Prsentation PowerPoint

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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
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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)
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Chronic respiratory infections in CF
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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
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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
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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 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
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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 a 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
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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
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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 l 3.9 probe
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Chronic respiratory infections in CF
Collaboration with Renée Sabran Hospital
(Giens) - Transversal study (210
patients) 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
Angers
Giens
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 37C for 7
days
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Scedosporium apiospermum and cystic fibrosis
Transversal Longitudinal study 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
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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
Origin of the contamination of the patients ?
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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 (patients 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
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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
P1 P2 P3 P4 P5 P6 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
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Scedosporium apiospermum and cystic fibrosis
Adherence to the host tissues
Evasionto the host immune response
Invasionof the host tissues
Subtilisin-like serine protease Larcher et al.,
Biochem J, 1994 Cu,Zn cytoplasmic superoxide
dismutase Lima et al., manuscript in preparation
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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 4
S1
S2
S3
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Scedosporium apiospermum and cystic fibrosis
Usually no clinical signs Cerebral or
disseminated mycoses in immunocompromised
patients Allergic broncho-pulmonary
mycoses Cimon et al., Eur J Clin Microbiol
Infect Dis, 2000 Even in the absence of
respiratory infection, one cant disregard the
colonization of the airways by S. apiospermum -
contributes to the inflammatory reaction -
"contra-indication" to lung transplantation
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Scedosporium apiospermum and cystic fibrosis
CF patient, 26-year old, chronically colonized by
S. apiospermum
In April 1999, double-lung transplantation Antibac
terial 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
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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
7-11 sputum samples 7 8 9 1999, April 4
10 1999, April 7 11 1999, April 16 12-13
vitreal fluid 1999, May
1997 1999, April 1

UBC 701
UBC 703
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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 )
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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
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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
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Acrophialophora fusispora and cystic fibrosis
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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,
1991 15.7Blaschke-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
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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
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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 speciesisolated
from respiratory secretions from patients with
bronchiectasis Biomarkers of a CF-related
disease
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Chronic respiratory infections in CF
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/