Abstract
BackgroundThe number of patients with yeast infection has increased during the last years. Also the variety of species of clinical importance has increased. Correct species identification is often important for efficient therapy, but is currently mostly based on phenotypic features and is sometimes time-consuming and depends largely on the expertise of technicians. Therefore, we evaluated the feasibility of PCR-based amplification of the internally transcribed spacer region 2 (ITS2), followed by fragment size analysis on the ABI Prism 310 for the identification of clinically important yeasts.ResultsA rapid DNA-extraction method, based on simple boiling-freezing was introduced. Of the 26 species tested, 22 could be identified unambiguously by scoring the length of the ITS2-region. No distinction could be made between the species Trichosporon asteroides and T. inkin or between T. mucoides and T. ovoides. The two varieties of Cryptococcus neoformans (var. neoformans and var. gattii) could be differentiated from each other due to a one bp length difference of the ITS2 fragment. The three Cryptococcus laurentii isolates were split into two groups according to their ITS2-fragment lengths, in correspondence with the phylogenetic groups described previously. Since the obtained fragment lengths compare well to those described previously and could be exchanged between two laboratories, an internationally usable library of ITS2 fragment lengths can be constructed.ConclusionsThe existing ITS2 size based library enables identification of most of the clinically important yeast species within 6 hours starting from a single colony and can be easily updated when new species are described. Data can be exchanged between laboratories.
Highlights
The number of patients with yeast infection has increased during the last years
The existing internally transcribed spacer region 2 (ITS2) size based library enables identification of most of the clinically important yeast species within 6 hours starting from a single colony and can be updated when new species are described
We report an evaluation and extension of the technique published by Turenne et al.[9], whereby the rRNA Internally Transcribed Spacer Region 2 (ITS2), i.e. the region in between the fungal 5.8S and 28S rRNA genes, is amplified and its length is determined by fragment analysis on an ABI Prism 310 capillary electrophoresis system
Summary
The number of patients with yeast infection has increased during the last years. We evaluated the feasibility of PCR-based amplification of the internally transcribed spacer region 2 (ITS2), followed by fragment size analysis on the ABI Prism 310 for the identification of clinically important yeasts. Rapid and correct identification of the different clinically relevant yeast species has become more important because of several reasons. The impact and frequency of yeast infections has gained importance mainly due to an increased number of immunocompromised patients [1]. An increasing number of non-C. albicans yeast species are considered as potential agents of clinical infections [2]. A more broadly applicable approach is based on PCR with universal fungal primers (directed towards conserved regions in the ribosomal region) and followed by either restriction analysis [7], sequencing [8] or size determination of the amplified fragment(s) [9,10]
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