Evaluation of spectrophotometric reading of EUCAST antifungal susceptibility testing of Aspergillus fumigatus

G. Jan1 , L. Kristensen2 , I. Bach Bentsen2 , M. Nielsen2

Author address: 

1Department Of Clinical Microbiology, Aarhus University Hospital, Aarhus N, Denmark, 2Department Of Clinical Microbiology, Aarhus University Hospital, Aarhus, Denmark

Abstract: 

Objectives: The rise in azole resistance in Aspergillus fumigatus1 highlights the importance of antifungal susceptibility testing. While azole containing agar plates have been shown to have excellent sensitivity and specificity in detecting azole resistance2 , access to MIC testing is still important. As commercially available methods have serious limitations reference broth microdilution is the only feasible option at present. The EUCAST method for antifungal susceptibility testing of moulds relies on visual reading of MICs which is laborious and time-consuming. A recent study3 suggests that spectrophotometric determination of MICs of azoles may improve objectivity and allow automatisation of the EUCAST E.Def 9.3 method. The scope of this study is to evaluate the spectrophotometric metod in our center using seven antifungal drugs.

Methods: 22 clinical isolates from A. fumigatus species complex were selected for the study. 9 isolates were resistant to at least one azole while 13 were wild type. All isolates were tested against itraconazole, voriconazole, isavuconazole, posaconazole, amphotericin B, natamycin and terbinafine using EUCAST E.Def 9.3. Visually determined MICs were read at complete inhibition of growth. Spectrophotometric reading was performed using a Thermo Multiskan FC microplate reader with a 492 nm filter. The OD values of the negative control wells were subtracted from all wells and MICs were determined as the lowest concentration corresponding to either 5% growth compared to the drug free control or as 0.02 OD values above background. Spectrophotometrically determined MICs were compared to visually determined MICs. For all drugs the quantitative agreement within 0, 1 and 2 two-fold dilutions was calculated. For itraconazole, voriconazole, isavuconazole, posaconazole and amphotericin B categorical agreement was calculated using EUCAST Clinical breakpoints for fungi v. 9.0.

Results: Results are shown in Table 1. When using the 5% growth endpoint we found a high level of essential agreement (95-100%) for all drugs and a high level of categorical agreement (86-95%) for itraconazole, voriconazole, isavuconazole, posaconazole and amphotericin B. Of importance no major or very major errors was observed. When using the 0.02 OD values above background endpoint however we found only 64% essential agreement for terbinafine and a categorical agreement of 68% for posaconazole including 5% very major errors.

Conclusion: Spectrophotometric determination of MICs using the EUCAST E-Def 9.3 for A. fumigatus species complex appears to be a promising method for all tested drugs. Highest agreement between visual and spectrophotometric MICs is obtained using the 5% growth endpoint. References 1. van der Linden J et al. Prospective Multicenter International Surveillance of Azole Resistance in Aspergillus fumigatus. Emerg Infect Dis. 2015;21(6):1041-1044. 2. Arendrup MC et al; Multicentre validation of 4-well azole agar plates as a screening method for detection of clinically relevant azoleresistant Aspergillus fumigatus, Journal of Antimicrobial Chemotherapy, Volume 72, Issue 12,Pages 3325–3333 3. Meletiadis, J. et al., Spectrophotometric reading of EUCAST antifungal susceptibility testing of Aspergillus fumigatus, Clinical Microbiology and Infection, Volume 23, Issue 2, 98 - 103

Tables: 

2019

abstract No: 

P053

Full conference title: 

9th Trends in Medical Mycology Conference 2019
    • TIMM (2019)