Simple and highly discriminatory microsatellite-based multiplex PCR for Aspergillus fumigatus strain typing.

The answers to important questions concerning Aspergillus fumigatus pathogenicity, transmissions routes and efficacy of treatments require highly discriminating and reproducible genotyping methods. The present study was aimed at improving microsatellite methodology for A. fumigatus typing by reducing the task of strain identification to a single multiplex reaction and by selecting highly accurate short tandem repeat polymorphisms. A set of eight primer pairs was used for the genotype determination of 116 clinical isolates of A. fumigatus obtained from three healthcare centres. A new, automated and highly discriminatory typing method is described for A. fumigatus strains. The optimized multiplex PCR was successfully performed with all tested clinical strains and showed a discriminatory power of 0.9997 among presumably unrelated isolates. The comparison of groups of strains from different health centres showed that 99.6% of the genotypic variation was present within groups. Strains with the same genotype were isolated from the same patient, sometimes recovered more than 1 year later. A few cases of patients at the same clinic unit carrying strains of identical genotype strongly suggested colonization by A. fumigatus during their hospitalization. Specific measures must therefore be taken in order to prevent and restrict such incidents.