Aspergillus fumigatus variant with decreased susceptibility to multiple antifungals

Abstract

Isolates of Aspergillus fumigatus that demonstrate resistance to itraconazole (ITZ) have been described previously; however, the prevalence and clinical significance of ITZ resistance are not completely understood. In this study we assessed the ITZ susceptibilities of 128 A. fumigatus isolates that caused invasive infection in 82 stem cell transplant patients before and after the use of ITZ in our institution (study period, 1991 to 2000). The MICs for 10 isolates obtained from seven patients were high, > or 1 microg/ml. The average ITZ MIC increased after institutional use of the drug began in 1995. The majority of the isolates for which MICs were high (6 of 10) and one isolate for which the MIC was low (0.06 microg/ml) demonstrated an unusual phenotype, appearing as predominantly white colonies. For all seven atypical isolates, voriconazole MICs were high (> or = 2 microg/ml), and minimal effective concentrations of caspofungin were high (> or 4 microg/ml). For two of the seven atypical isolates, amphotericin B MICs were high (> or 2 microg/ml). The isolates appeared white due to slow sporulation; however, after prolonged incubations, the isolates sporulated with no difference in conidial color or conidiophore morphology compared with typical isolates. Randomly amplified polymorphic DNA-PCR patterns of these isolates were distinct compared with those of other A. fumigatus isolates. Sequencing of 18S rRNA genes confirmed that all were A. fumigatus; however, the mitochondrial cytochrome b gene sequences of all the atypical isolates were unique. These data suggest the potential presence of a genetically unique, poorly sporulating variant of A. fumigatus that demonstrates decreased susceptibilities to several antifungals.

FIG. 1.

(a) Typical blue-green sporulating colonies of A. fumigatus Fresenius (left panel) are shown compared to fluffy white colonies of the poorly sporulating variant, which are composed of predominantly sterile hyphae (right panel). Both pictures were taken after 7 days of incubation on CZD at 35°C. (b) Comparison of the radial growth of the atypical and wild-type A. fumigatus strains, with both isolates growing on CZD. Each gray square represents the average radius of colony growth of seven atypical A. fumigatus isolates, and the white triangles represent the average radius of colony growth of A. fumigatus wild-type isolates from three individual experiments (± the standard deviation). Radial growth was measured as the radius of the expanding colony in millimeters (y axis) relative to hours of incubation (x axis). Growth rates during a 37°C incubation are shown. Growth rates did not differ when the isolates were incubated at 25°C or when they were grown on multiple media (not shown). (c) Reduction of the XTT dye by the atypical (white triangles) and wild-type (gray squares) isolates over time. The XTT dye was used as an indicator of fungal burden.

FIG. 2.

RAPD-PCR profiles. Profiles of several typical A. fumigatus isolates and one atypical variant (indicated by *) are shown. Primers utilized for each reaction are indicated. DNA molecular weight markers are shown in the leftmost lane of each panel.

FIG. 3.

Comparison of mitochondrial subunit b gene sequences. Sequences of genes from atypical isolates are compared with A. fumigatus Fresenius, other known Aspergillus variants, and Neosartorya species (29). Only nucleotides that differ from those of A. fumigatus Fresenius (top) are shown. The sequences of the atypical isolates differed from those of A. fumigatus (indicated by arrows), A. fumigatus var. neoellipticus (indicated by *), and Neosartorya species.