Yeast Identification Methods Research Articles

Phenotypic Characterization of Candida species in Tertiary Care Hospital of Eastern Nepal

Background: Candida albicans is one of the most frequently isolated yeast in clinical laboratories and studies have shown that it accounts for up to 80% of the yeast recovered from the site of infection. Candida species have emerged as significant opportunistic fungal pathogens and the conventional methods of yeast identification are reported to be cumbersome with delayed diagnosis and initiation of treatment. Thus rapid identification and speciation of Candida species is essential in clinical laboratories.
 Materials and Methods: A hospital based cross sectional study was carried out in the department of microbiology, Nobel medical college from January 2020 to December 2020. Approval was acquired from the Institutional Review Committee. Various clinical specimens were obtained and identification as per the standard microbiological procedures. Data were analyzed by SPSS, version 20.
 Results: A total of 62 Candida species were isolated out of which 65% were from male and 35% were from female patients. Candida albicans was found to be the most common species with 68%. The highest incidence was seen in the age group below 20 years and 21 – 40 years with 32.25% cases each followed by 41-60 years with 20.96% cases. Among 62 specimens, urine samples yielded the highest number of Candida species 48.38% followed by sputum samples with 29.03% Candida species. High vaginal swab (HVS) yielded 12.9% Candida species and blood samples yielded 9.67% Candida species.
 Conclusion: Candida albicans is still the most significant clinically but other non albicans are also emerging significant pathogens and warrant routine discrimination in clinical laboratories.

Multiple region high resolution melting-based method for accurate differentiation of food-derived yeasts at species level resolution

Yeasts comprise a divergent group of microorganisms playing an important role in foods production. To monitor the production processes, validate authenticity and safety of foods, reliable methods of yeast identification and differentiation are needed. Nowadays, traditional PCR/sequencing-based methods are replaced by rapid techniques not requiring post-PCR processing. In the present study, we developed a three region-based qPCR-HRM protocol, for rapid differentiation of yeast species isolated from food products. The three targeted fragments (26S rDNA, 18S rDNA, ITS) were carefully analyzed to dock the primers at inter-species conservative regions, flanking polymorphic regions of ∼200 bp. Thirty eight yeast strains were used as a training material. The collection of yeast spanned Pichia, Clavispora, Candida, Yarrowia, Kluyveromyces, Saccharomyces, and Wickerhamomyces genera. MALDI-TOF mass spectrometry and conventional rDNA sequencing were used for validation. Conducted studies demonstrated that when used individually, each of the three regions analyzed by qPCR-HRM possessed its own yeast species-specific limitations, sometimes leading to inaccurate taxonomic classification. On the other hand, simultaneous analysis of the three proposed regions resulted in rapid and adequate differentiation of all the yeast strains at species-level resolution.

2045. Pitfalls in the Use of MALDI TOF Mass Spectrometry for the Identification of Problematic Yeast Isolates from a Historical Collection

BackgroundThe identification of yeast traditionally entails macroscopic/microscopic findings and biochemical testing. Recently, MALDI TOF MS has replaced traditional methods for identification as a proposed new standard. We performed identification of previously unidentifiable yeasts from a collection in the United States.MethodsThe Mycoses Study Group (MSG) collected 2,947 Candida isolates from 1,911 patients as part of two U.S. studies between 1995 and 1999. The identification of the isolates was done in 2002 using API 20 c aux with supplemented standard mycological and biochemical test (corn meal agar, germ tube and the Murex ID system). Ninety-four isolates could not be identified at that time. For this study, the isolates were defrosted, plated on Sabureau Dextrose agar, and incubated at 30°C for 48 hours. We then sub-cultured again in Blood Agar. Isolates when then tested by MALDI TOF MS following the methodology for the Bruker MALDI biotyper.ResultsIn the first attempt, 65/94 (69%) isolates were identified. The remaining 29 samples were re-tested with a yield of 21/29 (72.4%) identified isolates. The remaining isolates had to be identified with another round of MALDI TOF and further biochemical testing. The table below shows the final identification of the isolates.ConclusionMALDI TOF MS is rapidly becoming a reference method for yeast identification. However, in a historical collection of yeast that could not be identified by conventional biochemical methods, it took up to three rounds of MALDI TOF MS with a yield of ~70% per round, and additional biochemical testing, for identification of all isolates. Continuing validation of MALDI TOF MS for identification of difficult yeast isolates is warranted.SpeciesNumberPercentage C. albicans 2021 C. glabrata 2223.40 C. parapsilosis 3436.10 C. tropicalis 88.50 C. krusei 22.10 C. orthopsilosis 33.10 C. nivariensis 11.06 C. Kefyr 11.06 Pichia norvegensis 11.06 C. lusitane 11.06 Trichosporum spp.11.06Total94100Disclosures All authors: No reported disclosures.

Development and validation of a TaqMan RT-PCR method for identification of mayonnaise spoilage yeast Pichia kudriavzevii

Food spoilage and its contamination with yeast and mold is a serious problem of food industry. Despite the high fat content, mayonnaise is an attractive substrate for food spoilage microorganisms. The aim of this study was to develop a method for yeast identification in mayonnaise and to test commercially available mayonnaises for the presence of these contaminating microorganisms. Based on the sequencing of intergenic regions ITS1 and ITS2, we identified a yeast microorganism that causes mayonnaise spoilage. We found that DNA sequences were more than 99% identical to the GenBank DNA sequences from Pichia kudriavzevii. We developed a specific to P. kudriavzevii TaqMan probe and primers. The reaction conditions were optimized regarding to the components concentration and temperature cycle. The minimum amount of P. kudriavzevii DNA that could be detected by developed method was 50 fg. The minimal number of P. kudriavzevii cells that could be detected by developed method without pre-enrichment was 50. We tested verified method with DNAs from microorganisms of different taxonomic groups that were obtained from three collections of microorganisms. Finally, we analyzed 20 different brands of mayonnaise from 14 producers and 10 different brands of mayonnaise sauce from seven producers. We determined the Cq parameter that characterizes transition of the fluorescence curve to the logarithmic phase and, therefore, correlates with the extent of sample contamination with P. kudriavzevii yeast. P. kudriavzevii was detected in six analyzed samples of mayonnaise and one sample of mayonnaise sauce.

MALDI-TOF MS Supplementary database for species identification employing the yeast diversity encountered on southern Brazil grapes.

The study of grape microflora is of interest when autochthonous yeasts, which are related to typical wine characteristics, are intended to be used in winemaking. The election of matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) as the first method for yeast identification was based on its accuracy and rapidity compared to alternative laboratory protocols for identification. The aims of this study are to consolidate the MALDI-TOF MS Supplementary database for environmental yeasts already constructed, to expand it through the addition of standard spectra of not included yeast species, and to discuss the grape microflora encountered in Southern Brazil. A total of 358 strains, isolated from grape berries, were submitted to protein profiling employing Biotyper and Supplementary database. Molecular biology techniques were used as alternatives to identify 6.4% of strains not promptly designated by protein profiling. These strains corresponded to the species Candida californica, Zygoascus meyerae, Candida akabanensis, Candida azyma, and Hanseniaspora vineae. The MALDI-TOF MS spectra of the identified species were added to Supplementary database. The presented results strengthen the need for further expansion of the mass spectra database to broaden its microbiological application.

Yeast species associated with diseased fish: Occurrence, identification, experimental challenges and antifungal susceptibility testing

The effort to characterize yeasts in fish is still meager, and identification of yeasts from aquacultured diseased fish could be a good strategy to identify those yeast species which have a role in overall fish health. Yet, knowledge on their identification using Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is lacking. Hence, this study investigates the diversity of yeasts residing in the skin, gills, intestine and internal organs of three economically important diseased freshwater fish species. We carried out a comparative evaluation of the performance of the biochemical identification kit RapID Yeast Plus system and MALDI-TOF MS, with amplification/sequencing of Internal Transcribed Spacer (ITS1–ITS2) regions of rDNA as the reference method for yeast identification. Antifungal susceptibility pattern of yeast isolates was assessed using Vitek2 compact. Among 420 samples from Oreochromis species, Clarias gariepinus and Mugil cephalus, 215 yeast isolates (51.2%) were detected, with 7 genera: Candida, Cryptococcus, Debaryomyces, Rhodotorula, Trichosporon, Geotrichum and Pichia identified. Candida spp. accounted for 65.12% of the isolates and Rhodotorula spp. 28.84%. Overall, the yeast belonging to Ascomycota was higher than the Basidiomycota yeast (P < 0.001). There is statistically highly significant difference between incidences of yeast species in different fish. The intraperitoneal-challenged fish groups with Cryptococcus uniguttulatus, Candida lipolytica, Candida albicans and Trichosporon mucoides exhibited the highest cumulative mortality percentages (100, 80.95, 71.43 and 61.90%, respectively). C. uniguttulatus challenged group exhibited exophthalmia, opacity in the lens and yellow discoloration of the body and internal organs, meanwhile C. albicans group showed red mouth. The RapID Yeast Plus system identified the tested isolates at the species and genus level with a percentage of 30% for each. The MALDI-TOF MS accurately identified 75% of the isolates to the species level. Of the isolates (25%) not present in the MALDI-TOF MS database at the species level, 10% were ascribed to the genus level and an incorrect identification was given for 15%. Voriconazole, amphotericin B and caspofungin were the most active drugs against all the species tested, while fluconazole demonstrated poor activity. The diseased freshwater fish species may harbor potential clinically relevant yeast species, including C. albicans, non-albicans Candida species, Trichosporon, Cryptococcus and Rhodotorula species. MALDI-TOF MS offers an accurate and cost-effective alternative to the conventional and molecular methods for yeast identification. Subsequently, implementation of the VITEK MS with a more extended database is required.

Approach to the Investigation and Management of Patients With Candida auris, an Emerging Multidrug-Resistant Yeast.

Candida auris is an emerging, multidrug-resistant yeast that can spread in healthcare settings. It can cause invasive infections with high mortality and is difficult to identify using traditional yeast identification methods. Candida auris has been reported in more than a dozen countries, and as of August 2017, 112 clinical cases have been reported in the United States. Candida auris can colonize skin and persist in the healthcare environment, allowing for transmission between patients. Prompt investigation and aggressive interventions, including notification to public health agencies, implementation of contact precautions, thorough environmental cleaning and disinfection, infection control assessments, contact tracing and screening of contacts to assess for colonization, and retrospective review of microbiology records and prospective surveillance for cases at laboratories are all needed to limit the spread of C. auris. This review summarizes the current recommended approach to manage cases and control transmission of C. auris in healthcare facilities.

Utilization of size polymorphism in ITS1 and ITS2 regions for identification of pathogenic yeast species

Despite the existence of a variety of available yeast-identification strategies, easier and more cost-effective methods are required for routine use in clinical laboratories. The internal transcribed spacer (ITS) regions of fungal rRNA genes exhibit variable sizes depending on the yeast species. In the present study, fragment size polymorphism (FSP) analysis of the ITS1 and ITS2 regions for identification of the clinically most important yeast species was assessed. The ITS1 and ITS2 regions of 190 strains, including isolates of 31 standard strains and 159 clinical isolates, were separately PCR amplified with two primer sets: ITS1-ITS2 and ITS3-ITS4. PCR products were mixed and the two-band electrophoretic pattern of each sample was analysed according to the size of the ITS regions as predicted from the GenBank database. Using this method and avoiding expensive tools such as sequencing or capillary electrophoresis, we were able to differentiate nearly all pathogenic yeast species, including Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida krusei, Candida guilliermondii, Candida kefyr, Candida lusitaniae, Candida rugosa, Cryptococcus neoformans and Saccharomyces cerevisiae. The method showed limited discriminatory power to differentiate species of the Candida parapsilosis complex. Differentiation of Candida albicans and Candida tropicalis needs already identified controls. FSP method benefits from advantages such as lower cost, higher speed and wider range of species than some commercial yeast-identification methods. We consider this method as one of the easiest molecular approaches for identifying a wide range of human pathogenic yeast species, applicable to both diagnostic and epidemiological purposes.

Auxanographic Carbohydrate Assimilation Method for Large Scale Yeast Identification.

The auxanographic carbohydrate assimilation had been an important method for differentiation of yeasts. Prevailing methods described in the literature for carbohydrate assimilation has limited scope for use in large scale yeast identification. To optimize the large scale auxanographic carbohydrate assimilation method for yeast identification. A modified auxanographic carbohydrate assimilation method was developed and a total of 35 isolates of Candida species comprising of four ATCC (American Type Culture Collection) Candida strains (Candida albicans ATCC 90028, Candida tropicalis ATCC 90018, Candida parapsilosis ATCC 750, Candida krusei ATCC 6258) and 31 clinical isolates of Candida tropicalis (n=13), Candida krusei (n=7), Candida glabrata (n=3), Candida kefyr (n=3), Candida albicans (n=5) were validated. The carbohydrates tested were Glucose, Sucrose, Maltose, Lactose, Cellubiose, Raffinose, Trehalose, Xylose, Galactose and Dulcitol. A total of 35 Candida species were tested for their carbohydrate assimilative property and the results were consistent with the existing standard protocols. A well circumscribed opaque yeast growth indicated assimilation of the test carbohydrate and translucent to opalescent growth with the outline of initial inoculum alone indicated lack of assimilation. The control plate indicated no growth of the Candida species. The carbohydrate assimilation tests finds utility for yeast diversity studies exploring novel ecological niches. The technique described here facilitates testing of an extended range of carbohydrates and yeasts in a cost effective manner.

Isolation and identification of Candida and Non albicans Candida species using chromogenic medium

Background: Identification of Candida to species level is required to reduce morbidity & mortality as non albicans candida species are being recovered with greater frequency nowadays.CHROM Agar has the advantage of rapid identification and speciation of Candida by making use of chromogenic substrates. Material & Methods: A prospective study was carried out from February 2014 to May 2014. The material for the study constituted various clinical samples that were sent routinely to the microbiology laboratory for culture and sensitivity testing. CHROM Agar Candida was used to differentiate Candida species along with standard yeast identification methods and antifungal susceptibility testing. Results: A total of 45 Candida species isolates were recovered in which Non albicans candida 35(77.78%) remained higher in comparison to C. albicans 10(22.22%) with C. krusei 15(33.3%), C.glabrata 13(28.9%) and C.tropicalis 7(15.6%).C.albicans was isolated mainly from respiratory medicine ward (5), C. troplicalis from medical intensive care unit (03), C. glabrata and C.krusei with eleven isolates each from neonatal intensive care unit. C.krusei (26.67%) and C.glabrata (26.67%) remained predominant in blood, C.tropicalis (13.33%) in urine and C.albicans (8.89%) in sputum.C.albicans sensitivity to Fluconazole, Voriconazole and Amphotericin B was 70%, 70% and 90% respectively. C.krusei remained resistant to Voriconazole (60%) but susceptible to Amphotericin B (80%) whereas C. tropicalis depicted sensitivity of 71% to both Fluconazole and Amphotericin B. Conclusion: CHROM Agar Candida shows good potential in identification of azole and amphotericin B resistant candida species, which in combination with local antifungal susceptibility pattern shall help to optimize the initiation of antifungal therapy and further avoid treatment failures.

The first isolation of Trichosporon coremiiforme from soil in Iran.

Background and Purpose: Trichosporon is a genus of anamorphic basidiomycetous yeast which is widely distributed in nature and is found in tropical and temperate areas. The aim of this work was to study the isolation, identification and molecular analysis of Trichosporon species in soil.Materials and Methods:In order to isolate and identify Trichosporon species in soil, 30 samples were collected from 30 different locations across Iran. The isolates were identified by means of the standard methods of yeast identification. To confirm morphological identification, genomic DNA was extracted and the hypervariable D1/D2 domain of the large-subunit (LSU) ribosomal DNA (rDNA) gene was amplified by polymerase chain reaction (PCR), using primer pair NL-1/NL-4, and then the sequences were analyzed. Results:According to the morphological and physiological assessments, isolates were identified as T. coremiiforme. The isolates formed chlamydospore after one week on yeast-malt (YM) agar medium. Using Blast program, we found that the D1/D2 sequences of the T. coremiiforme isolates from Iran (accession no: KP055040 and KP055041) showed 99% homology with the T. coremiiforme deposited in GenBank. All the T. coremiiforme isolates placed in the Ovoides cluster were well-supported by bootstrap values.Conclusion:The present study is the first attempt to survey Trichosporon in soil of Iran. To the best of our knowledge, this is the first investigation of T. coremiiforme in Iran.

Comparison of the yeast microbiota of different varieties of cool-climate grapes by PCR-RAPD

The yeast microbiota occurring on different varieties of grapes grown in cool-climate is not completely researched. Therefore, its identification is important to research. On the other hand, yeasts occurring in these fruits can be potentially used as starter cultures to obtain particularly demanded features in the production of wine. In addition, rapid methods for yeast identification allow to eliminate the contamination with pathogenic yeasts, which could cause the loss of wine production. The aim of the study was to isolate and identify the yeasts occurring on the surface of the different varieties of white and red grapes, grown in cool-climate of Poland. Also, the aim was to compare the qualitative and quantitative composition of yeasts on the tested grapes. The 84 cultures of yeasts were isolated, that were initially macroscopic and microscopic analyzed and the purity of cultures was rated on the WL medium. Identification of yeasts by PCR-RAPD was carried using the M13 primer. In the PCR-RFLP method ITS1 and ITS4 primers, as well as restriction enzymes HhaI, HinfI, HaeIII, were used. Preliminary identification of yeasts by standard methods produced results very different from the results obtained by molecular methods. Among the isolated microorganisms yeasts were dominating, but bacteria and molds were also present. Using the PCR-RAPD method most strains of yeasts were identified. Yeast microflora of different varieties of white and red grapes was very similar as the same species of yeasts were identified. Yeasts of the genus Saccharomyces were present in all varieties of grapes. The Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Metschnikowia pulcherrima, Rhodotorula minuta, Pichia kluyveri, Hanseniaspora uvarum and Rhodotorula mucilaginosa were identified by PCR-RAPD. 4 of the 33 tested strains of yeasts were identified by PCR-RFLP. By PCR-RAPD only Hanseniaspora uvarum was identified. The quantity and quality of microorganisms living on the surface of grape fruits is very important for the process of winemaking. Yeasts influence the course of alcoholic fermentation, the flavor, aroma, and thus the quality of the produced wine. To a large extent their presence depends on the condition of the surface of the fruit. Many researchers reported significant differences between yeast microflora in grapes of Mediterranean and cool-climate vineyards. As they are expected to affect the final wine properties precise researching of the microflora of cool-climate grapes may lead to the isolation of new species of yeasts and thus the wines with unique characteristics can be obtained.

Are the Conventional Commercial Yeast Identification Methods Still Helpful in the Era of New Clinical Microbiology Diagnostics? A Meta-Analysis of Their Accuracy

Accurate identification of pathogenic species is important for early appropriate patient management, but growing diversity of infectious species/strains makes the identification of clinical yeasts increasingly difficult. Among conventional methods that are commercially available, the API ID32C, AuxaColor, and Vitek 2 systems are currently the most used systems in routine clinical microbiology. We performed a systematic review and meta-analysis to estimate and to compare the accuracy of the three systems, in order to assess whether they are still of value for the species-level identification of medically relevant yeasts. After adopting rigorous selection criteria, we included 26 published studies involving Candida and non-Candida yeasts that were tested with the API ID32C (674 isolates), AuxaColor (1,740 isolates), and Vitek 2 (2,853 isolates) systems. The random-effects pooled identification ratios at the species level were 0.89 (95% confidence interval [CI], 0.80 to 0.95) for the API ID32C system, 0.89 (95% CI, 0.83 to 0.93) for the AuxaColor system, and 0.93 (95% CI, 0.89 to 0.96) for the Vitek 2 system (P for heterogeneity, 0.255). Overall, the accuracy of studies using phenotypic analysis-based comparison methods was comparable to that of studies using molecular analysis-based comparison methods. Subanalysis of studies conducted on Candida yeasts showed that the Vitek 2 system was significantly more accurate (pooled ratio, 0.94 [95% CI, 0.85 to 0.99]) than the API ID32C system (pooled ratio, 0.84 [95% CI, 0.61 to 0.99]) and the AuxaColor system (pooled ratio, 0.76 [95% CI, 0.67 to 0.84]) with respect to uncommon species (P for heterogeneity, <0.05). Subanalysis of studies conducted on non-Candida yeasts (i.e., Cryptococcus, Rhodotorula, Saccharomyces, and Trichosporon) revealed pooled identification accuracies of ≥98% for the Vitek 2, API ID32C (excluding Cryptococcus), and AuxaColor (only Rhodotorula) systems, with significant low or null levels of heterogeneity (P > 0.05). Nonetheless, clinical microbiologists should reconsider the usefulness of these systems, particularly in light of new diagnostic tools such as matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, which allow for considerably shortened turnaround times and/or avoid the requirement for additional tests for species identity confirmation.

Comparison between MALDI-TOF MS and FilmArray Blood Culture Identification panel for rapid identification of yeast from positive blood culture

In this study we evaluated MALDI-TOF MS and FilmArray methods for the rapid identification of yeast from positive blood cultures. FilmArray correctly identified 20/22 of yeast species, while MALDI-TOF MS identified 9/22. FilmArray is a reliable and rapid identification system for the direct identification of yeasts from positive blood cultures.

Yeast Identification Algorithm Based on Use of the Vitek MS System Selectively Supplemented with Ribosomal DNA Sequencing: Proposal of a Reference Assay for Invasive Fungal Surveillance Programs in China

Sequence analysis of the internal transcribed spacer (ITS) region was employed as the gold standard method for yeast identification in the China Hospital Invasive Fungal Surveillance Net (CHIF-NET). It has subsequently been found that matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is potentially a more practical approach for this purpose. In the present study, the performance of the Vitek MS v2.0 system for the identification of yeast isolates collected from patients with invasive fungal infections in the 2011 CHIF-NET was evaluated. A total of 1,243 isolates representing 31 yeast species were analyzed, and the identification results by the Vitek MS v2.0 system were compared to those obtained by ITS sequence analysis. By the Vitek MS v2.0 system, 96.7% (n = 1,202) of the isolates were correctly assigned to the species level and 0.2% (n = 2) of the isolates were identified to the genus level, while 2.4% (n = 30) and 0.7% (n = 9) of the isolates were unidentified and misidentified, respectively. After retesting of the unidentified and misidentified strains, 97.3% (n = 1,209) of the isolates were correctly identified to the species level. Based on these results, a testing algorithm that combines the use of the Vitek MS system with selected supplementary ribosomal DNA (rDNA) sequencing was developed and validated for yeast identification purposes. By employing this algorithm, 99.7% (1,240/1,243) of the study isolates were accurately identified with the exception of two isolates of Candida fermentati and one isolate of Cryptococcus gattii. In conclusion, the proposed identification algorithm could be practically implemented in strategic programs of fungal infection surveillance.

Recent Taxonomic Developments with Candida and Other Opportunistic Yeasts.

Increases in susceptible patient populations and advances in identification methods have resulted in the continued recognition of novel yeasts as agents of human infection. Most of these agents are members of the well-recognized genera Candida, Cryptococcus, Trichosporon, and Rhodotorula. Some of these agents are "cryptic species," members of species complexes, and may not be detectable using classical carbohydrate assimilation-based methods of yeast identification. Such species require DNA- or MALDI-based methods for correct identification, although sporadic isolates may not routinely require delineation to the individual species level. The coming end of the fungal taxonomy rules requiring separate names for sexual and asexual forms of the same fungus will hopefully allow greater clarity, as names for medically important yeast can now be based on the needs of the medical mycology community and the common goal of better communication between laboratory and clinician.

Comparison of nosocomial candidemia of pediatric and adult cases in 2-years period at a Turkish university hospital.

Although there are limited numerous reports of candidemia in adults, data on paediatrics are stil limeted. The aim of the present study was to compare the aetiology and risk factors of nosocomial candidemia among the paediatric and adults in our hospital. This study includes the patients hospitalised and diagnosed as fungemia at Ondokuz Mayis University Hospital between June 30, 2007 and June 30, 2009 whose blood cultures sent to our microbiology laboratory. After fungal growth was observed in blood cultures, the yeast cells were inoculated onto Saboraud glucose agar. The colonies were identified by conventional yeast identification methods and ID 32C yeast identification system according to the manifacturer's instructions. During this period 51 paediatric and 69 adults were studied. The most common yeast form was Candida albicans (43.3%) followed by C. parapsilosis (25.0%) and C. tropicalis (17.5%). Although the non-albicans Candida species represent more than half (56.7%) of all candidemic cases C. albicans was the most common frequent etiologic agent. There was no statistically significant difference between patient age (paediatric and adult) and distribution of Candida species (p>0.05) Neoplasia (in adults) and prematurity (in paediatrics) were the main underlying diseases. Predisposing factors and mortality rates were not different among paediatrics and adults. We reinforce the necessity of continous epidomiologic surveillance to follow the dynamics of candidemia.

Improved Identification of Yeast Species Directly from Positive Blood Culture Media by Combining Sepsityper Specimen Processing and Microflex Analysis with the Matrix-Assisted Laser Desorption Ionization Biotyper System

Current methods for identification of yeast from blood cultures may take several days after these microorganisms have been observed by Gram stain smears from positive blood cultures. We explored the use of a matrix-assisted laser desorption ionization (MALDI) Biotyper system in combination with Sepsityper specimen processing and Microflex analysis for improved detection and identification of yeast species directly from positive blood culture specimens demonstrating yeast-like organisms by Gram stain. The limit of detection of yeast species in blood culture medium was determined to be 5.9 × 10(5) CFU, with intra- and interstrain coefficients of variation of 1.8 to 3.6% and 2.9%, respectively. A total of 42 yeast-containing positive blood culture specimens were processed, and the identification results were compared to those obtained by routinely used phenotypic methods. Specimens with discrepant results between the Biotyper and phenotypic methods were identified on the basis of internal transcribed spacer region sequencing. The MALDI Biotyper system correctly identified the 42 specimens to species level, including 28 (66.7%) Candida albicans, 8 (19.0%) Candida parapsilosis, and 5 (11.9%) Candida tropicalis isolates and 1 (2.4%) Cryptococcus neoformans isolate. The entire procedure, from specimen extraction to final result reporting, can be completed within 1 h. Our data indicated that the Sepsityper specimen processing and Microflex analysis by the MALDI Biotyper system provide a rapid and reliable tool for yeast species identification directly from positive blood culture media.

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

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.

DNA Pyrosequencing–Based Identification of Pathogenic Candida Species by Using the Internal Transcribed Spacer 2 Region

Abstract Context.—The incidence of infections due to diverse Candida species is increasing, with correspondingly different antifungal susceptibility patterns. Routine yeast identification methods cause significant delays in appropriate patient management. Objective.—A DNA pyrosequencing strategy was evaluated for identification of pathogenic Candida species associated with human infections. Design.—Clinical (n = 51) and commercial (n = 9) Candida isolates were identified in a blinded, parallel study consisting of routine fungal cultures and biochemical analyses in comparison with DNA pyrosequencing. Results.—DNA pyrosequencing yielded species-level identification of all 60 Candida isolates, and sequencing interpretations agreed in all cases with results of biochemical and morphologic testing. Different Candida species were identified, such as C albicans, C dubliniensis, C glabrata, C guilliermondii, C krusei, C lusitaniae, C parapsilosis, and C tropicalis. Automated and manual approaches to DNA sequence i...