Status of susceptibility testing in Aspergillus

1. Status of susceptibility testing in Aspergillus Cornelia Lass-Flörl Medical University of Innsbruck, Austria Department of Hygiene and Medical Microbiology AAA 2006 Athens

2. …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

3. M38-A reference method for filamentous fungi, published by the Clinical Laboratory Standard Institute (CLSI) E test MTT XTT Flow cytometry …… Methods for susceptibility testing

4. 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 CLSI M38-A

5. 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

6. 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

7. E-test MIC is influenced by the choice of growth medium, RPMI-based agars seem to be the most useful.

8. 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

9. FUN 1 Viability testing Lass-Flörl 2003 Viable Aspergillus Dead Aspergillus, 16 µg/ml Voriconazole

10. 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;

11. and had elevated itraconazole MICs. Test conditions • 106 conidia as inoculum • 2% 1640 RPMI supplemented with glucose • 48 h/no growth Denning, 1997;

12. 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;

13. 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).

14. 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

15. 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.

17. 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). Interlaboratory evaluation of the EUCAST-AST-ASPERGILLUS. The table summarizes the results of reproducibility per Aspergillus strain in ICC terms.

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

19. 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

20. 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

21. Itraconazole resistance

22. EUCAST Discussion document E.Dis.7.1 for fermentative yeasts www.escmid.org/EUCAST/documents EUCAST-AST-ASPERGILLUS draft under evaluation