17-Regine Horre - ISHAM

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
• Cetrimide agar
• Candi-select agar
• Sabouraud glucose agar
(2d, 37 °C) Gram- bacteria
(2d, 37 °C) Pseudomonas spp.
(2d, 37 °C) Candida spp.
(10d, 30 °C) other fungi

Two different studies
Study 1: & ECA & paraffin enrichment
Study 2: & SceSel+ & wooden stick
Specimen
Study
part
Duration
Number of
patients
In total
Oral /
nasal
swabs
Sputum
samples
Trachealaspirates
1
18
months
81
439
122 / 1
315
1
2
24
months
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
Manufacturer
Incubation time
Incubation temperature
Selective property
Erythritol-
Chloramphenicol
agar
Becton Dickinson,
Sigma;
self production
28-30 d
35-37 °C
E. dermatitidis;
growing as yeast-like
brownish colonies
Paraffin on Sabouraud-
Glucose broth
self production
28-30 d
35-37 °C
Phaeohyphomycetes;
visible by colour change
of the paraffin from hyalin
to brownish

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

Results of study 1
50
Number of isolates
40
30
20
10
0
Candida albicans (236)
Non-albicans-Candida spp. (40)
36
28
26
14
8
5 5
3
3
3
0 0
0 0 1 0 0 1 0
Exophiala dermatitidis (5)
Trichosporon/Geotrichum spp. (5)
Aspergillus spp. (103)
Penicillium spp. (31)
Microascus cinereus (1)
Pseudallescheria boydii (3)
OS: oral swabs; SS: sputum samples
Cladosporium spp. (3)
Other Hyphomycetes (13)
Aureobasidium pullulans (1)
13
OS
SS

Results of study 1
Trichosporon/Geotrichum
spp.
E. dermatitidis
A. fumigatus
other Aspergillus spp.
Penicillium spp.
P. boydii complex
Microascus cinereus
Cladosporium spp.
Aureobasidium pullulans
Other Hyalohyphomycetes
Other Phaeohyphomycetes
CS/SGA 5 1 45 30 12 2 0 0 0 9 1
ECA 0 5 23 17 18 3 1 3 1 2 2
Paraffin 0 3 0 0 0 0 1 2 1 0 13*
In total 5 5 57 38 28 3 1 3 1 10 13
* sterile fungi

Study 2
42 patients, 150 samples (149 SS, 1 TS)
Additional media used
in Study part 2
Manufacturer
Incubation time
Incubation temperature
SceSel+ agar
self production
28-30 d
35-37 °C
Wooden stick in BHI
self production
28-30 d
35-37 °C
Selective property
Scedosporium spp.,
Pseudallescheria spp.;
visible as cotton wooly
greyish colonies
Hyphomycetes;
growing on the part of the
wood prodruding out of the
liquid

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.

fungal growth =>
Wooden stick (WS)

Results of study 2
C. albicans
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 56 24 0 3 38 2 1 1 3 0 0 0 0 0 48
SceSel+ NT NT 2 0 0 0 0 0 7 1 0 0 0 2 12
WS NT NT 1 1 38 3 2 2 4 1 2 2 1 5 62
In total 56 24 2 3 38 3 2 3 7 1 2 2 1 5 69

Results of Scedosporium positive patients
8 isolates in 6 patients
positive
cultures
SGA
SceSel+
WS
Number of isolates
8
3
8
5
Number of isolates in %
100
37.5
100
62.5
Positive samples (N = 150)
8
3
8
5
Positive samples (N = 150) in %
5.3
2.0
5.3
3.3
Positive patients (N = 42)
6
2
6
4
Positive patients (N = 42) in %
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 !