Identification of Clinical Mold Isolates by Sequence Analysis of the Internal Transcribed Spacer Region, Ribosomal Large-Subunit D1/D2, and β-Tubulin
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Background: The identification of molds in clinical laboratories is largely on the basis of phenotypic criteria, the classification of which can be subjective. Recently, molecular methods have been introduced for identification of pathogenic molds in clinical settings. Here, we employed comparative sequence analysis to identify molds. Methods: A total of 47 clinical mold isolates were used in this study, including Aspergillus and Trichophyton. All isolates were identified by phenotypic properties, such as growth rate, colony morphology, and reproductive structures. PCR and direct sequencing, targeting the internal transcribed spacer (ITS) region, the D1/D2 region of the 28S subunit, and the ß-tubulin gene, were performed using primers described previously. Comparative sequence analysis by using the GenBank database was performed with the basic local alignment search tool (BLAST) algorithm. Results: For Aspergillus, 56% and 67% of the isolates were identified to the species level by using ITS and ß-tubulin analysis, respectively. Only D1/D2 analysis was useful for Trichophyton identification, with 100% of isolates being identified to the species level. Performances of ITS and D1/D2 analyses were comparable for species-level identification of molds other than Aspergillus and Trichophyton. In contrast, the efficacy of ß-tubulin analysis was limited to genus identification because of the paucity of database information for this gene. Conclusions: The molecular methods employed in this study were valuable for mold identification, although the different loci used had variable usefulness, according to mold genus. Thus, a tailored approach is recommended when selecting amplification targets for molecular identification of molds.
Abstract
Background: The identification of molds in clinical laboratories is largely on the basis of phenotypic criteria, the classification of which can be subjective. Recently, molecular methods have been introduced for identification of pathogenic molds in clinical settings. Here, we employed comparative sequence analysis to identify molds. Methods: A total of 47 clinical mold isolates were used in this study, including Aspergillus and Trichophyton. All isolates were identified by phenotypic properties, such as growth rate, colony morphology, and reproductive structures. PCR and direct sequencing, targeting the internal transcribed spacer (ITS) region, the D1/D2 region of the 28S subunit, and the ß-tubulin gene, were performed using primers described previously. Comparative sequence analysis by using the GenBank database was performed with the basic local alignment search tool (BLAST) algorithm. Results: For Aspergillus, 56% and 67% of the isolates were identified to the species level by using ITS and ß-tubulin analysis, respectively. Only D1/D2 analysis was useful for Trichophyton identification, with 100% of isolates being identified to the species level. Performances of ITS and D1/D2 analyses were comparable for species-level identification of molds other than Aspergillus and Trichophyton. In contrast, the efficacy of ß-tubulin analysis was limited to genus identification because of the paucity of database information for this gene. Conclusions: The molecular methods employed in this study were valuable for mold identification, although the different loci used had variable usefulness, according to mold genus. Thus, a tailored approach is recommended when selecting amplification targets for molecular identification of molds.
참고문헌 (19)
- Nucci M, "Emerging fungal diseases", Clin Infect Dis, 41, pp.521~526 (2005)
- Malani AN, "Changing epidemiology of rare mould infections: implications for therapy", Drugs, 67, pp.1803~1812 (2007)
- Nucci M, "Emerging moulds: Fusarium, Scedosporium and Zygomycetes in transplant recipients", Curr Opin Infect Dis, 16, pp.607~612 (2003)
- Summerbell RC, "Microcoding: the second step in DNA barcoding", Philos Trans R Soc Lond B Biol Sci, 360, pp.1897~1903 (2005)
- Balajee SA, "Sequence-based identification of Aspergillus, fusarium, and mucorales species in the clinical mycology laboratory: where are we and where should we go from here?", J Clin Microbiol, 47, pp.877~884 (2009)
- Ciardo DE, "Identification of moulds in the diagnostic laboratory--an algorithm implementing molecular and phenotypic methods", Diagn Microbiol Infect Dis, 59, pp.49~60 (2007)
- Ciardo DE, "Systematic internal transcribed spacer sequence analysis for identification of clinical mold isolates in diagnostic mycology: a 5-year study", J Clin Microbiol, 48, pp.2809~2813 (2010)
- Iwen PC, "Utilization of the internal transcribed spacer regions as molecular targets to detect and identify human fungal pathogens", Med Mycol, 40, pp.87~109 (2002)
- Atkins SD, "Fungal molecular diagnostics: a mini review", J Appl Genet, 45, pp.3~15 (2004)
- Li DM, "Coniosporium epidermidis sp. nov., a new species from human skin", Stud Mycol, 61, pp.131~136 (2008)
- Barton RC, "Isolation of the fungus Geosmithia argillacea in sputum of people with cystic fibrosis", J Clin Microbiol, 48, pp.2615~2617 (2010)
- Giraud S, "Geosmithia argillacea: an emerging pathogen in patients with cystic fibrosis", J Clin Microbiol, 48, pp.2381~2386 (2010)
- Hinrikson HP, "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", J Clin Microbiol, 43, pp.2092~2103 (2005)
- Graser Y, "Molecular taxonomy of Trichophyton mentagrophytes and T. tonsurans", Med Mycol, 37, pp.315~330 (1999)
- Sun PL, "Molecular characterization of dermatophytes of the Trichophyton mentagrophytes complex found in Taiwan with emphasis on their correlation with clinical observations", Br J Dermatol, 163, pp.1312~1318 (2010)
- Heidemann S, "Signature polymorphisms in the internal transcribed spacer region relevant for the differentiation of zoophilic and anthropophilic strains of Trichophyton interdigitale and other species of T. mentagrophytes sensu lato", Br J Dermatol, 162, pp.282~295 (2010)
- Clinical and Laboratory Standards Institute, "Clinical and Laboratory Standards Institute, Interpretive criteria for identification of bacteria and fungi by DNA target sequencing: approved guideline", ~ (2008)
- Balajee SA, "Aspergillus lentulus sp. nov., a new sibling species of A. fumigatus", Eukaryot Cell, 4, pp.625~632 (2005)
- Montenegro G, "Phenotypic and genotypic characterization of Aspergillus lentulus and Aspergillus fumigatus isolates in a patient with probable invasive aspergillosis", J Med Microbiol, 58, pp.391~395 (2009)