Identification of clinically relevant yeast species by DNA sequence analysis of the D2 variable region of the 25–28S rRNA gene

Putignani, Lorenza; Paglia, Maria Grazia; Bordi, E.; Nebuloso, E.; Pucillo, Leopoldo Paolo; Visca, Paolo

doi:10.1111/j.1439-0507.2007.01472.x

Cited by 39 publications

(32 citation statements)

References 52 publications

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“…Sequence analysis of the D1/D2 and ITS2 regions provided identifications of only 87% and 79% of the isolates to the species level, respectively. The percentages of identification for both the D1/D2 region and ITS regions are lower than previous reports for human clinical isolates (6,13,24,26,33,34). However, this may be attributed to the variety of yeasts isolated from animal sources, as well the number of uncommon isolates found in a veterinary population.…”

Section: Discussioncontrasting

confidence: 48%

Molecular Identification of Veterinary Yeast Isolates by Use of Sequence-Based Analysis of the D1/D2 Region of the Large Ribosomal Subunit

et al. 2010

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Conventional methods of yeast identification are often time-consuming and difficult; however, recent studies of sequence-based identification methods have shown promise. Additionally, little is known about the diversity of yeasts identified from various animal species in veterinary diagnostic laboratories. Therefore, in this study, we examined three methods of identification by using 109 yeast samples isolated during a 1-year period from veterinary clinical samples. Comparison of the three methods-traditional substrate assimilation, fatty acid profile analysis, and sequence-based analysis of the region spanning the D1 and D2 regions (D1/D2) of the large ribosomal subunit-showed that sequence analysis provided the highest percent identification among the three. Sequence analysis identified 87% of isolates to the species level, whereas substrate assimilation and fatty acid profile analysis identified only 54% and 47%, respectively. Less-stringent criteria for identification increased the percentage of isolates identified to 98% for sequence analysis, 62% for substrate assimilation, and 55% for fatty acid profile analysis. We also found that sequence analysis of the internal transcribed spacer 2 (ITS2) region provided further identification for 36% of yeast not identified to the species level by D1/D2 sequence analysis. Additionally, we identified a large variety of yeast from animal sources, with at least 30 different species among the isolates tested, and with the majority not belonging to the common Candida spp., such as C. albicans, C. glabrata, C. tropicalis, and the C. parapsilosis group. Thus, we determined that sequence analysis of the D1/D2 region was the best method for identification of the variety of yeasts found in a veterinary population.In both veterinary and human diagnostic laboratories, correct identification of yeasts is important for the care of patients. Traditionally, yeast identification has been performed using biochemical analysis, substrate assimilation methods, morphological examination, or various combinations of the three. To increase the ease of identification, commercial tests that use these methods have been created, but despite the convenience provided by these methods, identification of yeasts by these conventional applications can still be timeconsuming and difficult. In addition, considerable variability in the efficacy of these methods has been reported for identification of clinically important yeast (11; also reviewed in references 12, 32, 36, and 42), attributed primarily to the limitations of the databases used for the comparison of clinical isolates, as well as the subjectivity involved in the interpretation of results. Recent studies have also examined the effectiveness of various molecular identification methods for yeasts by the use of rRNA genes, with the internal transcribed spacer 1 (ITS1) and ITS2 regions and the region spanning the D1 and D2 regions (D1/ D2) shown to be the most useful for species-level identification of yeasts, as a result of the variability within...

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Section: Discussioncontrasting

confidence: 48%

Molecular Identification of Veterinary Yeast Isolates by Use of Sequence-Based Analysis of the D1/D2 Region of the Large Ribosomal Subunit

et al. 2010

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“…33,34 To assess this notion, clinical implementation of methods facilitating reliable identification of these species at an adequate level of sensitivity would be required. A number of methods for rapid typing of fungal pathogens have been published, [35][36][37][38][39][40][41][42][43][44][45] and the development of novel and expectedly more potent methods is currently underway, including also approaches based on the exploitation of nano-technological devices, as currently pursued in our laboratory. Rapid and sensitive techniques for the identification of putatively non-or low-pathogenic environmental fungal species that may cause clinically silent fungemia could improve the interpretation of results obtained by broad-spectrum PCR screening methods.…”

Section: Discussionmentioning

confidence: 99%

Diagnosis of invasive fungal infections by a real-time panfungal PCR assay in immunocompromised pediatric patients

Landlinger¹,

Preuner²,

Bašková³

et al. 2010

Leukemia

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Invasive fungal disease (IFD) is a life-threatening event in immunocompromised patients, and there is an urgent need for reliable screening methods facilitating rapid and broad detection of pathogenic fungi. We have established a two-reaction real-time PCR assay permitting highly sensitive detection of more than 80 fungal pathogens, covering a large spectrum of moulds, yeasts and Zygomycetes. To assess the clinical potential of the assay, more than 600 consecutive specimens from 125 pediatric patients carrying a high risk of IFD were analyzed. An excellent correlation between PCR positivity and the presence of proven, probable or possible fungal infection according to the European Organization for Research and Treatment of Cancer criteria was demonstrated, as revealed by the sensitivity of the assay of 96% (95% CI: 82-99%). The negative predictive value of the panfungal PCR assay presented was 98% (95% CI: 90-100%), while the specificity and the positive predictive value were 77% (95% CI: 66-85%) and 62% (95% CI: 47-75%), respectively. The results indicate that molecular screening of patients during febrile neutropenic episodes by the assay presented could help prevent unnecessary toxicity resulting from empirical antifungal treatment in individuals who may not be at risk of imminent fungal disease. Our observations raise the possibility that rapid species identification may be required to increase the positive predictive value for impending fungus-related disease.

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“…33 For the reference strains and some clinical isolates for which the preceeding technique failed to give positive PCR, genomic DNA was extracted as previously described. 39 …”

Section: Fungal Dna Extractionmentioning

confidence: 99%

“…39 Thus, PCR/restriction fragment length polymorphism (RFLP) fingerprints or sequencing of the IGS domain 39 can be used as alternative or adjunct to D1D2 sequence (26S rDNA) 40,41 or ITS sequencing. 30,33,42 Here, we selected primers for partial amplification of the IGS (IGS2) and we established the specific patterns of C. …”

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

Cornet

Sendid

Fradin

et al. 2011

The Journal of Molecular Diagnostics

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Recent changes in the epidemiology of candidiasis highlighted an increase in non-Candida albicans species emphasizing the need for reliable identification methods. Molecular diagnostics in fungal infections may improve species characterization, particularly in cases of the closely related species in the Candida complexes. We developed two PCR/restriction fragment length polymorphism assays, targeting either a part of the intergenic spacer 2 or the entire intergenic spacer (IGS) of ribosomal DNA using a panel of 270 isolates. A part of the intergenic spacer was used for discrimination between C. albicans and C. dubliniensis and between species of the C. glabrata complex (C. glabrata/C. bracarensis/C. nivariensis). The whole IGS was applied to C. parapsilosis, C. metapsilosis, and C. orthopsilosis, and to separate C. famata (Debaryomyces hansenii) from C. guilliermondii (Pichia guilliermondii) and from the other species within this complex (ie, C. carpophila, C. fermentati and C. xestobii). Sharing similar biochemical patterns, Pichia norvegensis and C. inconspicua exhibited specific IGS profiles. Our study confirmed that isolates of C. guilliermondii were frequently misidentified as C. famata. As much as 67% of the clinical isolates phenotypically determined as C. famata were recognized mostly as true P. guilliermondii. Conversely, 44% of the isolates initially identified as C. guilliermondii were corrected by the IGS fingerprints as C. parapsilosis, C. fermentati, or C. zeylanoides. Current changes in the epidemiology of invasive mycoses highlighted a shift in the Candida species involved with a reduced proportion of C. albicans and an increase in non-C. albicans species. [1][2][3][4] In the most recent series, including the large cohort of 2019 patients with candidemia enrolled from 2004 through 2008, C. albicans accounts for less than one half of the isolates.3,5-12 Although C. albicans antifungal susceptibility remains the rule, and reports on resistant isolates are very scarce, other species such as C. krusei, C. glabrata, C. bracarensis, C. nivariensis, C. parapsilosis, and C. guilliermondii are either innately resistant or show decreased susceptibility patterns to azoles, amphotericin B, or echinocandins.13-21 Thus, the therapeutic impact of this shift might be critical and should be considered in patient management. Consistent with this trend, the recent revision of the consensus guidelines actually recommends an adjustment of the treatment according to the isolated Candida species. 22 In yeasts, there is no transfer of resistance between cells and acquisition of resistance, which is mainly observed in restricted clinical settings such as allogeneic blood marrow transplant or AIDS patients under sustained prolonged azole treatment. 5,14,23 Therefore, species identification remains basically predictive of drug susceptibility. Current methods for yeast identification in clinical practice are based on phenotypic features and carbohydrate assimilation tests that require 2 to 5 days or even longer in the case ...

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Identification of clinically relevant yeast species by DNA sequence analysis of the D2 variable region of the 25–28S rRNA gene

Cited by 39 publications

References 52 publications

Molecular Identification of Veterinary Yeast Isolates by Use of Sequence-Based Analysis of the D1/D2 Region of the Large Ribosomal Subunit

Molecular Identification of Veterinary Yeast Isolates by Use of Sequence-Based Analysis of the D1/D2 Region of the Large Ribosomal Subunit

Diagnosis of invasive fungal infections by a real-time panfungal PCR assay in immunocompromised pediatric patients

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

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