Filamentous fungi and chronic respiratory infections in cystic fibrosis Jean-Philippe Bouchara,...
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Transcript of Filamentous fungi and chronic respiratory infections in cystic fibrosis Jean-Philippe Bouchara,...
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 25-29th
Chronic respiratory infections in CF
Due to mutations in the gene CFTR(Cystic Fibrosis Transmembrane Conductance Regulator)
The most common genetic inhereted disease in the European Caucasian population
The third orphan disease in France
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 aeruginosaBurkholderia cepacia
Lm Fn
Fg
Fungalspores
neutrophils
Inflammatoryreaction
Elastase
Bacterial proteases
Bouchara et al., Contrib Microbiol, 1999
Lm Fn
Aspergillus fumigatus and cystic fibrosis
The major causative agent of colonizationof the airways
9 to 57% of the patientsBecker 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 10.0%
Simmonds et al., Arch Dis Child, 1990 5.8%
Marchant et al., Thorax, 1994 6.8%
Mroueh et Spock, Chest, 1994 6.5%
Becker et al., Chest, 1996 1.9%
Hutcheson et al., Chest, 1996 5.1%
Cimon et al., J Mycol Med, 2000 3.9%
Skov et al., Allergy, 2000 10.9%
Great variations in the criteria used for diagnosis of the disease
Aspergillus fumigatus and cystic fibrosis
Cystic Fibrosis Foundation Consensus ConferenceJune 2001 (Bethesda, Ma)
Minimal diagnostic criteriaAcute 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 IgE concentration (> 500 UI/ml)
+ immediate cutaneous reactivity to Aspergillus
or presence of specific serum IgE
+ presence of specific serum IgG
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 DR2, DR5 (and DR4 or DR7) contribute to susceptibility to APBA, while HLA-DQ2 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 chain (substitution I75V) 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 -1082GG 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 NS3 and NS7)
Recently colonized patients
Huge diversity of genotypes even in the same 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 (EcoRI) followed by hybridization with the repetitive 3.9 probe
Some genotypes seem to be selected with the ageing colonization
Mechanisms of this genotype selection ?
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Collaboration with Renée Sabran Hospital (Giens)
- Transversal study (210 patients)
Mycological examination of sputum samplesand search for precipitating antibodies directed towards pathogenicaspergilli 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
Chronic respiratory infections in CF
Angers
Giens
Procedure used for mycological examination of sputum samples :Inoculation of the sample on two YPDA plates supplemented, for oneplate, with 0.1% cycloheximideIncubation at 37°C for 7 days
Transversal Longitudinalstudy study
Aspergillus fumigatus 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
- 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 49th among 52 genera identified, 1 CFU among 20,000)
Beguin and Nolard, Aerobiologia, 1994
Scedosporium apiospermum and cystic fibrosis
Origin of the contamination of the patients ?
Air (patient’s bedroom, bathroom and livingroom) : 2 x 1 m3 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
Environmental study at home of 6 patients with CF colonized by
S. apiospermum, and followed in Angers University Hospital
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 :
38 out of the 55 potted plants studied were positive
P1 P2 P3 P4 P5 P6
Potted plants 9 5 10 7 13 11
Positive samples 8 4 5 6 7 8
Scedosporium apiospermum and cystic fibrosis
Colonizationand/or infection
Adherenceto the host tissues
Subtilisin-like serine proteaseLarcher et al., Biochem J, 1994
Cu,Zn cytoplasmic superoxide dismutaseLima et al., manuscript in preparation
Invasionof the host tissues
Evasionto the host immune response
Scedosporium apiospermum and cystic fibrosis
Scedosporium apiospermum and cystic fibrosis
Responsible for a chronic colonization of the airways
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
Unrelated isolates Patient 4S1 S2 S3
Usually no clinical signs
Cerebral or disseminated mycoses in immunocompromised patients
Allergic broncho-pulmonary mycoses
Cimon et al., Eur J ClinMicrobiol Infect Dis,
2000
Even in the absence of respiratory infection, one can’t disregardthe colonization of the airways by S. apiospermum
- contributes to the inflammatory reaction
- "contra-indication" to lung transplantation
Scedosporium apiospermum and cystic fibrosis
Pre
cip
itin
lin
es (
)
FE
V1
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Eos
inop
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ells
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1500
1000
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80
60
40
20
0
8
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Itra Itra
Scedosporium apiospermum and cystic fibrosis
CF patient, 26-year old, chronically colonized by S. apiospermum
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
In April 1999, double-lung transplantation
Antibacterial and antifungal prophylaxis (itraconazole orally and aerosols of amphotericin B)
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
UBC 701
UBC 703
M 1 2 3 4 5 6 7 8 9 10 11 12 13 M
7-11: sputum samples
7:
8:
9: 1999, April 4
10: 1999, April 7
11: 1999, April 16
12-13: vitreal fluid 1999, May
Unrelated isolates Patient with CF
1997
1999, April 1
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 )
Chronic respiratory infections in CF
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 patientGonzález-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., 20th EWGCF, Brussels, June 1995
7.5% Haase et al., Lancet, 1990
9.1% Haase et al., Mycoses, 199115.7%Blaschke-Hellmessen et al., Mycoses, 19946.2% 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 signsPneumonia 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 growthof 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 2003and 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
L206W (exon 6A) / V754M (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
Host-Parasite Interaction Study Group, Angers
Véronique Apaire Bernard Cimon
Dominique Chabasse Gérald Larcher
Patricia Licznar Guy Tronchin
PhD 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/
Chronic respiratory infections in CF