Sequence-based identification of filamentous basidiomycetous fungi from clinical specimens: a cautionary note

Abstract

The species-level identification of sterile and/or arthroconidium-forming filamentous fungi presumed to be basidiomycetes based upon morphological or physiological features alone is usually not possible due to the limited amount of hyphal differentiation. Therefore, a reliable molecular approach capable of the unambiguous identification of clinical isolates is needed. One hundred sixty-eight presumptive basidiomycetes were screened by sequence analysis of the internal transcribed spacer (ITS) and D1/D2 ribosomal DNA regions in an effort to obtain a species identification. Through the use of this approach, identification of a basidiomycetous fungus to the species level was obtained for 167/168 of the isolates. However, comparison of the BLAST results for each isolate for both regions revealed that only 28.6% (48/168) of the isolates had the same species identification by use of both the ITS and the D1/D2 regions, regardless of the percent identity. At the less stringent genus-only level, the identities for only 48.8% (82/168) of the isolates agreed for both regions. Investigation of the causes for this low level of agreement revealed that 14% of the species lacked an ITS region deposit and 16% lacked a D1/D2 region deposit. Few GenBank deposits were found to be complete for either region, with only 8% of the isolates having a complete ITS region and 10% having a complete D1/D2 region. This study demonstrates that while sequence-based identification is a powerful tool for many fungi, sequence data derived from filamentous basidiomycetes should be interpreted carefully, particularly in the context of missing or incomplete GenBank data, and, whenever possible, should be evaluated in light of compatible morphological features.

FIG. 1.

Typical morphological features of basidiomycetes in culture. The morphological features that basidiomycetes may display in culture are shown. Microscopic features include chlamydoconidia (A), arthroconidia (B), spicules (open arrow) and clamp connections (solid arrow) of Schizophyllum commune (C), macroscopic basidiocarp of a dikaryotic Schizophyllum commune isolate (D), setal hyphae of Inonotus (Phellinus) tropicalis (E), and conidia of the Hormographiella anamorph of a Coprinus sp. (F). (A, B, C, E, F) Magnifications, ×880; (D) magnification, ×5.

FIG. 2.

Comparison of ITS and D1/D2 sequence lengths. (A) Comparison of ITS lengths to ITS lengths in GenBank. The sequence lengths of the ITS regions of our isolates were compared to those found in GenBank. The six species represented here were chosen on the basis of being the most redundant among the results from the BLASTn search. (B) Comparison of D1/D2 lengths to D1/D2 lengths in GenBank. The sequence lengths of the D1/D2 regions of our isolates were compared to those found in GenBank.