Nucleic acid sequence-based amplification (NASBA) detection of medically important Candida species.

Nucleic Acid Sequence Based Amplification (iNASBA), an isothermal amplification technique for nucleic acids, was evaluated for the identification of medically important Candida species using primers selected from 18S rRNA sequences conserved in fungi. An RNA fragment of 257 nucleotides was amplified for Candida albicans. Nineteen different fungi were tested for rRNA amplification with the NASBA. All were positive when analyzed on agarose gel, whereas human RNA was negative. For the identification of Candida species, NASBA amplification products were analyzed in an enzyme bead-based detection format, using species-specific biotinylated probes and a generic Candida HRPO probe or a membrane-based system using biotinylated probes and avidin-HPRO. Discrimination of the major human pathogenic Candida spp. was based on a panel of biotinylated probes for C. krusei, C. tropicalis, C. albicans, C. glabrata, and C. lusitaniae. Using rRNA dilutions obtained from pure cultures of C. albicans, the combination of NASBA and the enzymatic bead-based detection yielded a sensitivity equivalent to 0.01 CFU. In a model system using 1 ml of artificially contaminated blood as few as 1-10 CFU of C. albicans could be detected. Testing of 68 clinical blood samples from patients suspected of candidemia showed that eight samples were positive for C. albicans and one for C. glabrata. Testing of 13 clinical plasma samples from patients suspected of fungemia identified the presence of C. albicans in two specimens. The whole procedure of sample preparation, amplification and identification by hybridization can be performed in 1 day. This speed and the observed sensitivity of the assay make the NASBA a good alternative to PCR for the detection of candidemia.     

Characterization of Candida Species from Different Populations in Taiwan

The opportunistic Candida species existing as part of commensal microbiota in humans are usually the etiological agents causing infections. We investigated whether isolates collected from different age groups, hospital units, and sources have distinct characteristics. A total of 913 isolates comprising 395 Candida albicans, 230 Candida tropicalis, 202 Candida glabrata, 62 Candida parapsilosis, 13 Candida krusei, and 11 of other six species were analyzed. Urine was the most common source (41.2%), followed by sputum (16.3%), blood (15.2%), and others (27.3%). Candida albicans and C. parapsilosis were more prevalent in the working group [from 19 to 65 years], whereas C. tropicalis and C. glabrata were more prevalent in the elder one (≥ 66 years). We found that the age of patients and the source of isolates affect the distribution of species. On the other hand, the drug susceptibility of isolates was associated with fungal species and whether patients were hospitalized.     

Cell-surface hydrophobicity of Candida species as determined by the contact-angle and hydrocarbon-adherence methods

Cell-surface hydrophobicities of six Candida species were studied by two methods: measurement of the contact angle, and partitioning with aqueous-hydrocarbon (n-octane, n-hexadecane and p-xylene) mixtures. C. tropicalis, C. glabrata and C. krusei adhered better to the hydrocarbons than did C. albicans, C. stellatoidea and C. parapsilosis. Contact angles for the less adherent species were smaller than those for the more adherent species. Thus the two methods gave results that were similar overall and indicated that C. tropicalis, C. glabrata and C. krusei have greater cell-surface hydrophobicities than C. albicans, C. stellatoidea and C. parapsilosis.     

Candida biotypes isolated from clinical specimens in Malaysia

The distribution of Candida species was examined using 1114 yeasts isolated from various clinical specimens. The isolates were identified by germ tube test, hyphal/pseudohyphae and chlamydoconidia production and carbohydrate assimilation test using ten carbohydrates (glucose, sucrose, trehalose, cellobiose, arabinose, galactose, mannitol, raffinose, lactose and maltose). Among the 1114 isolates studied, 9 species of Candida were identified and the relative frequency of isolation was C. albicans (44.2%), C. parapsilosis (26.0%), C. tropicalis (17.7%), C. glabrata (9.6%), C. krusei (1.2%), C. rugosa (0.6%), C. guilliermondii (0.2%), C. lusitaniae (0.08%) and C. kefyr (0.08%). Non- C. albicans was the most common Candida species isolated from blood, respiratory system, urine and skin. The isolate from vaginal swabs was predominantly C. albicans. 82.2% of C. glabrata and 64.2% of C. krusei isolated in this study were from vaginal swabs. This revised version was published online in August 2006 with corrections to the Cover Date.     

Comparison of a randomly amplified polymorphic DNA (RAPD) analysis and ATB ID 32C system for identification of clinical isolates of different Candida species

The objective of this work was to compare the usefulness of a randomly amplified polymorphic DNA (RAPD) assay to that of the ATB ID32C kit (bioMérieux, France) for identification of different species of Candida isolated from clinical specimens. The RAPD-PCR patterns obtained with OPE-18 primer for identification of clinical isolates were consistent, and the different independent assays revealed reproduction of the band patterns. RAPD with the OPE-18 primer is a very specific and sensitive method for identification of Candida glabrata, Candida guilliermondii, Candida tropicalis, Candida pelliculosa, Candida albicans, Candida krusei, and Candida lusitaniae.     

Molecular typing of Candida spp. by random amplification of polymorphic DNA and analysis of restriction fragment length polymorphism of ribosomal DNA repeats

Random amplification of polymorphic DNA (RAPD) using an arbitrary oligonucleotide primer (5'-CGGTGCGACG) and analysis of restriction fragment length polymorphism of ribosomal DNA (rDNA-RFLP) after digestion of genomic DNA with restriction endonuclease EcoRI were investigated as tools for genotypic delineation beyond the species level of 91 Candida clinical isolates and four reference strains including 33 Candida albicans, 19 Candida tropicalis, 22 Candida krusei and 21 Candida (Torulopsis) glabrata. Results indicated that both techniques can be useful for typing isolates of the above species, although showing a variable discriminative potential with different species. As compared to RAPD fingerprinting, the discriminative potential of rDNA-RFLP appeared to be highest for C. albicans and lowest for C. glabrata, being overall similar for C. krusei and identical for C. tropicalis. A comparative analysis of the results obtained with the two typing techniques showed that, except for C. tropicalis, they were able to provide non-redundant information, and that their use in combination could enhance the discriminative potential for delineation among C. glabrata and C. krusei isolates.     

Evaluation of a new chromogenic medium (Candida ID) for the isolation and presumptive identification of Candida albicans and other medically important yeasts

Candidiasis is a frequent human infection caused mainly by Candida albicans. However, other species are emerging as important pathogens, as Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei or Candida guilliermondii. Rapid identification of clinical isolates could facilitate diagnosis and treatment. Candida ID (bioMerieux, Spain) is a new medium for the isolation and presumptive identification of yeasts: C. albicans grows as blue colonies, and C. tropicalis, C. guilliermondii, Candida kefyr and Candida lusitaniae as pink ones. The utility of Candida ID was evaluated with more than 700 clinical isolates and type culture collection strains from different genera including Candida, Cryptococcus, Saccharomyces, and Rhodotorula. Presumptive identification was confirmed by germ tube test, microscopic morphology and chlamydoconidia production on corn meal agar and carbohydrate assimilation on API-ATB ID 32C or Vitek (bioMerieux). Growth on Candida ID was rapid (18-24 h) for most of the yeast strains tested. Sensitivity and specificity of identification of C. albicans was significantly high (>98%), since a very low number of isolates were found to be false negative or false positive. A better result was obtained for species growing as pink colonies (>99.5%). Detection of different species of medical important yeasts was easy with Candida ID, as perfectly distinct colors and textures of colonies were observed on this medium. Candida ID allowed the discrimination between C. glabrata (creamy and smooth) and C. krusei (rough and white) colonies. Other species showed different colony textures and colours, white being the predominant colour. Candida ID was very useful for the presumptive identification C. albicans isolates.     

Use of the BIOMIC Video System to evaluate the susceptibility of clinical yeast isolates to fluconazole and voriconazole by a disk diffusion method

The ARTEMIS Global Antifungal Susceptibility Program provides the collection of epidemiological data and the results of the fluconazole and voriconazole susceptibility testing of yeast isolates. Participating in this study, a total of 7318 clinical yeast isolates were tested from different geographical areas in Hungary in the period 2001 to 2003. The species isolated most frequently was C. albicans (68.8%), followed by C. glabrata (11.8%), C. tropicalis (5.7%) and C. krusei (4.6%). Isolates of C. albicans, C. kefyr, C. lusitaniae, C. tropicalis and C. parapsilosis were highly susceptible to fluconazole (78.9-100%). The rates of isolation of fluconazole-resistant C. glabrata and C. krusei were higher in our study than the global mean in 2001 (28.2% and 87.5% vs. 18.3% and 70.2%, respectively). Differences were detected in the distribution of fluconazole-susceptibility data of C. glabrata isolates in the different counties of Hungary: most of the resistant isolates were observed in the eastern part of the country.     

米诺环素对临床几种常见假丝酵母菌体外药敏的初步探究

探究米诺环素在体外对常见酵母菌的药敏特点。采用Rosco纸片扩散法对168株常见的几种假丝酵母菌进行米诺环素的药敏试验。米诺环素有抑菌环的比率:白假丝酵母菌41%,光滑假丝酵母菌1.2%,热带假丝酵母菌和克柔假丝酵母菌没有抑菌环。在常见几种酵母菌中,米诺环素几乎只对白假丝酵母菌在体外有抗菌活性。     

26S rDNA单链构象多态性分析在临床酵母菌菌种鉴定中的应用

摘要： 【目的】探讨酵母菌临床分离株26S rDNA D1/D2区序列种内相似性和种间差异性的快速检测方法,为临床酵母菌菌种鉴定方法的改进奠定基础。调查北京地区临床酵母菌的种群多样性,为国内酵母菌感染的流行病学研究提供新的基础数据。【方法】用5种常见临床酵母菌种的模式和权威菌株作为标准参考菌株,从北京四家综合性医院收集临床酵母菌260余株,PCR扩增其26S rDNA D1/D2区,对扩增产物进行单链构象多态性（Single-Strand Conformation Polymorphism,SSCP）分析和序列测定分析。【结果】常见病原酵母菌26S rDNA D1/D2区的SSCP图谱具有明显的种间差异性和种内相似性,可以通过该方法对菌株进行初步的菌种鉴定。D1/D2-SSCP和序列分析相结合,对260余株临床酵母菌进行了菌种鉴定,共鉴定有10个属20个种,优势属为念珠菌属(Candida),优势种及其所占比例分别是：C. albicans (57.7%), C. parapsilosis (10.0%), C. tropicalis (9.2%), C. glabrata (6.7%)和C. krusei (5.8%),并发现过去从未或很少报道致病的酵母菌种,愈来愈多地出现在临床分离菌株中。【结论】 26S rDNA D1/D2区的SSCP图谱分析为临床酵母菌株的快速鉴定提供了新的方法;北京地区酵母菌临床分离株呈种群多样性分布,C. albicans虽然仍占优势,但其它念珠菌种的比例已达42%。     

Evaluation of a rapid,quantitative real-time PCR method for enumeration of pathogenic Candida cells in water

Quantitative PCR (QPCR) technology, incorporating fluorigenic 5' nuclease (TaqMan) chemistry, was utilized for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C. lusitaniae) in water. Known numbers of target cells were added to distilled and tap water samples, filtered, and disrupted directly on the membranes for recovery of DNA for QPCR analysis. The assay's sensitivities were between one and three cells per filter. The accuracy of the cell estimates was between 50 and 200% of their true value (95% confidence level). In similar tests with surface water samples, the presence of PCR inhibitory compounds necessitated further purification and/or dilution of the DNA extracts, with resultant reductions in sensitivity but generally not in quantitative accuracy. Analyses of a series of freshwater samples collected from a recreational beach showed positive correlations between the QPCR results and colony counts of the corresponding target species. Positive correlations were also seen between the cell quantities of the target Candida species detected in these analyses and colony counts of Enterococcus organisms. With a combined sample processing and analysis time of less than 4 h, this method shows great promise as a tool for rapidly assessing potential exposures to waterborne pathogenic Candida species from drinking and recreational waters and may have applications in the detection of fecal pollution.     

Distribution and susceptibility of Candida species isolated in the Medical University of Debrecen

Data of Candida albicans and non-albicans Candida species isolated during the 1997-2000 period in the Medical and Health Science Center of the University of Debrecen are analysed. The number of yeast isolates increased from 408 to 1213 per year during this period. Dominance of C. albicans has been persistent, but a slight increase of C. glabrata and C. krusei could be observed. Distribution of different Candida species isolated from 16 body sites indicates that C. albicans seems to be still the most aggressive Candida species. Investigation of 244 urinary Candida isolates (parallel with bacterial cultures) suggests that tha aetiological role of Candida species in the pathogenesis of urinary tract infections can be hypothesized if colony forming unit (CFU) number of yeasts is higher than 10(4)/ml and bacteria are present in low CFU number or are absent. Antifungal susceptibility testing of C. albicans, C. glabrata, C. tropicalis and C. krusei against Flucytosine, Amphotericin-B, Miconazole, Ketoconazole and Fluconazole suggests that Amphotericin-B is still the most effective antifungal agent. Finally, the problems in judging the aetiological role of isolated Candida species in the pathogenesis of different types of diseases are critically discussed.     

Rapid identification of Candida glabrata using a new commercial kit

Candida glabrata is an emergent pathogen with diminished susceptibility to azoles, thus a rapid identification of this yeast could be of help to choose the appropriate treatment. GLABRATA RTT (Fumouze Diagnostics, France) is a new C. glabrata identification test. To evaluate its utility in the clinical laboratory daily routine, we prospectively tested 168 yeasts isolated in our hospital. GLABRATA RTT results had a sensitivity of 98.4% and a specificity of 100%. The combination of CHROMagar Candida isolation medium and GLABRATA RTT test allowed the identification of the four most common species in the clinical practice (Candida albicans, Candida tropicalis, Candida krusei and C. glabrata).     

In Vitro Evaluation of Putative Virulence Attributes of Oral Isolates of <Emphasis Type="Italic">Candida</Emphasis> spp. Obtained from Elderly Healthy Individuals

Identification of Candida isolates obtained from oral cavity of elderly healthy individuals revealed the predominance of non-albicans Candida species (88.9%) compared to Candida albicans (11%). CHROMagar Candida differential medium and PCR revealed the presence of Candida tropicalis (33.3%), Candida glabrata (27.8%), and Candida krusei (16.7%). We investigated the presence of virulence attributes in a total of 18 isolates, including acid protease and phospholipase production, hemolytic activity, and biofilm production. Extracellular protease was found in five isolates (27.8%) whereas extracellular phospholipase was found in three isolates (17%). All isolates showed hemolytic activity. About 56% of the isolates were weakly positive for biofilm formation (score +) whereas a minority (5.6%) of them showed strong biofilm formation (score 4+). Susceptibility in vitro of the isolates to fluconazole was carried out by microdilution method. Fluconazole showed a strong inhibition against most buccal isolates. The resistant isolates were 2 C. tropicalis, 2 C. glabrata, and 1 C. krusei.     

Alveolar macrophage reaction to candida species

Candida species are increasingly important fungal pathogens. The reaction of rat alveolar macrophages (AM) to Candida albicans was compared with that to C. glabrata and C. krusei . Phagocytosis of C. glabrata was similar to that of C. albicans , but significantly slower for C. krusei due to reduced attachment. After opsonization, attachment of C. albicans and C. krusei to AM was significantly increased and there was no significant difference between the two species. The oxidative metabolism of AM with candida species was two to three times higher than that of the resting AM both during and 24 h after the phagocytosis. All three species showed a considerable fraction (5–10%) of phagolysosomes with pH ≥ 6·5 after 3 h and a smaller percentage (1%) after 24 h.     

Identification and antifungal susceptibility of Candida isolated from intensive care unit patients

Candida was isolated in 205 of 1060 clinical specimens (19.33%) in our laboratary sent from the intensive care unit for mycological investigation between January 98-December 99. All isolated strains were identified to species level using the API Candida system (Bio-Meieux, France) as follows; Candida albicans (n:115, 56.09%), Candida tropicalis (n:23, 11.21%), Candida parapsilosis (n:21, 10.24%), Candida glabrata (n:12, 5.83%). Candida kefyr (n:9, 4.39%), Candida lusitaniae (n:7, 3.41%), Candida famata (n:6, 2.92%), Candida krusei (n:6, 2.92%), Candida guilliermondii (n:6, 2.92%). These stains were identified using congo-red-glucose-brain-heart-infusion agar and slime production was determined in Candida albicans 53.91% and 67.77% in other than Candida species. In the present study, E test (AB Biodisk, Solna, Sweeden) was used to test antifungal susceptibility. The resistance to amphotericin B was 19.51%, to fluconazole 27.31% and to flucytosine 20.00%.     

Evolutionary relationships among pathogenic Candida species and relatives.

Small subunit rRNA sequences have been determined for 10 of the most clinically important pathogenic species of the yeast genus Candida (including Torulopsis [Candida] glabrata and Yarrowia [Candida] lipolytica) and for Hansenula polymorpha. Phylogenetic analyses of these sequences and those of Saccharomyces cerevisiae, Kluyveromyces marxianus var. lactis, and Aspergillus fumigatus indicate that Candida albicans, C. tropicalis, C. parapsilosis, and C. viswanathii form a subgroup within the genus. The remaining significant pathogen, T. glabrata, falls into a second, distinct subgroup and is specifically related to S. cerevisiae and more distantly related to C. kefyr (psuedotropicalis) and K. marxianus var. lactis. The 18S rRNA sequence of Y. lipolytica has evolved rapidly in relation to the other Candida sequences examined and appears to be only distantly related to them. As anticipated, species of several other genera appear to bear specific relationships to members of the genus Candida.     

男性尿道炎和包皮龟头炎致病真菌的分布与药敏分析

目的了解男性念珠菌性尿道炎和包皮龟头炎的菌群分布及体外抗真菌药敏试验情况。方法菌株分离均来自复旦大学附属华山医院皮肤性病门诊临床症状轻重不一、真菌直接镜检阳性的61例患者。用科玛嘉念珠菌显色培养基及API 20C AUX鉴定系统进行菌种鉴定;采用CLSIM27-A2肉汤微量稀释法对61株临床分离念珠菌作了氟康唑、两性霉素B、氟胞嘧啶、伊曲康唑、伏立康唑、特比萘芬6种抗真菌药物敏感性测定。结果对培养阳性的61例菌株,通过科玛嘉念珠菌显色培养基及API 20C AUX鉴定系统作菌种鉴定,白念珠菌52例（85.2%）,近平滑念珠菌3例,光滑念珠菌2例,热带念珠菌2例,季也蒙念珠菌1例,克柔念珠菌1例。对52株白念珠菌的药敏试验显示氟康唑98.1%敏感,1.9%剂量依赖性敏感;氟胞嘧啶96.2%敏感,3.8%耐药;伊曲康唑44.2%敏感,40.5%剂量依赖性敏感,15.3%耐药;伏立康唑84.6%敏感,15.4%耐药;两性霉素B全部敏感;特比萘芬的MIC范围为1-64μg/ml,MIC50和MIC90皆为64μg/ml。结论在男性念珠菌性尿道炎和包皮龟头炎中,白念珠菌仍是第一位致病菌,体外药敏试验显示氟康唑、伏立康唑、氟胞嘧啶、两性霉素B对男性念珠菌性尿道炎均有较好的敏感性。     

临床分离161株念珠菌菌种鉴定及氟康唑药敏试验分析

目的调查临床分离的念珠菌种类及其对氟康唑的敏感性。方法采用科玛嘉念珠菌显色培养基和YBC平板对山东大学齐鲁医院细菌室分离到的161株念珠菌进行鉴定,并对分离出的白念珠菌分别采用NCCLS推荐的微量稀释法和ROSCO药敏纸片法对氟康唑进行药物敏感性试验。结果在161株念珠菌中白念珠菌为69．57％,热带念珠菌为19．88％,近平滑念珠菌为4．97％,克柔念珠菌为2．48％,光滑念珠菌为1．86％,其他念珠菌为1．24％。两种方法药敏结果显示：112株白念珠菌对氟康唑的敏感率分别为96．43％和97．32％,仅有2株耐药。结论白念珠菌仍然是我院分离率最高的念珠菌,其次是热带念珠菌和近平滑念珠菌。白念珠菌对氟康唑仍敏感,耐药菌株极少。     

Comparison between conventional methods, ChromAgar Candida® and PCR method for the identification of Candida species in clinical isolates

The increase in the incidence of yeast species causing fungemia in susceptible immunocompromised patients in the last two decades and the low sensitivity of conventional blood culture has led to the need to develop alternative approaches for the early detection and identification of causative species. The aim of this study was to compare the usefulness of molecular testing by the polymerase chain reaction (PCR) and conventional methods to identify clinical isolates of different species, using the ID32C ATB system (bioMérieux, France), chromogenic culture Chromagar Candida? (CHROMagar, France) and morphogenesis in corn meal agar. We studied 79 isolates, in which the most prevalent species using the system ID32C and PCR was C. albicans, followed by C. tropicalis, C. glabrata and C .krusei. PCR patterns obtained for the identification of clinical isolates were stable and consistent in the various independent studies and showed good reproducibility, concluding that PCR with species-specific primers that amplify genes ITS1 and ITS2 for rRNA or topoisomerase II primers is a very specific and sensitive method for the identification of C. glabrata, C. krusei, C. albicans, and with less specificity for C. tropicalis.