Morbidity and mortality caused by
Aspergillus species have increased dramatically in recent years, particularly in patients who receive cytotoxic chemotherapy or hematopoietic stem cell transplantation (SCT). In vitro resistance to itraconazole (ITZ) and amphotericin B (AMB), antifungals used to treat or prevent invasive aspergillosis (IA), has been reported; however, the clinical significance of antifungal drug resistance among
Aspergillus species is not presently understood (
13).
Most
Aspergillus infections in humans appear to be caused by typical
A. fumigatus isolates that have green, echinulate conidia (
A. fumigatus Fresenius) (
24,
25). However, infections with atypical variants that differ based on minor morphological features have been reported; for instance,
A. fumigatus var.
ellipticus has smooth, ellipsoid conidia, and
A. fumigatus var.
albus has white conidia (
17,
23,
29). While variants have identical 18S rRNA gene sequences, they differ based on unique mitochondrial cytochrome
b gene sequences (
9,
12,
29). To date, there has been no indication that the variants have different antifungal susceptibility patterns or that antifungal-resistant
A. fumigatus isolates are unique variants.
This study was performed to assess the ITZ susceptibilities of a large number of A. fumigatus isolates obtained from SCT patients at the Fred Hutchinson Cancer Research Center (FHCRC) during the 1990s. The results suggest that ITZ resistance is infrequent, but MICs increased in years subsequent to the start of institutional use of the drug. Most of the A. fumigatus isolates for which ITZ MICs were high had a variant phenotype and a unique genotype. Several also displayed decreased susceptibilities to other antifungals (AMB, ITZ, voriconazole [VRZ], and caspofungin [CAS]).
DISCUSSION
The resistance of
Aspergillus to antifungals has been recognized previously, with most recent studies suggesting that ITZ and AMB resistance among
Aspergillus species may, at least in part, contribute to therapeutic failures (
5,
11). We have observed a time-dependent increase in ITZ MICs for a large number of invasive
Aspergillus isolates recovered from patients with infection in our center. The relative increase in MICs appears to be associated with infections caused by phenotypically and genotypically unique
A. fumigatus isolates for which MICs of ITZ and other antifungals with different modes of action are frequently high.
A. fumigatus is identified in the laboratory predominantly by the characteristic green echinulate conidia, produced basipetally in chains from greenish uniseriate phialides. The conidial state of
A. fumigatus is morphologically very similar to that of
N. fischeri, an ascomycete that has been considered by some taxonomists to be the putative sexual stage of, or at least to be identical to, an ancestor of
A. fumigatus (
7). In the laboratory,
N. fischeri appears at first as white, velvety colonies, which later become granular with formation of the teleomorphic state (ascomata). Although all of our typical isolates initially grew as white colonies (sterile hyphae), they never produced any cleistothecia and all were genotypically distinct from
Neosartorya species, differing both in rRNA gene and mitochondrial cytochrome
b gene sequence.
A. fumigatus as a species is morphologically heterogeneous, which has led to the description of several varieties of the species, including
A. fumigatus var.
albus, var.
acolumnaris, var.
brevipes, var.
phialiseptus, var.
ellipticus, and var.
sclerotiorum. In most of these fungi the distinctions are based on only slight morphological differences, like color of the conidia and septation of the phialides. However, the species distinction for these variants of
A. fumigatus is controversial, largely because of similar secondary-metabolite profiles and DNA complementarity (
19,
29). The pathogenic potentials and antifungal susceptibilities of these fungi have not been compared. The results of our study suggest that the slowly sporulating isolates of
A. fumigatus (all clinical samples), which had unique RAPD-PCR patterns and a distinct mitochondrial cytochrome
b gene sequence, have the potential of being less susceptible to several antifungals. A new variant designation for these isolates is being presented, and a taxonomic description will follow in a future publication. Correct identification and characterization of these atypical isolates may have clinical and epidemiological significance.
MICs of multiple antifungal agents are higher for atypical isolates than for the wild-type
A. fumigatus isolates evaluated in our laboratory (data not shown) and the average wild-type isolates evaluated in prior studies (
3,
5). Growth rates alone are not likely to explain the high MICs measured for the atypical isolates. Whether the high MICs for the atypical
A. fumigatus isolates signify true resistance to antifungal drugs is not clear, as breakpoints have not been generated from large studies and in vivo responses have not yet been assessed. Although cross-resistance of
A. fumigatus to different azoles has been reported (
30), there have been no reports of high MICs of multiple classes of drugs for
A. fumigatus. Recently, an isolate of
Candida albicans with resistance to both ITZ and CAS was described; overexpression of an ABC transporter was implicated as a potential mechanism explaining resistance to both drugs (
26). Transporters or differences in the compositions of cell walls and/or membranes may explain the high MICs for our isolates. Alternatively, the high MICs may be associated with other factors that impact the relative fitness of these isolates upon ex vivo exposure to antifungals. The mechanism of resistance, stability of the phenotype, and significance of the decreased susceptibilities in vivo are currently being determined.
It is not clear whether the high MICs found in the present study correlate to innate or acquired traits of the isolates, although innate resistance seems less likely given the variable frequency of high MICs. Another explanation for the apparent resistance could be selection pressure that might occur during azole therapy, as has been described with
C. albicans (
16). Although none of the patients from whom variants were isolated had prior exposure to ITZ or CAS, all had prior exposure to an azole drug (FLU) and AMB, and one patient had received VRZ.
Whether the atypical isolates described are ubiquitous or represent a strain unique to our institution is not known. The finding that the infected patients were not clustered with regard to either time or place suggests that this isolate may be prevalent in the environment and not acquired from a single source of exposure. Also, there is indirect evidence in the literature that similar isolates have caused disease in other patients. Dannaoui and coworkers reported the isolation of four slowly sporulating
A. fumigatus isolates that had similar RAPD patterns among themselves but whose patterns differed from those of other clinical isolates of
A. fumigatus (
4). These strains also demonstrated an acquired resistance to ITZ, but no attempt was made by these investigators to further characterize the organism. Recently, in an outbreak of
A. fumigatus infections among renal transplant recipients (
21), the majority of the isolates recovered demonstrated an atypical, slow-sporulation phenotype (A. Panackal and M. Brandt, verbal communication). It will be necessary to document the presence of this organism among other culture collections using molecular studies in order to further assess the clinical significance of our observations.
This is the first report of what appears to be a distinct variant of A. fumigatus that causes invasive infection and demonstrates decreased susceptibilities to multiple antifungal drugs. Further studies are necessary to define the clinical significance, prevalence, and mechanisms of resistance of these atypical isolates of A. fumigatus.