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.

Discovering Potential Pathogens among Fungi Identified as Nonsporulating Molds

“…Often fungi cannot be characterized fully because the mold does not sporulate, making identification by microscopic morphology not possible and potentially increasing the time to report an inconclusive result to 21 days. While many laboratorians and clinicians assume that these fungal isolates are environmental organisms and not clinically significant, to our knowledge no study has systematically attempted to classify these previously unidentifiable fungi in a clinical microbiology laboratory.Amplification and sequencing of target regions within the ribosomal DNA gene complex has emerged as a useful, adjunctive tool for the identification of fungi and does not depend on mold sporulation for identification (3,5,9,11). The internal transcribed spacer (ITS) regions 1 and 2 located between the highly conserved small (18S) and large (28S) ribosomal subunit genes in the rRNA operon are known to have sufficient sequence variability to allow identification to the species level for many fungi (2,3,5,9,11,15).…”

“…The internal transcribed spacer (ITS) regions 1 and 2 located between the highly conserved small (18S) and large (28S) ribosomal subunit genes in the rRNA operon are known to have sufficient sequence variability to allow identification to the species level for many fungi (2,3,5,9,11,15). The goals of this study were to determine if sequencing the ITS 1 and 2 regions of nonsporulating molds (NSM) could identify fungi that were not identifiable by conventional methods and could serve as an approach to detect clinically relevant pathogens.…”

Fungal Keratitis: Update for 2014

“…Previous PCR techniques were unable to distinguish between filamentous and yeast species and required complex steps for amplification [27][28][29]. Real-time Taqman PCR is able to better characterize the fungal species, but is expensive, due in part to the requirement for a unique probe to search for a single fungal type per reaction [30].…”

Rapid molecular identification of fungal pathogens in corneal samples from suspected keratomycosis cases

“…Several potentially discriminating, species-specific regions that are possibly useful for molecular diagnosis have been described in the genome of fungi (Biswas et al, 2003;Einsele et al, 1997;Hennequin et al, 1999;Makimura et al, 1994;Rakeman et al, 2005;Reiss et al, 2000). The internal transcribed spacer (ITS) 1 and ITS2 rDNA sequences offer several advantages in this perspective (Iwen et al, 2002;Leinberger et al, 2005;Pryce et al, 2003).…”