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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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slide1

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

slide2

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

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)

slide3

Staphylococcus aureus, Pseudomonas aeruginosa

Burkholderia cepacia

Fungal

spores

Bacterial proteases

Fn

Lm

Fn

Lm

Elastase

neutrophils

Inflammatory

reaction

Fg

Bouchara et al., Contrib Microbiol, 1999

Chronic respiratory infections in CF

slide4

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

slide5

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

slide6

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

slide7

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

slide8

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

slide9

10

8

6

Number of isolates

4

2

0

05.18

01.10

03.21

07.29

11.29

02.10

03.07

05.30

09.25

12.13

01.15

02.15

03.21

04.23

05.10

06.07

06.17

09.26

03.02

04.01

04.21

05.10

06.04

07.10

10.23

01.26

03.05

03.13

1993

1994

1995

1996

Some genotypes seem to be selected with the ageing colonization

Mechanisms of this genotype selection ?

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

slide10

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 37°C for 7 days

slide11

Scedosporium apiospermum and cystic fibrosis

Transversal Longitudinal

study study

Aspergillus fumigatus21.446.1

Scedosporium apiospermum3.38.6

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

slide12

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 ?

slide13

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 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

slide14

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

slide15

Colonizationand/or infection

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

slide16

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

slide17

Itra

Itra

8

80

1500

6

60

1000

Eosinophils (cells/mm3 )

Precipitin lines ( )

FEV1 (% )

4

40

500

20

2

0

0

0

0

10

20

30

40

50

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 can’t disregard

the colonization of the airways by S. apiospermum

- contributes to the inflammatory reaction

- "contra-indication" to lung transplantation

slide18

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

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)

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

slide19

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

slide20

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 )

slide21

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

slide22

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

slide23

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

Acrophialophora fusispora and cystic fibrosis

slide24

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

15.7%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

slide25

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

slide26

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. dermatitidisor some Aspergillus or Scedosporium speciesisolated from respiratory secretions from patients with bronchiectasis

Biomarkers of a CF-related disease

slide27

Chronic respiratory infections in CF

Host-Parasite Interaction Study Group, Angers

Véronique ApaireBernard 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/