17-Regine Horre

Detection of Pseudallescheria / Scedosporium spp. and
E. dermatitidis in the upper respiratory tract
of patients with cystic fibrosis
in Bonn, Germany
Günter Marklein
Regine Horré
Federal Institute for Drugs and Medical Devices
The BfArM is a Federal Institute within the portfolio of the Federal Ministry of Health
Methods used in routine diagnostics
• Columbia 5% sheep-blood agar (2d, 37 °C) Gram+ bacteria
• McConkey‘s agar
(2d, 37 °C) Gram- bacteria
• Cetrimide agar
(2d, 37 °C) Pseudomonas spp.
• Candi-select agar
(2d, 37 °C) Candida spp.
• Sabouraud glucose agar
(10d, 30 °C) other fungi
Two different studies
Study 1:
Study 2:
&
&
ECA
SceSel+
&
&
paraffin enrichment
wooden stick
Specimen
Study
part
1
2
Duration
18
months
24
months
Number of
patients
In total
Oral /
nasal
swabs
Sputum
samples
Trachealaspirates
81
439
122 / 1
315
1
42
150
none
149
1
Study 1
81 patients; 439 samples (122 OS, 1 NS, 315 SS, 1 TS)
Additional media used
in Study part 1
ErythritolChloramphenicol
agar
Paraffin on SabouraudGlucose broth
Manufacturer
Becton Dickinson,
Sigma;
self production
self production
Incubation time
28-30 d
28-30 d
Incubation temperature
35-37 °C
35-37 °C
E. dermatitidis;
growing as yeast-like
brownish colonies
Phaeohyphomycetes;
visible by colour change
of the paraffin from hyalin
to brownish
Selective property
ECA: E. dermatitidis select agar
E. dermatitidis on SGA
overgrown by Candida
E. dermatitidis on ECA
incubation: 40°C; 7– 28 days
De Hoog GS, Haase G. Antonie Van Leeuwenhoek 1993; 64: 17–26.
Isolation in liquid paraffin
<= liquid
paraffin
<= enrichment
of melanised
fungi
Tr
ic
-a
A
OS: oral swabs; SS: sputum samples
er
H
iu
ho
m
yp
sid
et
es
(1
ns
p.
)
)
3
3)
(1
(3
)
)
0
yc
la
sp
(1
(3
1)
0 1
m
ii
us
yd
re
llu
m
ne
iu
pu
or
ci
bo
(3
3
th
ba
sp
s
a
0
O
eo
do
cu
ri
p.
)
)
3
ur
as
he
sp
03
(5
0
la
ro
sc
um
(1
s
)
26
C
ic
lle
lli
p.
di
(5
0)
)
5
M
da
ci
sp
iti
p.
(4
36
0
eu
ni
s
at
sp
p.
(2
5
Ps
llu
rm
m
sp
ns
0
gi
de
hu
da
ca
10
Pe
a
er
al
ic
di
bi
20
sp
hi
tr
an
al
Number of isolates
30
A
op
eo
C
da
40
Ex
s-
di
/G
an
an
on
ic
C
or
lb
sp
on
ho
N
Results of study 1
50
36
28
14
13
8
0 1
0
OS
SS
5
0
0
5
1
5
3
5
P. boydii complex
Microascus cinereus
Cladosporium spp.
45 30 12
23 17 18
0 0 0
57 38 28
2
3
0
3
0
1
1
1
0
3
2
3
Other Phaeohyphomycetes
Other Hyalohyphomycetes
Aureobasidium pullulans
other Aspergillus spp.
Penicillium spp.
A. fumigatus
E. dermatitidis
CS/SGA
ECA
Paraffin
In total
Trichosporon/Geotrichum
spp.
Results of study 1
0 9 1
1 2 2
1 0 13*
1 10 13
* sterile fungi
Study 2
42 patients, 150 samples (149 SS, 1 TS)
Additional media used
in Study part 2
SceSel+ agar
Wooden stick in BHI
Manufacturer
self production
self production
Incubation time
28-30 d
28-30 d
Incubation temperature
35-37 °C
35-37 °C
Scedosporium spp.,
Pseudallescheria spp.;
visible as cotton wooly
greyish colonies
Hyphomycetes;
growing on the part of the
wood prodruding out of the
liquid
Selective property
SceSel+: Scedosporium select agar
SceSel+
contains:
antibiotics
benomyl
Candida sp.
dichloran
Additionally, some Candida, Mortierella, Doratomyces, Microascus,
Scopulariopsis spp., and other Microascaceae grow at 25 °C
Rainer J, Kaltseis J, de Hoog GS, Summerbell RC. Antonie Van Leeuwenhoek 2008; 93: 315-322.
Wooden stick (WS)
fungal growth =>
other Candida
spp.
Trichosporon
spp.
Geotrichum
spp.
A. fumigatus
A. flavus
A. niger
Penicillium
spp.
P. boydii
complex
S. prolificans
E. dermatitidis
Cladosporium
spp.
Phoma sp.
other
hyphomycetes
Total number of
non- Candida
strains isolated
CS/SGA
SceSel+
WS
In total
C. albicans
Results of study 2
56
NT
NT
56
24
NT
NT
24
0
2
1
2
3
0
1
3
38
0
38
38
2
0
3
3
1
0
2
2
1
0
2
3
3
7
4
7
0
1
1
1
0
0
2
2
0
0
2
2
0
0
1
1
0
2
5
5
48
12
62
69
Results of Scedosporium positive patients
8 isolates in 6 patients
Number of isolates
Number of isolates in %
Positive samples (N = 150)
Positive samples (N = 150) in %
Positive patients (N = 42)
Positive patients (N = 42) in %
positive
cultures
SGA
SceSel+
WS
8
3
8
5
100
37.5
100
62.5
8
3
8
5
5.3
2.0
5.3
3.3
6
2
6
4
14.2
4.8
14.2
9.5
These studies were performed with
several colleagues from
the BfArM* and
Med. Microbiology, University of Bonn°
especially:
Soo-Mi Reiffert°, Elisabeth Müller°, Rüdiger Siekmeier*°,
Sabine Nidermajer*, Tom Grüger*, Norbert Schnitzler*,
Josef Zündorf*, Michaela Lackner*
I am proud, that Michaela asked me
to present you her following NEWS.
Therefore, please keep listening for a few minutes.
Recently developed !
ITS tree of
Pseudallescheria, Scedosporium
and close relatives, based on
more than 700 ITS sequences
Barcoding of Pseudallescheria and Scedosporium species
What is ‘DNA-Barcoding’?
&
=?
What is ‘DNA-Barcoding’?
‘Internal Transcribed Spacer’
was internationally defined as the
‘Barcode of Life’
Based on > 700 ITS sequences including all known haplotypes,
all Pseudallescheria and Scedosporium spp. can be differentiated
on the basis of ITS based RFLP (AluI, Sau3AI, HinfI, HaeIII, RsaI, HindIII)
With knowledge of restriction sites,
species-specific primers were developed
and already
protected by a patent
The primers will be proven for direct detection of these species in
sputum probes of CF patients
M. Lackner, J. Kaltseis, A.H.G. Gerrits van den Ende, S. de Hoog
Thank you very much
for
your attention !