Molecular Identification of Unusual Pathogenic Yeast Isolates by Large Ribosomal Subunit Gene Sequencing: 2 Years of Experience at the United Kingdom Mycology Reference Laboratory

Abstract

Rapid identification of yeast isolates from clinical samples is particularly important given their innately variable antifungal susceptibility profiles. We present here an analysis of the utility of PCR amplification and sequence analysis of the hypervariable D1/D2 region of the 26S rRNA gene for the identification of yeast species submitted to the United Kingdom Mycology Reference Laboratory over a 2-year period. A total of 3,033 clinical isolates were received from 2004 to 2006 encompassing 50 different yeast species. While more than 90% of the isolates, corresponding to the most common Candida species, could be identified by using the AUXACOLOR2 yeast identification kit, 153 isolates (5%), comprised of 47 species, could not be identified by using this system and were subjected to molecular identification via 26S rRNA gene sequencing. These isolates included some common species that exhibited atypical biochemical and phenotypic profiles and also many rarer yeast species that are infrequently encountered in the clinical setting. All 47 species requiring molecular identification were unambiguously identified on the basis of D1/D2 sequences, and the molecular identities correlated well with the observed biochemical profiles of the various organisms. Together, our data underscore the utility of molecular techniques as a reference adjunct to conventional methods of yeast identification. Further, we show that PCR amplification and sequencing of the D1/D2 region reliably identifies more than 45 species of clinically significant yeasts and can also potentially identify new pathogenic yeast species.

Evaluation of Pyrosequencing® technology for the identification of clinically relevant non-dematiaceous yeasts and related species

“…The availability of conserved and highly variable regions within the rRNA and its presence in multiple copies in fungal cells ( Figure 1) allows high sensitivity as well as accurate molecular identification for most yeasts [10,12,13].…”

Efficient Identification of Clinically Relevant Candida Yeast Species by Use of an Assay Combining Panfungal Loop-Mediated Isothermal DNA Amplification with Hybridization to Species-Specific Oligonucleotide Probes

“…Yeasts of the genus Candida are the main etiologic agents of those infections, with a high prevalence of C. albicans. However, other species (e.g., C. krusei and C. glabrata) have emerged as opportunistic pathogens associated with systemic infections (14,21), posing difficulties due to the different susceptibilities of these yeasts to antifungal therapy. Sensitive, reliable, and rapid identification of these pathogenic yeasts is of paramount importance to improve disease management and enable the selection of adequate treatment.…”

Identification of Uncommon Clinically Important Yeasts and Moulds by the Bruker Biotyper Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) System in a Global Antifungal Surveillance Program

“…) New and emerging fungal pathogens include species of Candida and Aspergillus other than C. albicans and A. fumigatus; opportunistic yeast-like fungi such as Cryptococcus gattii, Trichosporon spp., Dipodascus capitatus, Rhodotorula spp., and Saceharomyces cerevisiae; the mucormycotes; hyaline moulds such as Fusarium, Acremonium, Scedosporium, Paecilomyces (Purpureocillium) and Trichoderma species; and a wide variety of demateaccores moulds (AlastrueyIzquierdo et al, 2013;Brown et al, 2012;Pfaller et al, 2012;Pfaller et al, 2013). Accurate identification (ID) of these organisms is important in guiding therapy and determining prognosis in these IFIs as well as in epidemiological surveys (Balajee et al, 2009;Borman et al, 2012;Brandt and Park, 2013;Linton et al, 2007;Pounder et al, 2007). Conventional methods of yeast and mould ID take into account biochemical and morphological characteristics and are both slow and labor intensive requiring considerable mycological expertise (Alastruey-Izquierdo et al, 2013;Balajee et al, 2009;Borman et al, 2012;CendejasBueno et al, 2010;Latouche et al, 1997;Meletiadis et al, 2011;Sanguinetti et al, 2007).…”