Occurrence and molecular characterisation of triazole-resistant Aspergillus fumigatus in outdoor and hospital environment in Kuwait

Khan, Z.(1)*; Ahmad, S.(1); Joseph, L.(1); Theyyathel, A.(1); Hagen, F.(2); Meis, J.F.(2); Meis, J.F.(3);


Objective: Aspergillus fumigatus, one of the most prevalent airborne fungal pathogen, causes invasive aspergillosis (IA), a life threatening infection in immunocompromised patients. Triazole resistance is an emerging problem in Aspergillus spp. which impacts the management of IA. Triazole-resistant A. fumigatus strains have also been isolated from the environment from several agriculturally important European and Asian countries and an environmental source of infection with triazole-resistant strains is increasingly being argued. This study describes the isolation and molecular characterization of triazole-resistant A. fumigatus strains from outdoor air and hospital environment in Kuwait. Methods: Cultures of outdoor air and indoor air samples from different wards of a major tertiary care hospital were obtained by exposing malt extract agar (MEA) medium-containing Petri plates. Water and cotton swab samples from different wards/units were also cultured and A. fumigatus colonies were identified by phenotypic and molecular methods. Drug susceptibility of A. fumigatus isolates to itraconazole, posaconazole and voriconazole was carried out by Etest. The dominant resistance mechanism involving TR34/L98H mutations in cyp51A was probed by mixed-format real-time (MF-rt)-PCR and/or multiplex-allele-specific (MAS)-PCR assays. Isolates were typed by nine-locus microsatellite analysis. Results: Of 72 A. fumigatus isolates grown from outdoor air and hospital environment during a 14-month study period, 8 isolates were resistant to itraconazole (MIC >2 mg/ml) with 4 isolates exhibiting additional resistance to voriconazole and posaconazole. MF-rt-PCR/MAS-PCR assays detected TR34 in promoter region of all itraconazole-resistant A. fumigatus isolates and L98H mutation in 7 of 8 isolates. One itraconazole-resistant A. fumigatus isolate contained both wild-type and mutant sequence at cyp51A98 (heteroresistance). None of the susceptible isolates contained these alterations. Results were confirmed by direct DNA sequencing of PCR amplicons. Three microsatellite patterns were observed among the resistant isolates. One clustered with Indian clinical and environmental isolates. Conclusions: This study has shown that triazole-resistant A. fumigatus isolates are also prevalent in Kuwait and, similar to other studies, the dominant resistance mechanism involved TR34/L98H in cyp51A. Presence of triazole-resistant A. fumigatus strains in the environment (including health care facilities) suggests that the possibility of susceptible individuals getting infected with such strains exists even in semi-arid, desert countries like Kuwait in the Arabian Peninsula. Supported by KURS grant MI 01/09.

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