Molecular studies reveal frequent misidentification of Aspergillus fumigatus by morphotyping

Abstract

Aspergillus fumigatus has been understood to be the most common cause of invasive aspergillosis (IA) in all epidemiological surveys. However, recent studies have uncovered a large degree of genetic heterogeneity between isolates morphologically identified as A. fumigatus, leading to the description of a new species, Aspergillus lentulus. Here, we examined the genetic diversity of clinical isolates identified as A. fumigatus using restriction enzyme polymorphism analysis and sequence-based identification. Analysis of 50 clinical isolates from geographically diverse locations recorded the presence of at least three distinct species: A. lentulus, Aspergillus udagawae, and A. fumigatus. In vitro, A. lentulus isolates demonstrated decreased susceptibility to antifungal drugs currently used for IA, including amphotericin B, voriconazole, and caspofungin; A. udagawae isolates demonstrated decreased in vitro susceptibility to amphotericin B. Results of the present study demonstrate that current phenotypic methods to identify fungi do not differentiate between genetically distinct species in the A. fumigatus group. Differential antifungal susceptibilities of these species may account for some of the reported poor outcomes of therapy in clinical studies.

FIG. 1.

ML tree of partial nucleotide sequence of rodA region of the Aspergillus isolates revealing three distinct clades: clade 1, A. fumigatus; clade 2, A. lentulus; and clade 3, A. udagawae. Sequences of isolates from section Fumigati were derived from the GenBank database and are denoted by an asterisk, and sequences of isolates derived from a previous study are shown in black (2). Shown in gray are sequences derived from this study. Bootstrap values generated from 1,000 pseudoreplicates are shown.

FIG. 2.

ML tree of Aspergillus benA nucleotide sequences, with the asterisk denoting sequences from the GenBank database (2). Two clades and species distinct from A. fumigatus are revealed: A. udagawae and A. lentulus. Sequences of isolates derived from a previous study are shown in black (2). Shown in gray are sequences derived from this study. Values above the node indicate bootstrap values, generated from 1,000 pseudoreplicates.

FIG. 3.

Analysis of a subclade of isolates that did not type as A. fumigatus or A. lentulus by the RFLP method. ML tree of rodA (a) and benA (b) nucleotide sequences of the subset. Values above the node indicate bootstrap values, generated from 1,000 pseudoreplicates.