Establishing in vitro-in vivo correlations for Aspergillus fumigatus: the challenge of azoles versus echinocandins

Abstract

Two clinical isolates of Aspergillus fumigatus, designated AT and DK, were recently obtained from patients failing caspofungin and itraconazole therapy, respectively. The isolates were tested by microdilution for susceptibility to itraconazole, voriconazole, posaconazole, ravuconazole, and caspofungin and by Etest for susceptibility to amphotericin B and caspofungin. Susceptibility testing documented that the DK isolate was azole resistant (itraconazole and posaconazole MICs, >4 microg/ml; voriconazole MIC, 2 microg/ml; ravuconazole MIC, 4 microg/ml), and the resistance was confirmed in a hematogenous mouse model, with mortality and the galactomannan index as the primary and secondary end points. Sequencing of the cyp51A gene revealed the M220K mutation, conferring multiazole resistance. The Etest, but not microdilution, suggested that the AT isolate was resistant to caspofungin (MIC, >32 microg/ml). In the animal model, this isolate showed reduced susceptibility to caspofungin. Sequencing of the FKS1 gene revealed no mutations; the enzyme retained full sensitivity in vitro; and investigation of the polysaccharide composition showed that the beta-(1,3)-glucan proportion was unchanged. However, gene expression profiling by Northern blotting and real-time PCR demonstrated that the FKS gene was expressed at a higher level in the AT isolate than in the susceptible control isolate. To our knowledge, this is the first report to document the presence of multiazole-resistant clinical isolates in Denmark and to demonstrate reduced susceptibility to caspofungin in a clinical A. fumigatus isolate with increased expression of the FKS gene. Further research to determine the prevalence of resistance in A. fumigatus worldwide, and to develop easier and reliable tools for the identification of such isolates in routine laboratories, is warranted.

FIG. 1.

Growth curves for the A. fumigatus DK (a) and AT (b) isolates determined by the EUCAST microdilution method after 2 days of incubation. POS, posaconazole (4 to 0.03 μg/ml); VOR, voriconazole (4 to 0.03 μg/ml); ITR, itraconazole (4 to 0.03 μg/ml); GCon, growth control (no antifungal).

FIG. 2.

Susceptibilities of the A. fumigatus DK (a) and AT (b) isolates to caspofungin (CS), determined by Etest after 2 days of incubation.

FIG. 3.

Survival curves for mice challenged with A. fumigatus DK (a) or A. fumigatus AT (b) on day zero and subsequently treated with caspofungin, low-dose posaconazole, high-dose posaconazole, or glucose (control group). For the AT isolate, the experiment was performed twice (dashed lines and solid lines indicate results for experiments 1 and 2, respectively), because the caspofungin-treated animals in the first experiment accidentally received one posaconazole dose on day 3.

FIG. 4.

Aspergillus GM antigen index values for surviving mice challenged with the A. fumigatus DK [Af (DK)] or A. fumigatus AT [Af (AT)] isolate. Individual values, medians, and interquartile ranges are displayed. Cas, caspofungin; Pos l, low-dose posaconazole; Pos h, high-dose posaconazole; 2, second (repeated) experiment.

FIG. 5.

Light microscopic MEC evaluation according to EUCAST methodology after 24 h at 35°C. Shown are changes in the A. fumigatus AT (A) and A. fumigatus DK (B) isolates under caspofungin (CAS) treatment. The positive control shows long, unbranched hyphal elements. CAS treatment at ≥0.25 μg/ml resulted in abnormal, short, and branched hyphal clusters in both the AT isolate (CAS resistant) and the DK isolate (CS sensitive).

FIG. 6.

Expression analysis of the fks gene in caspofungin-resistant (AT) and -susceptible (control) A. fumigatus isolates. Total RNA was extracted from mycelia grown for 24 h at 37°C in the absence (Co) and presence (CAS) of caspofungin. RNA (10 μg) was separated by agarose gel electrophoresis, blotted, and hybridized with an fks probe coding for the β-1,3-glucan synthase complex. Caspofungin treatment did not influence fks gene expression by the resistant A. fumigatus (AT) isolate relative to that by untreated fungi, whereas the susceptible A. fumigatus control isolate showed lower gene expression when cultured in the presence of CAS. Ethidium bromide staining of 26S and 18S rRNA was used as a loading control.

FIG. 7.

Evaluation of the radial growth rate of the A. fumigatus AT isolate in comparison with those of the caspofungin-susceptible control strains ATCC 13073 and A. fumigatus 293 and the caspofungin-resistant A. fumigatus strain EMFR-S678P on YPD agar (a) and minimal medium agar (b). The growth rate is expressed as the colony diameter (in centimeters) and the Kr (in centimeters per hour) after 24 to 96 h.