Species-specific polymorphisms in the noncoding internal transcribed spacer 2 (ITS2) region of the rRNA operon provide accurate identification of clinically significant yeasts. In this study, we tested the hypothesis that ITS1 noncoding regions contain diagnostically useful alleles. The length of ITS1 region PCR products amplified from 40 species (106 clinical strains, 5 reference strains, and 30 type strains) was rapidly determined with single-base precision by automated capillary electrophoresis. Polymorphisms in the PCR product length permitted 19 species to be distinguished by ITS1 alone, compared with 16 species distinguished by using only ITS2. However, combination of both ITS alleles permitted identification of 30 species (98% of clinical isolates). The remaining 10 species with PCR products of similar sizes contained unique ITS alleles distinguishable by restriction enzyme analysis. DNA sequence analysis of amplified ITS1 region DNA from 79 isolates revealed species-specific ITS1 alleles for each of the 40 pathogenic species examined. This provided identification of unusual clinical isolates, and 53 diagnostic ITS1 sequences were deposited in GenBank. Phylogenetic analyses based on ITS sequences showed a similar overall topology to 26S rRNA gene-based trees. However, different species with identical 26S sequences contained distinct ITS alleles that provided species identification with strong statistical support. Together, these data indicate that the analysis of ITS polymorphisms can reliably identify 40 species of clinically significant yeasts and that the capacity for identifying potentially new pathogenic species by using this database holds significant promise.The complexity of opportunistic fungal infections is increasing as more patients are adversely affected by an expanding diversity of yeasts. Invasive procedures or immunosuppression increases the risk of fungemia (24, 34), and drug-resistant strains have emerged (9, 21). Although Candida albicans is still the predominate agent of nosocomial infection, serious infections caused by other yeasts have increased in frequency (8,11,29,34). For example, species of Cryptococcus other than Cryptococcus neoformans, previously considered to be nonpathogenic saprophytes, have been reported to cause cryptococcosis (28). Adequate treatment of these infections depends on early detection and accurate identification of the pathogens. However, conventional identification by evaluation of morphological and physiological characteristics can be laborious, sometimes leads to incorrect classification and identification (2, 3, 13), and can be impeded by database limitations (4, 6).To develop a rapid molecular method for identifying yeasts, we recently analyzed the length and sequence polymorphisms in the DNA of noncoding internal transcribed spacer region 2 (ITS2) of the rRNA operon (2). ITS2 region polymorphisms permitted accurate identification of over 400 clinical strains representing 34 species of pathogenic yeasts. ITS2 region PCR product length determined by aut...

Identification of medically relevant yeasts can be time-consuming and inaccurate with current methods. We evaluated PCR-based detection of sequence polymorphisms in the internal transcribed spacer 2 (ITS2) region of the rRNA genes as a means of fungal identification. Clinical isolates (401), reference strains (6), and type strains (27), representing 34 species of yeasts were examined. The length of PCR-amplified ITS2 region DNA was determined with single-base precision in less than 30 min by using automated capillary electrophoresis. Unique, species-specific PCR products ranging from 237 to 429 bp were obtained from 92% of the clinical isolates. The remaining 8%, divided into groups with ITS2 regions which differed by <2 bp in mean length, all contained species-specific DNA sequences easily distinguishable by restriction enzyme analysis. These data, and the specificity of length polymorphisms for identifying yeasts, were confirmed by DNA sequence analysis of the ITS2 region from 93 isolates. Phenotypic and ITS2-based identification was concordant for 427 of 434 yeast isolates examined using sequence identity of >99%. Seven clinical isolates contained ITS2 sequences that did not agree with their phenotypic identification, and ITS2-based phylogenetic analyses indicate the possibility of new or clinically unusual species in the Rhodotorula and Candida genera. This work establishes an initial database, validated with over 400 clinical isolates, of ITS2 length and sequence polymorphisms for 34 species of yeasts. We conclude that size and restriction analysis of PCR-amplified ITS2 region DNA is a rapid and reliable method to identify clinically significant yeasts, including potentially new or emerging pathogenic species.

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.

Primarily saprophytic in nature, fungi of the genus Acremonium are a well-documented cause of mycetoma and other focal diseases. More recently, a number of Acremonium spp. have been implicated in invasive infections in the setting of severe immunosuppression. During the course of routine microbiological studies involving a case of fatal mycosis in a nonmyeloablative hematopoietic stem cell transplant patient, we identified a greater-than-expected variation among strains previously identified as Acremonium strictum by clinical microbiologists. Using DNA sequence analysis of the ribosomal DNA intergenic transcribed spacer (ITS) regions and the D1-D2 variable domain of the 28S ribosomal DNA gene (28S), the case isolate and four other clinical isolates phenotypically identified as A. strictum were found to have <99% homology to the A. strictum type strain, CBS 346.70, at the ITS and 28S loci, while a sixth isolate phenotypically identified only as Acremonium sp. had >99% homology to the type strain at both loci. These results suggest that five out of the six clinical isolates belong to species other than A. strictum or that the A. strictum taxon is genetically diverse. Based upon these sequence data, the clinical isolates were placed into three genogroups.