All Aspergillus spp. isolates cultured from clinical samples that were processed in the 7 university medical microbiology laboratories were routinely screened for the presence of azole resistance, irrespective of the clinical relevance of the culture result. The University Medical Centers are located in 7 different cities in the Netherlands, and clinical isolates were screened during a 20-month period (June 2007–January 2009). Patient data were recorded in a Web-based database. An online questionnaire was completed for every collected isolate. The questionnaire included questions about isolate characteristics (species identification and date of isolation) and patient characteristics (age, sex, and underlying disease).

Aspergillus spp. colonies that grew in primary cultures were subcultured on Sabouraud agar slants supplemented with 4 mg/L of ITZ and incubated at 35°C–37°C. The colonies’ ability to grow on the ITZ agar slants was assessed after 48 hours of incubation. For every isolate that was able to grow on the ITZ agar slants (ITZ positive), the primary culture isolate was sent to the Radboud University Nijmegen Medical Center for further analysis. Isolates that failed to grow on the ITZ agar slants (ITZ negative) were not analyzed. However, for all isolates that were screened, the online questionnaire was completed. At the screening sites, the Aspergillus spp. isolates were identified to species level by conventional methods, i.e., thermotolerance and macroscopic and microscopic assessment of culture morphologic features.

The ITZ agar slants were prepared at the Radboud University Nijmegen Medical Center and distributed to the other medical microbiology laboratories. Each center was provided with a protocol and a set of 6 A. fumigatus isolates. Four isolates were resistant to ITZ (MIC >16 mg/L), while 2 were susceptible (ITZ MICs 0.125 and 0.25 mg/L). The centers were blinded for the resistance profiles and were asked to determine the ability of the isolates to grow on the ITZ agar slants.

Every ITZ-positive isolate was analyzed for certain phenotypic and genotypic features. The phenotypic analysis included the morphologic features of the isolate and susceptibility testing according to established standards (32) by using a broth microdilution format. MICs were determined for ITZ, voriconazole, and posaconazole. Genotypic analyses were performed by complete sequencing of the cyp51A gene by using the reference sequence of strain no. AF338659 from GenBank.

For the confirmed ITZ-positive isolates, additional patient data that included azole exposure <3 months before the date of isolation of the resistant isolate and the presence of Aspergillus spp. disease were requested from the treating physician. Patients with cancer and invasive aspergillosis were classified according to the revised European Organization for Research and Treatment of Cancer/Mycoses Study Group definitions (33). In addition, patient data were collected on treatment and outcome 3 months after diagnosis.

Statistical analyses were performed by using SPSS version 17.0 (IBM, Somers, NY, USA). Analyses consisted of χ² tests and a calculation of the κ coefficient.

Four of 6 control A. fumigatus isolates were resistant to ITZ and expected to grow on the ITZ agar slants, while the remaining 2 isolates were ITZ susceptible and should not grow on the ITZ agar slants. The 4 isolates with the azole-resistant phenotype harbored the TR/L98H resistance mechanism. For 3 ITZ-positive and the 2 ITZ-negative isolates, 100% agreement was found between the 7 centers. For 1 ITZ-positive isolate, the assessment of growth on the ITZ agar slant was incorrect from 3 centers; this isolate was a slowly sporulating A. fumigatus isolate. The κ coefficient calculated to assess the reproducibility of the method was 0.81 (34).

During June 2007 through January 2009, we screened 2,062 Aspergillus spp. isolates from 1,385 patients for azole resistance using the ITZ agar slants. Most isolates were identified as A. fumigatus (1,792/2,062 [86.9%]) (Table 1). For 50 (2.5%) Aspergillus spp. isolates, species identification was not done. Most Aspergillus spp. isolates were isolated from respiratory samples; 1,461 of 2,062 (70.9%) were from sputum cultures, and 60 (2.9%) were from cultures derived from clinical specimens obtained from sterile sites (i.e., tissue specimens obtained through invasive procedures or at autopsy) (Table 1).

Ninety isolates were able to grow on the ITZ agar slants and were sent to the Radboud University Nijmegen Medical Center for further analyses. In vitro susceptibility testing showed that for 84 (93.3%) of 90 ITZ-positive isolates, the MIC of ITZ was >2 mg/L, which was considered resistant (20). Most ITZ-positive isolates also exhibited non–wild-type susceptibility profiles to voriconazole and posaconazole. A resistant phenotype for voriconazole (>2 mg/L) and posaconazole (>0.5 mg/L) was observed in 67/84 (79.8%) and 14/84 (16.7%) of ITZ-positive isolates, respectively. An intermediate susceptibility profile (2 mg/L for voriconazole and 0.5 mg/L for posaconazole) was observed in 12/84 (14.3%) and 44/84 (52.4%) of ITZ-positive isolates, respectively (Table 2) (20).

Sequence-based identification classified 82 ITZ-positive isolates as A. fumigatus and 2 as A. niger. Sequencing of the cyp51A gene showed a substitution of leucine for histidine at codon 98 in combination with a 34-bp tandem repeat in the gene promoter in 74 (90.2%) of 82 resistant A. fumigatus isolates (Table 2). Other cyp51A mutations were found in 3 isolates (substitutions G54W, P216L, and F219I) (Table 2) (7,8,20,21). In 1 isolate, a series of mutations was found, and in the remaining 4 azole-resistant A. fumigatus isolates (4.9%), no mutations were found in the cyp51A gene (8). Because A. niger has no known resistance mechanisms, the 2 azole-resistant A. niger isolates were not further analyzed.

A median of 100 isolates were screened each month, with a range of 78–140 isolates per month. The total number of screened clinical isolates per center ranged from 130 to 449 and were recovered from 84 to 238 patients. The collection of isolates over the study period and the distribution of the recovery of resistant isolates are shown in Figure 1. Overall, 82 (4.6%) of 1,792 screened A. fumigatus isolates were azole resistant. The per-patient analysis showed a prevalence of 5.3%. Figure 2 shows the prevalence of resistance in A. fumigatus per center. The prevalence of azole resistance in A. niger was 3.8%. No seasonal variation was observed.

The screened A. fumigatus isolates were cultured from 1,192 patients, while other Aspergillus spp. isolates were recovered from 193 patients. Among the patients who harbored A. fumigatus isolates, the predominant underlying diseases were cystic fibrosis (382/1,192 [32.1%]) and other pulmonary diseases (265/1,192 [22.2%]) Almost 12% (138/1,192) of the A. fumigatus isolates were from patients who had hematologic/oncologic diseases. The distribution of underlying diseases is shown in Table 3.

The 82 confirmed ITZ-resistant A. fumigatus isolates were recovered from 63 patients. The patients’ ages ranged from 1 to 85 years, with a mean age of 48.9 years. The sex distribution was equal.

Overall, the distribution of underlying diseases in patients with an ITZ-resistant isolate was similar to that observed in the group with ITZ-susceptible isolates, with 1 exception. In the group of patients with ITZ-resistant isolates, significantly more patients had hematologic/oncologic diseases than in the group that harbored ITZ-susceptible isolates (13/63 [20.6%] and 125/1,129 [11.1%], respectively; p<0.05; Table 3).

Aspergillus disease caused by an ITZ-resistant isolate was diagnosed in 23 patients (36%). Invasive aspergillosis was diagnosed in 8 patients (13%), 6 with confirmed and 2 with probable cases (Table 4). Four of these patients were azole naive, and 1 patient had been previously treated with fluconazole, which has no activity against Aspergillus species. All A. fumigatus isolates from patients with azole-resistant invasive aspergillosis harbored the TR/L98H resistance mechanism (Table 4). All 5 patients who were treated with voriconazole monotherapy died within 3 months of receiving a positive culture result. For 3 patients, voriconazole therapy was switched to another class of antifungal compounds, i.e., echinocandin, polyene, or both, but only 1 of those 3 patients survived. Overall, 7 (87.5%) of 8 patients with azole-resistant aspergillosis died within 3 months (Table 4).

Noninvasive Aspergillus spp. disease with an ITZ-resistant isolate was diagnosed in 15 patients. Five patients had cystic fibrosis and allergic bronchopulmonary aspergillosis. Six patients had an aspergilloma; of these patients, 2 were immunocompromised, 1 because of AIDS and 1 because of Job syndrome. Three patients sought treatment for otomycosis; 1 patient had Aspergillus spp. sinusitis. Data on previous exposure to azoles were available for 14 patients with noninvasive Aspergillus spp. disease; and 9 (64.2%) of these patients were azole naive. The TR/L98H mutation was the dominant resistance mechanism, because it was present in 12 (80%) of 15 isolates. Overall, 14 (64.3%) of 22 patients with azole-resistant Aspergillus spp. disease and known status of azole exposure were azole naive at the time the resistant isolate was cultured.