Sequencer-Based Capillary Gel Electrophoresis (SCGE) Targeting the rDNA Internal Transcribed Spacer (ITS) Regions for Accurate Identification of Clinically Important Yeast Species.

Xin Hou,Ying-Chun Xu,Jing-Wei Cheng,Xin Fan,Li Zhang,He Wang,Sharon C.-A Chen,Zhi-Peng Xu,Yu-Pei Zhao,Fanrong Kong,Meng Xiao,Patrick Cy Woo

doi:10.1371/journal.pone.0154385

Abstract

Accurate species identification of Candida, Cryptococcus, Trichosporon and other yeast pathogens is important for clinical management. In the present study, we developed and evaluated a yeast species identification scheme by determining the rDNA internal transcribed spacer (ITS) region length types (LTs) using a sequencer-based capillary gel electrophoresis (SCGE) approach. A total of 156 yeast isolates encompassing 32 species were first used to establish a reference SCGE ITS LT database. Evaluation of the ITS LT database was then performed on (i) a separate set of (n = 97) clinical isolates by SCGE, and (ii) 41 isolates of 41 additional yeast species from GenBank by in silico analysis. Of 156 isolates used to build the reference database, 41 ITS LTs were identified, which correctly identified 29 of the 32 (90.6%) species, with the exception of Trichosporon asahii, Trichosporon japonicum and Trichosporon asteroides. In addition, eight of the 32 species revealed different electropherograms and were subtyped into 2–3 different ITS LTs each. Of the 97 test isolates used to evaluate the ITS LT scheme, 96 (99.0%) were correctly identified to species level, with the remaining isolate having a novel ITS LT. Of the additional 41 isolates for in silico analysis, none was misidentified by the ITS LT database except for Trichosporon mucoides whose ITS LT profile was identical to that of Trichosporon dermatis. In conclusion, yeast identification by the present SCGE ITS LT assay is a fast, reproducible and accurate alternative for the identification of clinically important yeasts with the exception of Trichosporon species.

Highlights

Fungi are a major cause of human disease especially in patients with immune compromise and serious underlying disease [1]
As a potential alternative molecular methods, here we have developed a sequencer-based capillary gel electrophoresis (SCGE)-based approach for yeast identification and demonstrated that it can provide accurate and reproducible identification of major Candida, Cryptococcus and other rare yeast species with the exception of Trichosporon species
Both ITS1 and full length internal transcribed spacer (ITS) SCGE amplicon results in combination were needed to make these distinctions allowing for correct identification of all isolates of these genera

Summary

Introduction

Fungi are a major cause of human disease especially in patients with immune compromise and serious underlying disease [1]. RDNA gene complex sequencing targeted at the internal transcribed spacer (ITS) regions is considered as the “gold-standard” method for identifying yeast species [10,11,12,13] This approach requires time for the sequencing process, is expensive and dependent on public sequence repositories which are not curated and contain errors [14]. Other molecular identification methods include reverse line blot (RLB) assays, rolling circle amplification (RCA) [9, 15, 16] and pyrosequencing [17, 18] Proteomic approaches such as matrixassisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) lends itself as an identification tool [19]. As a simple and less expensive alternative to ITS sequencing, another molecular identification method, sequencer-based capillary gel electrophoresis (SCGE) systems, has been reported to have good accuracy, reproducibility and interlaboratory consistency, whilst retaining flexibility [20, 21]

Methods

Results

Conclusion