Status of susceptibility testing in Aspergillus Cornelia Lass-Flörl Medical University of Innsbruck, Austria Department of Hygiene and Medical Microbiology.

Status of susceptibility testing in Aspergillus

Cornelia Lass-FlörlMedical University of Innsbruck, AustriaDepartment 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 resistanceNo 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

Viable Aspergillus Dead Aspergillus, 16 µg/ml Voriconazole

Lass-Flörl 2003

• 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;

Test conditions

• 106 conidia as inoculum

• 2% 1640 RPMI supplemented with glucose

• 48 h/no growth

• and had elevated itraconazole MICs.

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 MembersMaiken 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 fumigatusAspergillus spp.

Inoculum 0.4-5x104 CFU/ml

1-2.5x105 CFU/ml

Inoculum standardization

Spectrophoto=metrically

Haemocytometer

Test medium RPMI 1640 RPMI 1640 G2%

Format Microdilution Microdiluation

Temperature 35°C 35°C

Duration of incubation

48h 48h

Endpoint No growth No growth

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.

Participant

Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 Lab 6

ICC 0.93 0.91 0.95 0.92 0.90 0.91

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).

Preliminary results

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

Interlaboratory evaluation of the EUCAST-AST-ASPERGILLUS. The table summarizes the ranges and percentages of MIC values (µg/ml)

EUCAST AST ASPERGILLUS

The method differentiated amphotericin B or itraconazole-resistant 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 resistanceisolates

Reference

Acquired Intrinsic

107 A. fumigatus 4 (3) 4 0 Chryssanthou 1997

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

EUCAST Discussion document E.Dis.7.1 for fermentative yeastswww.escmid.org/EUCAST/documents

EUCAST-AST-ASPERGILLUS draftunder evaluation