting of Stenotrophomonas maltophilia strains isolated from cystic fibrosis patients

Persons with CF can acquire Burkholderia cepacia complex (Bcc) infection through patient-to-patient contacts or from the environment. The former way of acquisition was well documented in the Prague CF centre where infection rate with a single B. cenocepacia strain ST32 reached over 30% in 1997–2003. The aim of this study was to compare epidemiological situation in the Centre in 2003 vs. 2010, and to evaluate efficacy of infection control in tackling the spread of ST32. System of surveillance exploiting typing techniques (RAPD and MLST) was set up in 2008. A total of 76 out of 211 patients examined by the end of 2003 (36.0%) were infected with Bcc; majority of them (78.9%) harboured epidemic strain ST32. Seven years later, Bcc positivity was detected in 72 out of 374 patients who attended the Prague CF clinic (19.3%). While 39 patients still suffered from infection with ST32 (54.2% of infected), a substantial portion of patients carried strains other than ST32 (see Table; note that ST number is specified only if more than two patients harboured the strain). Notably, only 2 patients within the ST32 group became positive after 2003, with the last case dated in May 2007. Epidemiological situation characterized by increasing heterogeneity of the Bcc population and no occurrence of new ST32 cases is a likely consequence of both healthcare workers’ and patients’ good compliance with strict infection control rules. Supported by NS10543−3, MSM0021620812 and MZ0FNM2005.