Rapid Molecular Identification of Pathogenic Yeasts by Pyrosequencing Analysis of 35 Nucleotides of Internal Transcribed Spacer 2

Abstract

Rapid identification of yeast species isolates from clinical samples is particularly important given their innately variable antifungal susceptibility profiles. Here, we have evaluated the utility of pyrosequencing analysis of a portion of the internal transcribed spacer 2 region (ITS2) for identification of pathogenic yeasts. A total of 477 clinical isolates encompassing 43 different fungal species were subjected to pyrosequencing analysis in a strictly blinded study. The molecular identifications produced by pyrosequencing were compared with those obtained using conventional biochemical tests (AUXACOLOR2) and following PCR amplification and sequencing of the D1-D2 portion of the nuclear 28S large rRNA gene. More than 98% (469/477) of isolates encompassing 40 of the 43 fungal species tested were correctly identified by pyrosequencing of only 35 bp of ITS2. Moreover, BLAST searches of the public synchronized databases with the ITS2 pyrosequencing signature sequences revealed that there was only minimal sequence redundancy in the ITS2 under analysis. In all cases, the pyrosequencing signature sequences were unique to the yeast species (or species complex) under investigation. Finally, when pyrosequencing was combined with the Whatman FTA paper technology for the rapid extraction of fungal genomic DNA, molecular identification could be accomplished within 6 h from the time of starting from pure cultures.

Epidemiology, Antifungal Susceptibility, and Pathogenicity of Candida africana Isolates from the United Kingdom

“…We have previously demonstrated that pyrosequencing of 35 bp of a portion of the internal transcribed spacer region 2 (ITS2) is sufficient to robustly identify the vast majority of pathogenic yeasts (27), including genetically closely related isolates within species complexes (28,29). This is also the case for the key species within the C. albicans species complex.…”

“…All 1,839 test isolates were subjected to pyrosequencing analysis of a portion of ITS2 exactly as described previously (27)(28)(29). For isolates identified by pyrosequencing as C. africana, identity was confirmed by PCR amplification and sequencing of a region of the large subunit gene (LSU) and the internal transcribed spacer 1 (ITS1) region using the primers described previously (30,31) and direct inoculation of PCR products with yeast suspensions as described in reference 32.…”

Assessment of Accuracy of Identification of Pathogenic Yeasts in Microbiology Laboratories in the United Kingdom

“…Non-Candida yeasts further contribute to an increasingly complicated clinical picture in which more than 150 yeast spp. from Candida and other genera have now been reported from mammalian infections (4,11,15; Mycology Reference Laboratory [MRL], unpublished data).…”

Biodiversity of Yeasts in the Gastrointestinal Ecosystem with Emphasis on Its Importance for the Host

“…The largest limitation for methods based on PCR is the low sensitivity, as the practice shows only 1-2% of the community can be detected in this way (Macnaughton et al, 1999). Furthermore, fingerprint methods have the bias combined to the fact that amplicons form different species with sequences of similar energetic profile may migrate to the same positions; multiple gene copies with slight sequence differences may give multiple bands for one strain or species; finally some species are phylogeneticaly very similar (Lachance et al, 2003;Janczyk et al, 2006;Borman et al, 2010). The design of probes for direct targeting needs knowledge on the sequence of the target gene and differences between species.…”