Azole containing agar plates as a screening tool for azole resistance of Aspergillus fumigatus.

J.W.M. Van der Linden1, M.C. Arendrup2, H.A.L. Van der Lee1, W.J.G. Melchers1, P.E. Verweij1

Author address: 

1Radboud University Medical Center, NIJMEGEN, The Netherlands 2Statens Serum Institut, COPENHAGEN, Denmark

Abstract: 

Objectives: Azole resistance is an increasing problem in the management of Aspergillus diseases, with resistant isolates commonly exhibiting an azole cross-resistant phenotype. Detection of resistance is problematic as cultures may remain negative especially in patients with invasive aspergillosis. But even when Aspergillus is cultured resistance may be missed due to the presence of different genotypes in respiratory cultures. Detection of azole resistant colonies amongst susceptible isolates may be feasible using agar supplemented with azole drugs. We investigated the performance of such an approach using a collection of well characterized Aspergillus fumigatus (Af) isolates. Methods: Four-well plates were prepared containing RPMI-1640 agar medium supplemented with respectively 4 mg/L itraconazole (ITZ), 1 mg/L voriconazole (VCZ) and 0.5 mg/L posaconazole (POS) and growth control in the fourth well. A collection of 70 clinical Af isolates was used to determine the performance of the 4-well plates. Thirty-five isolates were ITZresistant and 35 ITZ-susceptible (wild type) and phenotypic susceptibility profiles were determined using CLSI M38-A. The plates were inoculated using a swab and incubated for 48 hours at 35ºC. Growth on the plates was assessed twice, after 24 hours and 48 hours of incubation by three independent laboratory technicians. In the case of discrepancies between MIC and growth on the 4-well plate, the isolate growing on the agar was again analyzed for phenotypic resistance. Results: Fifty percent of isolates had MICs of >1 mg/L and >4 mg/L for resp. ITZ and VCZ. Five percent of the isolates had MIC of >0.5 mg/L for POS. In the well containing ITZ, 59% and 97% of isolates (MIC >4 mg/L) were able to grow after resp. 24 and 48 hours. In the well containing VCZ resp. 76% and 87% of isolates (MIC >1 mg/L) grew. Sixty percent of the isolates (MIC >0.5 mg/L) were able to grow on the POS containing medium after 48 hours. False-positive growth on the agar was present in 1% of isolates, none of which was due to development of resistance. Most disagreement in assessment of growth in the wells was seen after 24 hours (interreader variability: kappa coefficient 0.94), after 48 hours the interreader agreement was higher (kappa coefficient 0.98). Overall a sensitivity and specificity for detection of resistance was calculated of resp. 94% and 99%. Conclusion: Azole containing agar plates are sensitive and specific to detect ITZ and VCZ resistant isolates when read after 48 h of incubation. The sensitivity for POS resistance was lower but single resistance to POS has not been reported. There was no evidence for resistance induction due to exposure of the isolates to the azoles.
2009

abstract No: 

O1.1

Full conference title: 

4th Trends in Medical Mycology
    • TIMM 4th (2012)