Resistance to triazoles was recently reported in Aspergillus fumigatus isolates cultured from patients with invasive aspergillosis. The prevalence of azole resistance in A. fumigatus is unknown. We investigated the prevalence and spread of azole resistance using our culture collection that contained A. fumigatus isolates collected between 1994 and 2007. We investigated the prevalence of itraconazole (ITZ) resistance in 1,912 clinical A. fumigatus isolates collected from 1,219 patients in our University Medical Centre over a 14-y period. The spread of resistance was investigated by analyzing 147 A. fumigatus isolates from 101 patients, from 28 other medical centres in The Netherlands and 317 isolates from six other countries. The isolates were characterized using phenotypic and molecular methods. The electronic patient files were used to determine the underlying conditions of the patients and the presence of invasive aspergillosis. ITZ-resistant isolates were found in 32 of 1,219 patients. All cases were observed after 1999 with an annual prevalence of 1.7% to 6%. The ITZ-resistant isolates also showed elevated minimum inhibitory concentrations of voriconazole, ravuconazole, and posaconazole. A substitution of leucine 98 for histidine in the cyp51A gene, together with two copies of a 34-bp sequence in tandem in the gene promoter (TR/L98H), was found to be the dominant resistance mechanism. Microsatellite analysis indicated that the ITZ-resistant isolates were genetically distinct but clustered. The ITZ-sensitive isolates were not more likely to be responsible for invasive aspergillosis than the ITZ-resistant isolates. ITZ resistance was found in isolates from 13 patients (12.8%) from nine other medical centres in The Netherlands, of which 69% harboured the TR/L98H substitution, and in six isolates originating from four other countries. Azole resistance has emerged in A. fumigatus and might be more prevalent than currently acknowledged. The presence of a dominant resistance mechanism in clinical isolates suggests that isolates with this mechanism are spreading in our environment.

Fourteen Aspergillus fumigatus clinical isolates that exhibited a pattern of reduced susceptibility to triazole drugs were analyzed. The sequences of the cyp51A gene from all isolates showed the presence of a point mutation at t364a, which led to the substitution of leucine 98 for histidine (L98H), together with the presence of two copies of a 34-bp sequence in tandem in the promoter of the cyp51A gene. Quantitative expression analysis (real-time PCR) showed up to an eightfold increase in the level of expression of the cyp51A gene compared to that by the susceptible strain. Three PCR fragments of one azole-resistant strain (strain CM2627) that included the promoter with the tandem repeat and part of cyp51 A with the t364a mutation or PCR fragments with only one of the modifications were used to replace the cyp51A gene of an azole drug-susceptible A. fumigatus wild-type strain (strain CM237). Only transformants which had incorporated the tandem repeat in the promoter of the cyp51A gene and the L98H amino acid substitution exhibited similarly reduced patterns of susceptibility to all triazole agents and similarly increased levels of cyp51A expression, confirming that the combination of both alterations was responsible for the azole-resistant phenotype.

To investigate azole resistance in clinical Aspergillus isolates, we conducted prospective multicenter international surveillance. A total of 3,788 Aspergillus isolates were screened in 22 centers from 19 countries. Azole-resistant A. fumigatus was more frequently found (3.2% prevalence) than previously acknowledged, causing resistant invasive and noninvasive aspergillosis and severely compromising clinical use of azoles.

The use of voriconazole has become common for the management of invasive aspergillosis. However, therapy with voriconazole still sometimes fails, more often because of unresponsive underlying disease than because of resistance of the fungus. Since the first description of itraconazole resistance in Aspergillus fumigatus, ¹ three amino acid substitutions in the 14α-sterol demethylase cyp51A gene, which is the target site for azole drugs, have been described.²

Two cyp51 -related genes ( cyp51A and cyp51B ) encoding 14-α sterol demethylase-like enzymes were identified in the opportunistic human pathogen Aspergillus fumigatus . PCR amplification using degenerate oligonucleotides based on conserved areas of cytochrome P450 demethylases of other filamentous fungi and yeasts allowed the cloning and sequencing of two different homologue genes in A. fumigatus . Southern analysis confirmed that both genes hybridized to distinct genomic loci and that both are represented as single copies in the genome. Comparison of the deduced Cyp51A and Cyp51B proteins with the CYP51 proteins from Penicillium italicum, Aspergillus nidulans, Erysiphe graminis, Uncinula necator, Botrytis cinerea, Ustilago maydis, Cryptococcus neoformans, Candida albicans, Saccharomyces cerevisiae, Candida tropicalis , and Candida glabrata showed that the percentages of identity of the amino acid sequences (range, 40 to 70%) were high enough to consider Cyp51A and Cyp51B to be members of the fungal CYP51 family. Fragments from both genes were also cloned from other Aspergillus spp. ( A. flavus, A. nidulans , and A. terreus ). Phylogenetic analysis showed that, at least in the most pathogenic species of Aspergillus , there are two fungal CYP51 proteins. This is the first report of the existence of two homologue genes coding for 14-α sterol demethylase in the fungal kingdom. This finding could provide insights into the azole resistance mechanisms operating in fungi. The primers used here may be useful molecular tools for facilitating the cloning of novel 14-α sterol demethylase genes in other filamentous fungi.