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Development of azole resistance in a clinical A.fumigatus isolate with no mutations in the cyp51A gene

Abstract number: P1296

Arendrup M.C., Mortensen K.L., Melchers W., Frimodt-Møller N., Khan H., Verweij P.

Objectives:A. fumigatus triazole clinical resistance has been linked to cyp51A mutations with or without a concomitant tandem repeat in the gene promoter. We report the isolation over a 2.5 year period of 4 sequential A. fumigatus isolates from a CGD patient eventually failing azole and echinocandin combination therapy. The isolates were investigated phenotypically and genotypically and the in vivo efficacy of antifungal drugs was evaluated.

Methods: Susceptibility testing was performed using the EUCAST E.DEF 9.1 microdilution method for conidia-forming moulds. The entire coding region of the cyp51A gene from a susceptible and resistant A. fumigatus isolate was sequenced. Genotyping was performed using microsatellite typing. The in vivo efficacy of antifungal agents was investigated in an immunosuppressed haematogenous model using NRMI mice weighing 26–30 gram. The mice received intraperitoneal injection of 200 mg/kg cyclophosphamide, day -3 and 100 mg/kg day 0 to obtain prolonged neutropenia. Mice were inoculated i.v. with 0.2 ml of a105 CFU suspension day 0 and subsequently treated once daily at days 1–4 and 7–10 with either saline (control), anidulafungin (AND) 12 mg/kg, posaconazole (POS) 20 mg/kg, or both. Kidney burden and mortality were evaluated day 4, 8 and 11 of mice in groups of 6 or 10. The experiments were approved by the Danish Animal Experimentation Committee under the Ministry of Justice (number 2004/561–835).

Results: The POS MICs of the 4 consecutive isolates were 0.125 mg/ml, 0.125 mg/ml, 0.5 mg/ml, and 1 mg/ml, respectively. Caspofungin and AND MICs were stable (range 0.25–0.5 and 0.5–1 mg/ml, respectively). Genotyping showed the isolates were genetically identical and thus of clonal origin and sequencing of the Cyp51 genes revealed no mutations in the hot spots of the gene or its promoter. In the animal model AND alone and AND-POS combination therapy significantly reduced mortality (P < 0.0001) and kidney burden day 8 (P = 0.0121 and 0.0167, respectively) while POS monotherapy did not (P = 0.0856 comparing mortality and P = 0.1167 comparing kidney burden to control group).

Conclusion: This A. fumigatus isolate obtained from a patient failing 2 years of caspofungin+voriconazole combination treatment followed by 2 months of POS+caspofungin treatment showed POS resistance in the mouse model despite not possessing previously described resistance mechanisms. AND alone or in combination with POS was effective against azole resistant aspergillosis.

Session Details

Date: 16/05/2009
Time: 00:00-00:00
Session name: 19th European Congress of Clinical Microbiology and Infectious Diseases
Subject:
Location: Helsinki, Finland, 16 - 19 May 2009
Presentation type:
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