Assessment of Ribosomal Large-Subunit D1-D2, Internal Transcribed Spacer 1, and Internal Transcribed Spacer 2 Regions as Targets for Molecular Identification of Medically Important Aspergillus Species

Abstract

Molecular approaches are now being developed to provide a more rapid and objective identification of fungi compared to traditional phenotypic methods. Ribosomal targets, especially the large-subunit RNA gene (D1-D2 region) and internal transcribed spacers 1 and 2 (ITS1 and ITS2 regions), have shown particular promise for the molecular identification of some fungi. We therefore conducted an assessment of these regions for the identification of 13 medically important Aspergillus species: Aspergillus candidus, Aspergillus (Eurotium) chevalieri, Aspergillus (Fennellia) flavipes, Aspergillus flavus, Aspergillus fumigatus, Aspergillus granulosus, Aspergillus (Emericella) nidulans, Aspergillus niger, Aspergillus restrictus, Aspergillus sydowii, Aspergillus terreus, Aspergillus ustus, and Aspergillus versicolor. The length of ribosomal regions could not be reliably used to differentiate among all Aspergillus species examined. DNA alignment and pairwise nucleotide comparisons demonstrated 91.9 to 99.6% interspecies sequence identities in the D1-D2 region, 57.4 to 98.1% in the ITS1 region, and 75.6 to 98.3% in the ITS2 region. Comparative analysis using GenBank reference data showed that 10 of the 13 species examined exhibited a <1-nucleotide divergence in the D1-D2 region from closely related but different species. In contrast, only 5 of the species examined exhibited a <1-nucleotide divergence from sibling species in their ITS1 or ITS2 sequences. Although the GenBank database currently lacks ITS sequence entries for some species, and major improvement in the quality and accuracy of GenBank entries is needed, current identification of medically important Aspergillus species using GenBank reference data seems more reliable using ITS query sequences than D1-D2 sequences, especially for the identification of closely related species.Aspergillus species are an increasingly important cause of invasive fungal infections in immunocompromised patients (31, 59). Unfortunately, there are few specific clinical signs of invasive aspergillosis and current methods for laboratory diagnosis are less than ideal, particularly in the early stages of the disease (8,49). Given the recent reports of reduced antifungal drug susceptibilities among some Aspergillus species (21, 26, 50), the timely and accurate identification of aspergilli to the species level has become especially important (10). Species identification is also important for epidemiological purposes and as a guide to clinical management (29,47,48).The current laboratory identification of Aspergillus species is based on macroscopic colonial and microscopic morphological characteristics (7,20,45). Over 180 different species in at least 16 subgeneric groups or sections can be distinguished (35,37,38), including approximately 30 species which are recognized as opportunistic pathogens of humans (7). Many clinical laboratories use traditional phenotypic methods of identification and can differentiate only the more common Aspergillus species; the delineation of less common species must b...

Separation and Identification of Two Fungal Strains in Stored Maize

“…In addition, the number of ITS sequences available in the GenBank database has increased rapidly in recent years. The expanding database has improved the quality and accuracy of fungal identification (Hinrikson et al, 2005). The current study combined with previous research has proved that this method is effective and feasible for identifying fungal strains.…”

Mould and yeast identification in archival settings: Preliminary results on the use of traditional methods and molecular biology options in Portuguese archives

“…Because this is not the case with the ITS regions, the D1eD2 is sometimes used to confirm ITS1 or ITS2 results (Cano et al, 2004;Leaw et al, 2006). Nevertheless, and according to Hinrikson et al (2005), the current identification of some Aspergillus species, especially those closely related, is best achieved using the ITS query sequences rather than the D1eD2 regions of the 25e28S region of the genome. Also, ITS has recently been established as the bar-coding region of excellence (Rossman, 2007) as part of the European Consortium for the Barcode of Life initiative maintained by the Centraalbureau voor Schimmelcultures (CBS) and, therefore, an enrichment of the ITS region sequence database is to be expected.…”

“…ITS2 size determination has already been used successfully in the identification of Candida species using automated fluorescent capillary electrophoresis (AFCE), a method also being applied in this study but to the identification of culturable yeast colonies present in a mixture. However, when applied to a complex mixture of filamentous fungi e and given the universe of species one can find in the environment e it will be relatively easy to find different taxa with the same amplicon length, a fact that renders this technique insufficient (Fujita et al, 2001;Hinrikson et al, 2005).…”

Verification of Ribosomal Proteins of <i>Aspergillus fumigatus</i> for Use as Biomarkers in MALDI-TOF MS Identification

“…Identi cation of Aspergillus species has been reported using the internal transcribed spacer (ITS) region between the 18S, 5.8S, and 28S ribosomal RNA (rRNA) genes, 3) the D1/D2 region of the 28S rRNA gene 4) and the housekeeping genes such as β-tubulin 5) and calmodulin 6) genes. On the other hand, we have proposed a ribosomal protein based MALDI-TOF MS method for bacteria characterization.…”