Polymorphic Internal Transcribed Spacer Region 1 DNA Sequences Identify Medically Important Yeasts

Abstract

Species-specific polymorphisms in the noncoding internal transcribed spacer 2 (ITS2) region of the rRNA operon provide accurate identification of clinically significant yeasts. In this study, we tested the hypothesis that ITS1 noncoding regions contain diagnostically useful alleles. The length of ITS1 region PCR products amplified from 40 species (106 clinical strains, 5 reference strains, and 30 type strains) was rapidly determined with single-base precision by automated capillary electrophoresis. Polymorphisms in the PCR product length permitted 19 species to be distinguished by ITS1 alone, compared with 16 species distinguished by using only ITS2. However, combination of both ITS alleles permitted identification of 30 species (98% of clinical isolates). The remaining 10 species with PCR products of similar sizes contained unique ITS alleles distinguishable by restriction enzyme analysis. DNA sequence analysis of amplified ITS1 region DNA from 79 isolates revealed species-specific ITS1 alleles for each of the 40 pathogenic species examined. This provided identification of unusual clinical isolates, and 53 diagnostic ITS1 sequences were deposited in GenBank. Phylogenetic analyses based on ITS sequences showed a similar overall topology to 26S rRNA gene-based trees. However, different species with identical 26S sequences contained distinct ITS alleles that provided species identification with strong statistical support. Together, these data indicate that the analysis of ITS polymorphisms can reliably identify 40 species of clinically significant yeasts and that the capacity for identifying potentially new pathogenic species by using this database holds significant promise.The complexity of opportunistic fungal infections is increasing as more patients are adversely affected by an expanding diversity of yeasts. Invasive procedures or immunosuppression increases the risk of fungemia (24, 34), and drug-resistant strains have emerged (9, 21). Although Candida albicans is still the predominate agent of nosocomial infection, serious infections caused by other yeasts have increased in frequency (8,11,29,34). For example, species of Cryptococcus other than Cryptococcus neoformans, previously considered to be nonpathogenic saprophytes, have been reported to cause cryptococcosis (28). Adequate treatment of these infections depends on early detection and accurate identification of the pathogens. However, conventional identification by evaluation of morphological and physiological characteristics can be laborious, sometimes leads to incorrect classification and identification (2, 3, 13), and can be impeded by database limitations (4, 6).To develop a rapid molecular method for identifying yeasts, we recently analyzed the length and sequence polymorphisms in the DNA of noncoding internal transcribed spacer region 2 (ITS2) of the rRNA operon (2). ITS2 region polymorphisms permitted accurate identification of over 400 clinical strains representing 34 species of pathogenic yeasts. ITS2 region PCR product length determined by aut...

Metahyphopichia laotica gen. nov., sp. nov., a polymorphic yeast related to Hyphopichia

“…KP262069) of UFMG-CM-Y6070 differed from that of strain 11-1006 T at 10 positions which is close to the average intraspecific variability (2.51 % with a standard deviation of 4.57) determined by Nilsson et al (2008) for fungi, but higher than the usual variability within ascomycetous yeast species (e.g. Chen et al, 2001;Kurtzman, 2012). To further examine the relationship of 11-1006…”

Molecular Identification of Closely Related Candida Species Using Two Ribosomal Intergenic Spacer Fingerprinting Methods

“…Amplicon sizes may be estimated either by eye or digitally on the gel after normalization. However, some studies from Chen and colleagues 54 described automated capillary electrophoresis means to distinguish species of Candida according to the amplicon size, but later abandoned it due to lack of uniformity Recently, mass spectrometry using matrix assisted laser desorption/ionization-time of flight technology has been developed for microbiological identification and showed very promising results for rapid and reliable determination of the most important yeast species isolated in the clinical setting. [55][56][57] However, to date, mass spectrometry spectra of closely related species forming the major clinical Candida complexes and of unusual species have not been reported and have not been introduced into the reference databases.…”

In Vitro Antidrug Susceptibility Testing of Candida Species Isolated from Aseptic Body Fluids

“…and Ct2 (5'-TGGCCACTAGCAAAATAAGCGT-3') (11)(12)(13). PCR reactions were carried out in a GeneAmp PCR system 2400 (PerkinElmer, US) and performed in 0.2 mL microcentrifuge tubes with a final reaction mixture containing 2 µL of the prepared template, 12.5 µL of 2 × Taq PCR master mix (Tiangen Biotech (Beijing) Co., Ltd., China), 0.5 µL of each primer (10 mM)(BGI, China), and 7.5 µL ddH 2 O. Amplification conditions were as follows: 5 minutes initial denaturation at 96°C, repeated for 40 cycles of 30 seconds denaturation at 94°C, 30 seconds of primer annealing at 53°C, and elongation at 72°C for 1 minute.…”