Filamentous fungi and chronic respiratory infections in cystic

Filamentous fungi and chronic respiratory infections in cystic fibrosis Jean-Philippe Bouchara, Bernard Cimon and Dominique Chabasse Host-Parasite Interaction Study Group, UPRES-EA 3142 Angers University Hospital http: //www. med. univ-angers. fr/GEIHP/ XVI ISHAM Congress, Paris, June 25 -29 th

Chronic respiratory infections in CF The most common genetic inhereted disease in the European Caucasian population The third orphan disease in France Due to mutations in the gene CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) Modifications of the electrolytic exchanges Defect in the mucociliairy clearance and thickeness of the bronchial mucus Entrapment of the inhaled bacteria and fungal spores

Chronic respiratory infections in CF Staphylococcus aureus, Pseudomonas aeruginosa Burkholderia cepacia Fungal spores Bacterial proteases Lm Lm Fn Fn Elastase neutrophils Inflammatory reaction Fg Bouchara et al. , Contrib Microbiol, 1999

Aspergillus fumigatus and cystic fibrosis The major causative agent of colonization of the airways 9 to 57% of the patients Becker et al. , Chest, 1996 : 16% - USA Milla et al. , Pediatr Pulmonol, 1996 : 21. 2% - USA Burns et al. , Clin Infect Dis, 1998 : 24. 5% - USA Cimon et al. , Eur J Med Microbiol Infect Dis, 2000 : 46. 1% - France Bakare et al. , Mycoses, 2003 : 45. 7% - Germany Responsible for various diseases in the context of CF Asthma, bronchiitis and aspergilloma Invasive pulmonary aspergillosis (after lung transplantation) Allergic broncho-pulmonary aspergillosis or ABPA

Aspergillus fumigatus and cystic fibrosis Prevalence of ABPA : from 0. 9 to 11% of CF patients Laufer et al. , J Allergy Clin Immunol, 1984 Simmonds et al. , Arch Dis Child, 1990 Marchant et al. , Thorax, 1994 Mroueh et Spock, Chest, 1994 Becker et al. , Chest, 1996 Hutcheson et al. , Chest, 1996 Cimon et al. , J Mycol Med, 2000 Skov et al. , Allergy, 2000 10. 0% 5. 8% 6. 5% 1. 9% 5. 1% 3. 9% 10. 9% Great variations in the criteria used for diagnosis of the disease

Aspergillus fumigatus and cystic fibrosis Cystic Fibrosis Foundation Consensus Conference June 2001 (Bethesda, Ma) Minimal diagnostic criteria Acute or subacute clinical deterioration (cough, wheeze, exercise intolerance, exercise-induced asthma, change in pulmonary function, or increased sputum production) not attributable to another etiology + Elevated total serum Ig. E concentration (> 500 UI/ml) + immediate cutaneous reactivity to Aspergillus or presence of specific serum Ig. E + presence of specific serum Ig. G or new or recent abnormalities on chest radiography (infiltrates or mucus plugging) or chest CT (bronchiectasis) that are not cured with antibiotics Stevens et al. , Clin Infect Dis, 2003

Aspergillus fumigatus and cystic fibrosis Chauhan et al. , J Allegy Clin Immunol, 2000 HLA-DR molecules DR 2, DR 5 (and DR 4 or DR 7) contribute to susceptibility to APBA, while HLA-DQ 2 contributes to resistance Combination of genetic events determines the outcome of ABPA Knutsen et al. , Clin Mol Allergy, 2006 Polymorphism in the Il-4 receptor a chain (substitution I 75 V) constitutes a genetic risk for ABPA Brouard et al. , J Infect Dis, 2005 Polymorphism in the promoter region of Il-10 gene may influence the host response to A. fumigatus Relationship between the -1082 GG genotype and both ABPA and airway colonization by A. fumigatus

Aspergillus fumigatus and cystic fibrosis Genotype study of multiple and sequential isolates Sequence-specific DNA primer and RAPD (primers NS 3 and NS 7) Recently colonized patients Huge diversity of genotypes even in the sample (frequency and diversity of genotypes of A. fumigatus in the environment) With the ageing colonization A dominant genotype, common to the chronically colonized patients, tends to settle Associated with the presence of serum anti-A. fumigatus catalase antibodies (marker of altered lung function) Cimon et al. , J Med Microbiol, 2001

Aspergillus fumigatus and cystic fibrosis Neuvéglise et al. , Eur J Clin Microbiol Infect Dis, 1997 Restriction fragment length polymorphism (Eco. RI) followed by hybridization with the repetitive l 3. 9 probe 8 6 4 2 0 03. 02 04. 01 04. 21 05. 10 06. 04 07. 29 11. 29 01. 10 02. 10 03. 07 03. 21 05. 18 05. 30 07. 10 09. 25 10. 23 12. 13 01. 15 01. 26 02. 15 03. 05 03. 13 03. 21 04. 23 05. 10 06. 07 06. 17 09. 26 Number of isolates 10 1993 1994 1995 1996 Some genotypes seem to be selected with the ageing colonization Mechanisms of this genotype selection ?

Chronic respiratory infections in CF Collaboration with Renée Sabran Hospital (Giens) Angers - Transversal study (210 patients) Giens Mycological examination of sputum samples and search for precipitating antibodies directed towards pathogenic aspergilli or S. apiospermum Cimon et al. , J Mycol Méd, 1995 - Longitudinal study (128 patients followed during 5 years) Cimon et al. , Eur J Med Microbiol Infect Dis, 2000 Procedure used for mycological examination of sputum samples : Inoculation of the sample on two YPDA plates supplemented, for one plate, with 0. 1% cycloheximide Incubation at 37°C for 7 days

Scedosporium apiospermum and cystic fibrosis Transversal study Aspergillus fumigatus Longitudinal study 21. 4 46. 1 Scedosporium apiospermum 3. 3 8. 6 Aspergillus terreus 1. 9 6. 2 Up to 10% in Australia (Williamson et al. , J Clin Microbiol, 2001) Usually associated with A. fumigatus (10 out of 11 patients) Emerged subsequently to A. fumigatus in 9 patients (average delay : 14 months) Improvement of its detection by the use of cycloheximide

Scedosporium apiospermum and cystic fibrosis Origin of the contamination of the patients ? - Highly polluted soils and water - Rarely encountered in indoor environment Study of the air and surfaces in 130 dwellings in Brussels Never found from surface samples Recovered only once in the air (ranked 49 th among 52 genera identified, 1 CFU among 20, 000) Beguin and Nolard, Aerobiologia, 1994

Scedosporium apiospermum and cystic fibrosis Environmental study at home of 6 patients with CF colonized by S. apiospermum, and followed in Angers University Hospital Air (patient’s bedroom, bathroom and livingroom) : 2 x 1 m 3 per site Water (shower, bathtub) : 500 ml per sample Surface samples (bedroom, bathroom and livingroom) : 10 sites per house or flat Soil samples : soil from all potted plants present at home

Scedosporium apiospermum and cystic fibrosis 164 samples analyzed Air, water and surface samples : Only one (from a radiator in the bedroom of one patient) revealed the presence of Scedosporium apiospermum Soil samples : P 1 P 2 P 3 P 4 P 5 P 6 Potted plants 9 5 10 7 13 11 Positive samples 8 4 5 6 7 8 38 out of the 55 potted plants studied were positive

Scedosporium apiospermum and cystic fibrosis Adherence to the host tissues Invasion of the host tissues Evasion to the host immune response Colonization and/or infection Subtilisin-like serine protease Larcher et al. , Biochem J, 1994 Cu, Zn cytoplasmic superoxide dismutase Lima et al. , manuscript in preparation

Scedosporium apiospermum and cystic fibrosis Responsible for a chronic colonization of the airways Unrelated isolates S 1 Patient 4 S 2 S 3 Usually a unique genotype different from one patient to another, and conserved over time despite the antifungal treatment Sometimes associated with one or two other genotypes, found occasionally and closely related to the dominant genotype Defontaine et al. , J Clin Microbiol, 2002

Scedosporium apiospermum and cystic fibrosis Usually no clinical signs Cerebral or disseminated mycoses in immunocompromised patients Allergic broncho-pulmonary mycoses ) 60 6 FEV 1 (% Eosinophils (cells/mm 3 8 80 1500 4 1000 40 500 20 0 10 20 30 40 ) Itra 2 Precipitin lines ( ) Itra Cimon et al. , Eur J Clin Microbiol Infect Dis, 2000 0 50 Even in the absence of respiratory infection, one can’t disregard the colonization of the airways by S. apiospermum - contributes to the inflammatory reaction - "contra-indication" to lung transplantation

Scedosporium apiospermum and cystic fibrosis CF patient, 26 -year old, chronically colonized by S. apiospermum In April 1999, double-lung transplantation Antibacterial and antifungal prophylaxis (itraconazole orally and aerosols of amphotericin B) 4 weeks later: bilateral chorioretinitis and subcutaneous nodules Vitreal fluid and biopsies of the cutaneous lesions S. apiospermum Voriconazole (400 mg/day) during six months Two days after the end of the treatment, sub-acute meningitis Isolation of S. apiospermum from the cerebrospinal fluid Voriconazole again, but the patient died three weeks later Symoens et al. , J Heart Lung Transplant, 2006

Scedosporium apiospermum and cystic fibrosis Unrelated isolates Patient with CF M 1 2 3 4 5 6 7 8 9 10 11 12 13 M UBC 701 7 -11: sputum samples 7: 1997 8: 1999, April 1 9: 1999, April 4 10: 1999, April 7 11: 1999, April 16 UBC 703 12 -13: vitreal fluid 1999, May

Chronic respiratory infections in CF Aspergillus species : Aspergillus terreus A. flavus, A. niger, A. nidulans (transiently recovered) Scedosporium prolificans (mainly reported in Spain) Garcia et al. , Enferm Infecc Microbiol Clin, 1998 Del Palacio et al. , Mycoses, 2001 Lopez et al. , Enferm Infecc Microbiol Clin, 2001 Transient colonization Fetal invasive infection after lung transplantation (Vagefi et al. , Am J Ophthalmol, 2005 )

Penicillium emersonii and cystic fibrosis Biverticillate asymerical penicilli Phialides with a cylindrical base and a tapering tip Smooth cylindrical conidia Isolated repeatedly from sputum samples in a CF patient for nearly five years progressive sensitization to the fungus revealed by Western-blotting Cimon et al. , Med Mycol, 1999

Acrophialophora fusispora and cystic fibrosis In CF, transient colonization of the airways in a 11 -year old patient González-Escalada et al. Rev Iberoam Micol, 2000 Cimon et al. , J Clin Microbiol, 2005 Detected in 4 patients (a 26 -year old patient followed in Giens, and the others in Paris) All patients were also colonized by A. fumigatus, and A. fusispora was isolated repeatedly in one patient Prevalence low, maybe underestimated because of misidentifications with Scopulariopsis spp. , Paecilomyces spp. or S. prolificans Guarro & Gené, J Clin Microbiol, 2002; Sigler & Sutton, J Clin Microbiol, 2002

Acrophialophora fusispora and cystic fibrosis Pale buff colonies of 5 to 6 cm in diameter at day 7 Basally inflated phialides, arising mostly singly on vegetative hyphae Long chains of lemon-shaped or fusiform conidia, with ornamentations arranged in spiral bands

Exophiala dermatitidis and cystic fibrosis Mainly reported in Germany Low prevalence in The Netherlands (1. 8%) Mouton et al. , 20 th EWGCF, Brussels, June 1995 7. 5% Haase et al. , Lancet, 1990 9. 1% Haase et al. , Mycoses, 1991 15. 7%Blaschke-Hellmessen et al. , Mycoses, 1994 6. 2% Horre et al. , Respiration, 2004 Differences in geographic distribution of the fungus ? or lack of standardization of the mycological examination of sputum samples (slow growth: 1 - to 4 -week incubation) Transient or chronic colonization, usually without any clinical signs Pneumonia or invasive pulmonary mycoses Diemert et al. , Scand J Infect Dis, 2001 -Kusenbach et al. , Eur J Pediatr, 1992

Exophiala dermatitidis and cystic fibrosis Patient with bronchiectasis, and repeated episodes of bronchial infections requiring a lobectomy in June 1997 May 2003, hospitalization due to a new episode of hemoptysis July 2003, sputum sample exclusive and profuse growth of E. dermatitidis Similar results on successive samples (Dec 2003, Feb 2004, May 2004) Retrospective serological study (somatic extract): 3 to 6 precipitin lines by CIE since June 2003 and no reactivity with A. fumigatus extract Itraconazole treatment (200 mg/day from November 2003 to June 2004) Improvement of the patient which was cured in May 2004

Exophiala dermatitidis and cystic fibrosis The regular isolation of E. dermatitidis from this patient (65 -year old) with bronchiectasis and repeated episodes of bronchial infections led us to search a CF-related disease Sequencing of the gene CFTR revealed for both alleles minor mutations L 206 W (exon 6 A) / V 754 M (exon 13) Filamentous fungi like E. dermatitidis or some Aspergillus or Scedosporium species isolated from respiratory secretions from patients with bronchiectasis Biomarkers of a CF-related disease

Chronic respiratory infections in CF Host-Parasite Interaction Study Group, Angers Véronique Apaire Bernard Cimon Dominique Chabasse Gérald Larcher Patricia Licznar Guy Tronchin Ph. D students : Marc Pihet Post-doctoral fellows : Osana Lima, Rachid Zouhair Collaborations Pediatric and Adult CF Units of Angers University Hospital Renée Sabran Hospital (Giens, France) Françoise Symoens (IHEM, Brussels, Belgium) Alain Defontaine (University of Nantes, France) http: //www. med. univ-angers. fr/GEIHP/