Emergence of azole resistance in Aspergillus fumigatus and spread of a single resistance mechanism

Abstract

Background: 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.

Methods and findings: 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.

Conclusions: 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.

Figure 1. Epidemiology of ITZ Resistance in the A. fumigatus Isolates

Blue bars represent the number of patients with a positive A. fumigatus culture (left y-axis) and the red line represents the percentage of those patients with an ITZ+ isolate (right y-axis). The x-axis is the year.

Figure 2. Distribution of Proportions of MICs of 32 ITZ+ A. fumigatus Isolates (Blue Bars) and 115 ITZ− Controls (Red Bars) of ITZ (left upper panel), Voriconazole (right upper panel), Ravuconazole (left lower panel), and Posaconazole (right lower panel)

Left x-axis represents the proportion of isolates and on the right x-axis the MIC distribution is shown.

Figure 3. Genotypic Relatedness of 32 ITZ+ A. fumigatus Isolates and 32 ITZ− Controls

The numbers are the identification numbers of the individual isolates. The ITZ+ A. fumigatus with the L98H substitution and a tandem repeat in the promoter region of the cyp51A gene are underlined and printed in red. The ITZ+ isolates with other or unknown resistance mechanisms are underlined and printed in blue. The ITZ− isolates are printed in black.