4-Laurence Delhaes

1‐
Université
et
CHU
de
Lille
3‐
Université
et
CHU
de
Rouen
5‐
CHG
de
Dunkerque
7‐
Université
et
CHU
d’Angers
2‐
Université
et
CHU
de
Grenoble
4‐
Université
et
CHU
de
Bordeaux
6‐
Université
et
CHU
de
Dijon
Angers,
June
2009
Aims of the study
National Multicentre
Prospective study
 Epidemiology of fungal presence
in CF patients’sputum
 Species
 Quantity
 Determination of Risk Factors
associated with fungus
presence
Grants:
‐
PHRC
1902
(National
Grant)
‐
Vaincre
la
mucoviscidose
‐
Pfizer®
-In a large CF patient
population (300)
- From different French
geographic areas (see, cities,
mountain & country)
Methods of this prospective observational study
● 7 centres
Mycology Department + CF centre
● Clinical, radiological and
biological patterns
- Environmental factors
- CF pathology itself
- Bacteriological colonization
- ABPA criteria
-Therapies
● Mycological analysis: same protocol
- Serology
- Sputum’s cultures on semi-selective
growth media
- Sensibility to antifungal drugs
(E-test method)
300
patients
(>
6
y)
2007‐08
Data
at
inclusion
of
173
patients
Lung
graft
14
patients
on
graft
list
2
year‐
longitudinal
follow‐up
2010‐11
Results: Epidemiological data
Data
at
inclusion
of
173
patients
● 86.1% patients included during annual check-up
● 13.8% included during exacerbation
● 6.9% included during their annual check-up and with
exacerbation symptoms
Epidemiological Data
Clinical & radiological Data
Microbiological Data
Mycological Data
Therapeutic Data
 + Mycology during
the final year
 At inclusion: + DE
& presence of A.
fumigatus in sputum,
Statistical Analysis
p ? OD?
Continuous data
Categorical data
- Student’s test (normal distribution) - Chi square test (qualitative data)
- Wilcoxon’s test (non-parametric) - Fisher’s exact test (non-parametric)
Multivariate logistic regression model
Results: Epidemiological data
Data
at
inclusion
of
173
patients
29 under
18 years
Numbers
●
●
●
●
Mean of age = 29 ± 10 years
Sex Ratio = 1
Exocrine pancreatic insufficiency= 77.2%, <18 years= 82.8%
Mean of BMI =19.7, <18 years= 17.1

Whole
population
Female


Male
No significant association
Results: Epidemiological data
CFTR
characterization
F508del
Population
(%)
83.3
Genotype
F508del
/
F508del
40
F508del
/
Other
mutation
38.1
Other
/
Other
13.9
Genotype
incomplet
or
unknown
6.9
No
mutation
identified
1.1
(2
patients
without
mutations:
64
&
24
y‐old)
Homozygous / Yeast in sputum cultures during the final year
p= 0.0021
Registre français de la mucoviscidose bilan des données 2006 - Vaincre la
Mucoviscidose et Ined, Paris 2009
Results: Epidemiological data
● Environmental data
o Type of habitation
o Type of activity / hobbies
%
No significant association
Pet
52% (90)
Beach
4% (7)
Country
41%
(71)
Flat
37.7%
(66)
Town
57.8%
(100)
House
63.3%
(109)
Pot plant
52.6%
(91)
Garden
59.5%
(103)
Water
17.9% (31)
Animal
19.7% (34)
Results: Clinical data (whole population)
● Symptoms and clinical characters
o Cough / Dyspnoea (Sadoul’ scale)
o Pulmonary auscultation
● Radiology signs
Yeast in sputum cultures during the final year
p= 0.006
p= 0.014
Results: Clinical data
● Objective measures of lung function
o Median of FEV1: 61% [7-118%]
o Median of FVC: 77% [5-123%]
%
Yeast in sputum cultures during the final year
- Lower FVC % (67 ± 21 vs 82 ± 25) p<0.0001
- Lower FEV1 % (51 ± 25 vs 70 ± 28)
p<0.0001
FCV (%) median (-) mean (. . .)
FEV1 (%) median (-) mean (. . .)
Results: Clinical data
● Severity disease : Shwachman’score (Median)
Yeast in sputum cultures / Lower scores
P=
0.0001
<0.0001
0.041
0.009
0.0002
p<0.001
p<0.004
p<0.004
p<0.005
Results: Microbiological data
● Microbiology during the final year
o Bacterial presence
o Fungal presence
H. Influenzae 33.3%
 S. Aureus
0%
 P. aeruginosa 33.3%
23.1%
15.4%
15.4%
14.3%
9.5%
23.8%
10.9%
10.9%
61.8%
16.7%
13.3%
73.3%
13.6%
9.1%
72.7%
8.3%
16.7%
91.7%
35.3%
11.8%
41.2%
Results: Microbiological data
● Microbiology during the final year
o Bacterial presence
o Fungal presence
Pseudomonas presence / Yeast presence, p=0.0008
 Yeast
 Filamentous fungi
0%
0%
0%
0%
28.6%
47.6%
41.8%
45.5%
53.3%
26.7%
68.2%
45.5%
58.3%
16.7%
23.5%
29.4%
Results: Mycology data
● Chronic colonization
● Sputum at inclusion
Results: Mycology data
● Chronic colonization
● At inclusion
Yeast: 58.1% (18/31)
- 9.8 % without sputum
- Positive direct examination: 17.9%
Pseudo- or Hyphae: 67.7% (21/31)
+
+
p=0.037
-
p=0.0001
+
Results: Mycology data
9.5% A. fumigatus isolates have at
least 1 Azole-MIC elevated
Detail of filamentous fungi in sputum at inclusion
Antifungal susceptibility to azole of A. fumigatus’isolates
Pihet et al. 2009
Results: Therapeutic data
Inhaled treatment 93.3%
<18 y: 86.2% (25/29)
Corticoids: 57.8% - <18 y: 41.4% (12/29)
Under way: 54.3% - <18 y: 41.4% (12/29)
Moulds in sputum cultures, p=0.049
A. fumigatus in sputum
at inclusion, p=0.035
Systemic Corticoids: 30.1% - < 18 y: 24.1% (7/29)
Continued Corticoids: 2.3% - < 18y: 0%
0
1‐3
>3
Continued
Antibiotics
Whole
20.6%
64.4%
15%
15.6%
<
18
years
17.2%
65.5%
17.2%
17.2%
Population
Oral
Antibiotics
(N°
of
courses)
Azithromycin: 53.7% - < 18 y: 65.5% (19/29)
Not Significant
yeast in sputum
cultures, p=0.02
Results: Antifungal therapy data
o Population under antifungal drug during the last 6 months: 20.8%
o Population under antifungal at inclusion time: 14.4%
Conclusion: Significant associations
 Yeast meanly C. albicans:





F508del homozygote,
Pulmonary alteration (Dyspnea, Radiology, FCV, FEV)
Number of days on IV antibiotics, Shwachman Score
Azithromycin treatment
P. aeruginosa colonization
OD=2.4 [1.1-5.2] in logistic procedure including azithromycin,
Shwachman’ activity score, and days on IV antibiotics
 Moulds, and Aspergillus fumigatus:



Negative association with P. aeruginosa
Systemic corticoids OD=2.5 [1.3-5.0] , and OD=2.6 [1.2-5.9]
Positive association with inhaled corticoids
Conclusion
Mucofong program:
Conclusion: Significant associations in representative
population of CF French population
 Finalize the analysis of the whole data of the 300 CF
patients enrolled in the study:


Confirm these previous results,
Organize the follow-up
 ABPA / MBPA:


Complete the ABPA criteria analysis
Analyze which species are involved (A. fumigatus,
Scedosporium, …)
 Fungal risk in cystic fibrosis:


Azole previous treatment and resistant isolates / species
Is-it a major risk or not, when lung transplantation is done?
Conclusion
Mucofong program:
Emergence of Azole Resistance in A.
fumigatus and Spread of a Single
Resistance Mechanism. E Snelders, H van
der Lee, J Kuijpers, A Rijs, J Varga, R
Samson, E Mellado, A Donders, W Melchers,
and P Verweij; PLoSMed, 2008
Acknowledgements
L. Delhaes, E. Fréalle, Y. Lemeille,
B. Coltey, G. Gargala, S. Dominique,
I. Accoceberry, P. Domblides,
I. Durand-Joly, G-A. Loeuille, O. Vagner,
F. Dalle, A-L. Fanton, A. Boldron,
C. Pinel, C. Llerena, M. Pihet, J-L.
Giniès, J-P. Bouchara, C. Person, N.
Wizla,
C. Thumerelle, L. Favennec, C. Marguet,
S. Bui and S. Leroy
CIC (Pr Libersa, Dr Planque,
S. Surmont & S. Schilling)
SED: J. Sallerone & Dr F.
Richard: statistical analysis
Mycology protocol
Sputum
(+/‐
used
of
Digest‐EUR)
30
min
à
37°C
equal
volume
égal
of
a
DDT‐solution
(2,3‐dihydroxy‐1,4‐dithiolbutane
:
DDT
(Digest‐EUR;
Eurobio)
1/10
diluted
with
sterile
water
Direct
examination
Negative
Positive
(Yeast
and/or
filamentous
forms)
Centrifugation
of
the
unused
pellet,
conserved
at
‐20°
for
further
analysis
10µl/
plate
of
7
semi‐selective
growth
media
Cultures
negative
Cultures
positives
Macro‐
and
microscopic
identification
+/‐
molecular
identification
Sub‐culture
to
do
E‐test
analysis
and
to
conserve
isolates
Media
were
inspected
every
2
days,
during
15
days
Colony
number
was
expressed
per
µl
of
sputum
for
each
species
isolated
Importance of SceSel+ agar: Horé et al. 2009
only
when
the
fungus
was
isolated
chronically
or
in
high
quantity
Medium
‐1
Chromogenic
medium
Incubation
T°C
Specificity
37°C
Yeast
Isolation
‐1
Sabouraud
medium
37°C
Filamentous
fungi
A.
fumigatus
‐
2
YPD
+
cycloheximide
37°C
&
25°C
S.
apiospermum
‐1
in‐house‐prepared
dichloran‐rose
bengal‐
chloramphenicol
agar
supplemented
with
benomy
37°C
To
make
easier
isolation
of
other
Filamentous
fungi
(CHROMagar
Candida)
(slow
growing
fungus)
(DRBC
+
bénomyl
)
‐2
in‐house‐prepared
YNB
+
erythritol
agar
37°C
&
25°C
Exophiala
dermatitidis
(erythritol
=
specific
substrate)
Media
were
inspected
every
2
days,
during
15
days
Colony
number
was
expressed
per
µl
of
sputum
for
each
species
isolated
Mycology protocol
Serum
Antibodies
against
A.
fumigatus
‐
N°
of
precipine
‐IgG
quantification
(ELISA
Serion
IgG
Classic)
Antibodies
against
filamentous
fungi
according
to
culture
results:
‐
S.
apiospermum
‐
S.
prolificans
‐
A.
terreus
‐
A.
flavus
‐
Exophiala
dermatitidis,
…
0,5
ml
conserved
at
‐20°
for
further
analysis
Results: Antifungal therapeutic data 20.9%
Agent / Proportion
Name
Population
(%)
Antifungal drug
N° of days
in median
Under way
at inclusion
AMB
whole
<18y
1.2
0
14.5
[14‐15]
0
0
/
ITC
whole
<18y
13.9
13.8
180[20‐185]
180
[76‐180]
12.1
10.3
FLC
whole
<18y
2.3
3.4
29
[21‐30]
30
[/]
0
0
VRC
whole
<18y
2.3
3.4
180
[180‐183]
180
[/]
2.3
3.4
KCZ
whole
<18y
0.6
0.6
/
UNK
/
0
CPF
whole
<18y
0.6
0
21
[/]
/
0
/
Causes (%)
Continued
ABPA
Other
allergy
Infection
12.4
whole
63.9
whole
8.4
whole
22.2
whole
75
<18y
25
<18y
0
<18y
13.8
<18y
Shwachman
Score
Total
score
Points
General
activity
Physical
examination
Nutrition
X
ray
findings
Excellent
(86‐100)
25
normal
normal
normal
normal
Good
(71‐85)
20
lack
of
endurance
rare
cough,
no
clubbing,
minimal
hyperinflation
stools
slightly
abnormal
minimal
accentuation
of
bronchovascular
markings,
early
hyperinflation
15
tires
easily
after
exertion
occasional
cough
wheeze,
increased
respiratory
rate
early
clubbing
stools
often
abnormal,
minimal
abdo
distension,
reduced
muscle
mass
mild
hyperinflation,
patchy
atelectasis,
increases
bronchoalveolar
markings
10
home
teacher
dyspnoea
after
short
walk
frequent
cough,
clubbing,
moderate
hyperinflation
crackles
and
wheezes
wt
and
ht
below
the
3rd
centile,
offensive
stools,
reduced
muscle
mass
moderate
hyperinflation,
widespread
atelectasis
and
areas
of
infection
5
orthopnoea
stays
in
chair
or
bed
tachypnoea,
tachycardia,
extensive
crackles,
cyanosis,
severe
clubbing
marked
malnutrition,
rectal
prolapse,etc
severe
hyperinflation,
lobar
atelectasis
and
bronchiectasis.
nodules/cysts,
cardiac
enlargement
Mild
(56‐70)
Moderate
(41‐55)
Severe
<40
Results: ABPA criteria
● ABPA
o Documented ABPA: 27.2%
o Possible : 7.5%
o No ABPA: 62.4%
o Unknown : 2.9%
25.6% - 74.4%
41.7% - 58.3%
8.3% - 84.3%
● A. fumigatus-positive Ac
o Precipitins + / o IgG (ELISA)
● Presence of A. fumigatus in sputum
o Documented ABPA: 46.8% (22/47)
o Possible :
53.8% (7/13)
o No ABPA:
26.9% (31/115)
Results: Microbiological data
● Microbiology during the final year
o Bacterial presence
o Fungal presence
Results: Clinical data
● Disease severity evaluation (whole population)
o Number of days in hospital for respiratory exacerbation
Median 24 [1-71 days]
o Days on IV antibiotics for respiratory exacerbation Median 28 [0-126 d]
NumberYeast
of days
in sputum cultures / Elevated number of days on IV
antibiotics (36 ± 22 vs 28 ± 19) p=0.049
Canadian Trial 1997
Belgian Trial 2007
Median (__) and Mean (- -)of number of days in hospital; Median (__) and Mean (- -)of days on IV
antibiotics