Abstract
BackgroundNew fungal species that are morphologically similar to Aspergillus fumigatus were recently described and included in section Fumigati. Misidentification of such fungal species, particularly of the human pathogens, Aspergillus lentulus, Neosartorya fischeri, Neosartorya hiratsukae, Neosartorya pseudofischeri and Neosartorya udagawae, has been increasingly reported by numerous clinical labs. Nevertheless, A. fumigatus still accounts for more than 90% of all invasive aspergillosis cases. The purpose of the present study was to develop a rapid method for the molecular identification of A. fumigatus to distinguish it from other species within the section Fumigati.ResultsA multiplex PCR was developed using prior information based on β-tubulin (βtub) and rodlet A (rodA) partial gene sequences. PCR amplification of βtub and rodA fragments resulted in a distinctive electrophoretic pattern in A. fumigatus and N. udagawae. The polymorphisms found in the smallest amplified sequence of βtub (153 bp) and rodA (103 bp) genes were then compared among and within species of this taxonomic section. βtub was able to differentiate among 13 individual species and two groups of species that included the pathogenic fungus A. lentulus. A more limited number of sequences were available for rodA; nevertheless, we were able to distinguish Aspergillus viridinutans, N. hiratsukae and N. udagawae.ConclusionsThe assay described in the present study proved to be specific and highly reproducible, representing a fast and economic way of targeting molecular identification of the relevant mould, A. fumigatus, in clinical laboratories.
Highlights
IntroductionMisidentification of such fungal species, of the human pathogens, Aspergillus lentulus, Neosartorya fischeri, Neosartorya hiratsukae, Neosartorya pseudofischeri and Neosartorya udagawae, has been increasingly reported by numerous clinical labs
The assay described in the present study proved to be specific and highly reproducible, representing a fast and economic way of targeting molecular identification of the relevant mould, A. fumigatus, in clinical laboratories
Multiplex optimization The present strategy was proposed to simultaneously target btub and rodlet A (rodA) gene fragments that are specific to a single species (A. fumigatus) and other gene fragments that are common to a group of species
Summary
Misidentification of such fungal species, of the human pathogens, Aspergillus lentulus, Neosartorya fischeri, Neosartorya hiratsukae, Neosartorya pseudofischeri and Neosartorya udagawae, has been increasingly reported by numerous clinical labs. A few other techniques, such as random amplified polymorphic DNA [10,11], restriction fragment length polymorphisms [12] and a new proposed microsphere-based Luminex assay [13], may enable molecular identification of A. fumigatus without sequencing These methodologies are quite time consuming and labour demanding and are impractical in most clinical labs. They can be very expensive when employed to study collections of large numbers of isolates
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