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.

Quantifying the relative roles of selective and neutral processes in defining eukaryotic microbial communities

“…Unique sequences were identified and compared with a fungal reference database, and those not assigned to Fungi (6.76% of all unique sequences, 5.75% of reads) were removed. While there will likely be differential genetic diversity within fungal species, empirical studies suggest that multiple species of Ascomycota and Basidiomycota differ by less than 2% at the 26S rDNA gene (Kurtzman and Robnett, 2003;Romanelli et al, 2010). Thus, the remaining 486 279 reads were aligned against a fungal reference database and clustered into groups that share 498% identity.…”

Clinical Significance and Molecular Characterization of Nonsporulating Molds Isolated from the Respiratory Tracts of Bronchopulmonary Mycosis Patients with Special Reference to Basidiomycetes

“…This study demonstrates the relevance of NSMs in a clinical context. Previous series on the molecular characterization of NSMs did not consider the clinical backgrounds of patients (9,37). However, the authors of both of those series indicated that NSMs isolated from otherwise sterile sites could be potential pathogens (9,37).…”

“…Previous series on the molecular characterization of NSMs did not consider the clinical backgrounds of patients (9,37). However, the authors of both of those series indicated that NSMs isolated from otherwise sterile sites could be potential pathogens (9,37). Finally, patients with chronic pulmonary diseases for whom basidiomycetes are isolated from the lower respiratory tract should be thoroughly evaluated to rule out an allergic or invasive disease, and the basidiomycetes should not just be ignored as possible contaminants.…”

Disseminated Human Conidiobolomycosis Due to Conidiobolus lamprauges

“…Template DNA was isolated from PDA plates as described previously (12). PCR was performed using template DNA and primers ITS1 and NL4 to amplify a single contig containing the internal transcribed spacer (ITS) and D1-D2 regions, and the amplified material was then sequenced on both strands with primers ITS1, ITS4, NL1, and NL4 at the UTHSCSA Advanced Nucleic Acids Core Facility (12). The ITS and D1-D2 sequences were then used to search the GenBank database at the NCBI website (http://blast.ncbi .nlm.nih.gov/Blast.cgi) using the BLASTn algorithm.…”