Transcript Status of susceptibility testing in Aspergillus

Status of susceptibility
testing in Aspergillus
Cornelia Lass-Flörl
Medical University of Innsbruck, Austria
Department of Hygiene and
Medical Microbiology
AAA 2006 Athens
…the demand and interest for in vitro
antifungal susceptibility testing
increased.
• the increased incidence of invasive
infections due to Aspergillus
• the increased mortality
• the growing number of new antifungal
agents
Denning, 1996; Groll 1999; Bodey 1992
Methods for susceptibility
testing
• M38-A reference
method for
filamentous fungi,
published by the
Clinical Laboratory
Standard Institute
(CLSI)
• E test
• MTT
• XTT
• Flow cytometry
• ……
CLSI M38-A
Characteristics
• Suitable
• Inoculum
• Inoculum
Standardization
• Test medium
• Format
• Temperature
• Duration of
incubation
• Endpoint
CLSI M38A
• Conidium-and spore forming fungi
• 0.4x104-5x104 CFU/ml
• Spectrophotometrically
•
•
•
•
RPMI 1640
Microdilution
35°C
48h
• No growth
Limitations of susceptibility testing methods
(M38-A, …)
•
•
•
•
•
size of inoculum
the use of growth medium
the time of incubation
the inoculum preparation method
the use of Tween concentration
Lack of detection of amphotericin B resistance
No breakpoints
Rodriguez-Tudela, 2003; Denning, 1997; Gehrt 1995; Gomez-Lopez 2005
E-test
E-test is a commercially available method
for antimicrobial susceptibility testing.
This technique is based on a combination
of the concepts of dilution and diffusion
tests.
For Aspergillus spp., good correlations
with amphotericin B and itraconazole
Etest and M38-A method have been
demonstrated.
Espinel-Ingroff 2003; Pfaller 2000; Szekely 1999;Kontoyiannis 2004
E-test
MIC is influenced by the choice of growth medium, RPMI-based
agars seem to be the most useful.
• MTT, XTT, viability testing………………………
and several other antifungal susceptibility
testing methods for molds have been
developed
• all of these alternative methods correlate
more or less with the standard method
• each also has its own disadvantages:
– XTT or MTT method is cumbersome
– E test is relatively expensive
– Disk diffusion
– Viability tests are suitable for MFC
Ramani 2003; Espinel-Ingroff 1997; Balajee 2002; Lass-Flörl 2001
FUN 1 Viability testing
Lass-Flörl 2003
Viable Aspergillus
Dead Aspergillus,
16 µg/ml Voriconazole
• two isolates of A.
fumigatus were
collected from
patients who did
not respond to
therapy with
itraconazole
• these isolates were
resistant to
itraconazole in a
murine model of
invasive
aspergillosis
Denning, 1997;
• and had elevated itraconazole MICs.
Test conditions
• 106 conidia as inoculum
• 2% 1640 RPMI
supplemented with glucose
• 48 h/no growth
Denning, 1997;
Rodriguez-Tudela et al.
demonstrated that
conidia counting in
haemocytometer for
inoculum preparation is
an accurate,
reproducible and
universal procedure,
independent of the
colour and size of
conidia.
Rodriguez-Tudela 2003;
• The European Committee on Antimicrobial
Susceptibility Testing (EUCAST) has left
the
Subcommittee
on
Antifungal
Susceptibility Testing (AFST-EUCAST) in
charge of the preparation of guidelines for
in vitro susceptibility testing of antifungals
against Aspergillus spp.
• This committee adopted the M38-A
reference
method
and
developed
a
proposed EUCAST broth dilution method
for
susceptibility
testing
against
Aspergillus (EUCAST-AST-ASPERGILLUS).
EUCAST AST Members
Maiken Arendrup, Denmark
Malcolm Richardson (Executive)
Bertrand Dupont, France
Wolfgang Fegeler, Germany
Francesco Barchiesi, Italy
J Peter Donnelly, the Netherlands (Secretary)
Paul Verweij, the Netherlands (Executive)
Per Sandven, Norway
Juan Luis Rodriguez-Tudela, Spain (Chairman)
Manuel Cuenca-Estrella, Spain
Erja Chryssanthou, Sweden
Jacques Bille, Switzerland (Executive)
Caroline Moore, UK
David Denning, UK (Treasurer)
Cornelia Lass-Floerl, Austria
Aristia Velegraki, Greece
Lynda Fenelon, Ireland
Nikolay Klimko, Russia
EUCAST-AST-ASPERGILLUS
…a method to provide a valid, easy, rapid and
economic method for testing the susceptibility
to antifungal agents of Aspergillus spp.
…identify resistance
…to facilitate an acceptable degree of
conformity, e.g. agreement within specified
ranges and between laboratories in measuring
the susceptibility.
Characteristics
CLSI M38-A
EUCAST
Suitability
Conidium forming fungi
Aspergillus fumigatus
Aspergillus spp.
Inoculum
0.4-5x104
CFU/ml
1-2.5x105
CFU/ml
Inoculum
standardization
Haemocytometer
Test medium
Spectrophoto=
metrically
RPMI 1640
Format
Microdilution
Microdiluation
Temperature
35°C
35°C
Duration of incubation
48h
48h
Endpoint
No growth
No growth
RPMI 1640 G2%
Preliminary results
Intralaboratory evaluation of the EUCAST-AST-ASPERGILLUS draft. The table
summarizes the results of reproducibility per participant. The reproducibility was 0.90 in
ICC terms (P<0.01).
Participant Lab 1
Lab 2
Lab 3
Lab 4
Lab 5
Lab 6
ICC
0.91
0.95
0.92
0.90
0.91
0.93
Strain
ICC
A. fumigatus 1
0.85
A. fumigatus 2
0.99
A. terreus 3
0.86
A. flavus 4
0.85
A. flavus ATCC 22019
0.91
A. fumigatus ATCC 204304
0.91
Interlaboratory evaluation of the EUCAST-AST-ASPERGILLUS. The table summarizes
the results of reproducibility per Aspergillus strain in ICC terms.
Interlaboratory evaluation of the EUCAST-AST-ASPERGILLUS. The
table summarizes the ranges and percentages of MIC values (µg/ml)
A. fumigatus ATCC 204305
Drug
MIC range
MICs in the
range (%)
AMB
0.25-1.0
100
ITR
0.12-0.50
100
VOR
0.25-1.0
94.4
POS
0.03-0.25
90.3
A. flavus ATCC 204304
Drug
MIC range
MICs in the
range (%)
AMB
0.50-2.0
97.2
ITR
0.12-0.50
100
VOR
0.50-2.0
91.7
POS
0.12-0.50
91.7
EUCAST AST ASPERGILLUS
The method differentiated
amphotericin B or itraconazoleresistant Aspergillus strains in vivo
from the susceptible ones. The
MICs of amphotericin B and
itraconazole were > 2 and > 8 µg/ml,
respectively.
Gomez-Lopez 2005
Itraconazole resistance
• resistance of itraconazole in A. fumigatus is
detecable in vitro
• interpretations relate to in vivo, and
genotypic determinations of
resistance
• is present in wild type isolates
• several mechanisms responsible for resistance
• cross resistance between posaconazole and
itraconazole
• breakpoints
Denning 1997; Diaz-Guerra 2003; Osherov 2001; Chen 2005; Oakley, 1997
Itraconazole resistance
Number of total
isolates
Species
Number of resistant
isolates (patients)
ITR resistance
isolates
Reference
Acquired
Intrinsic
107
A. fumigatus
4 (3)
4
0
Chryssanthou
156
A. fumigatus
4 (3)
2
2
Dannaoui,
1999
17
A. nidulans
1 (1)
0
1
Dannaoui,
1999
150
A. fumigatus
0
0
0
Verweij,
1998
7
A. fumigatus
3 (2)
1
2
Denning,
1997
1997
EUCAST Discussion document E.Dis.7.1
for fermentative yeasts
www.escmid.org/EUCAST/documents
EUCAST-AST-ASPERGILLUS draft
under evaluation