Multilocus DNA sequence comparisons rapidly identify pathogenic molds

Abstract

The increasing incidence of opportunistic fungal infections necessitates rapid and accurate identification of the associated fungi to facilitate optimal patient treatment. Traditional phenotype-based identification methods utilized in clinical laboratories rely on the production and recognition of reproductive structures, making identification difficult or impossible when these structures are not observed. We hypothesized that DNA sequence analysis of multiple loci is useful for rapidly identifying medically important molds. Our study included the analysis of the D1/D2 hypervariable region of the 28S ribosomal gene and the internal transcribed spacer (ITS) regions 1 and 2 of the rRNA operon. Two hundred one strains, including 143 clinical isolates and 58 reference and type strains, representing 43 recognized species and one possible new species, were examined. We generated a phenotypically validated database of 118 diagnostic alleles. DNA length polymorphisms detected among ITS1 and ITS2 PCR products can differentiate 20 of 33 species of molds tested, and ITS DNA sequence analysis permits identification of all species tested. For 42 of 44 species tested, conspecific strains displayed >99% sequence identity at ITS1 and ITS2; sequevars were detected in two species. For all 44 species, identifications by genotypic and traditional phenotypic methods were 100% concordant. Because dendrograms based on ITS sequence analysis are similar in topology to 28S-based trees, we conclude that ITS sequences provide phylogenetically valid information and can be utilized to identify clinically important molds. Additionally, this phenotypically validated database of ITS sequences will be useful for identifying new species of pathogenic molds.

FIG. 1.

28S and ITS1/ITS2 DNA sequence-based phylogenetic trees of clinically significant molds. Trees were drawn using the neighbor-joining method with 1,000 bootstrap analyses. Numbers at the nodes indicate the bootstrap values, and the bars indicate relative genetic distance. The source of the organism or sequence is indicated in Table 2. The zygomycetes Mucor spp. and Rhizopus spp. were used as outgroups in both trees. The trees include organisms for which DNA from the type strain was sequenced in our laboratory or for which type strain sequence data were verified by identity to a DNA sequence generated in our laboratory from a conspecific isolate. (A) ITS-based tree of 32 molds. Analysis included partial 18S and 28S sequences and complete ITS1, 5.8S, and ITS2 sequences. (B) 28S-based tree of 27 molds. Analysis included the D1/D2 hypervariable region of the 28S rRNA gene.

FIG. 1.

28S and ITS1/ITS2 DNA sequence-based phylogenetic trees of clinically significant molds. Trees were drawn using the neighbor-joining method with 1,000 bootstrap analyses. Numbers at the nodes indicate the bootstrap values, and the bars indicate relative genetic distance. The source of the organism or sequence is indicated in Table 2. The zygomycetes Mucor spp. and Rhizopus spp. were used as outgroups in both trees. The trees include organisms for which DNA from the type strain was sequenced in our laboratory or for which type strain sequence data were verified by identity to a DNA sequence generated in our laboratory from a conspecific isolate. (A) ITS-based tree of 32 molds. Analysis included partial 18S and 28S sequences and complete ITS1, 5.8S, and ITS2 sequences. (B) 28S-based tree of 27 molds. Analysis included the D1/D2 hypervariable region of the 28S rRNA gene.