Intercellular matrix in colonies of Candida.

The histochemistry and fine structure of typical colonies of six species of Candida were studied, using a total of 31 clinical isolates. The colonies consisted of viable and degenerate cells which lay in an intercellular matrix. This matrix was made up of amorphous, granular, and fibrillar components, the relative proportions and total amount of which varied from species to species. The cells of all species were surrounded by a zone of homogeneous amorphus material, which may be a highly cross-linked carbohydrate. This separated intact cells from irregularly distributed granular debris derived from the cytoplasm of degenerate cells. Focal cellular degeneration and associated granular debris were present within the colonies of all species and were most common in the surface layers of cells of colonies of C. albicans and C. tropicalis. The large amounts of intercellular matrix in this region formed a surface coat on colonies of these two species. Intercellular strands of cell wall material, and to a lesser extent other membranous elements from degenerate cells, formed a prominent fibrillar meshwork in the colonies of C. albicans and C. tropicalis, but were less common in those of C. pseudotropicalis and C. guilliermondii and seldom seen in those of C. parapsilosis and C. krusei.     

Diabetes mellitus and candidiases

Patients in various clinical states of diabetes mellitus (according to the recommendation of the American Diabetes Association) as a primary diagnosis were examined for fungal infections by Candida species. Candida spp. were detected in urine, in the material taken from the mouth cavity, nails, skin lesions, ears and eyes, by cultivation on the Sabouraud agar, CHROMagar Candida, and by saccharide assimilation. In the group of diabetics with symptoms of oral candidiasis and denture stomatitis C. albicans was identified in 8 cases, C. tropicalis in 3, C. parapsilosis in 2; 1 strain of C. guilliermondii was also isolated. In patients with urinary tract infections the presence of C. albicans was shown in 12 cases; C. parapsilosis was detected in 6 cases and two strains of each C. tropicalis and C. krusei were also isolated. In patients with leg ulcers C. albicans (25 cases), C. parapsilosis (5), C. tropicalis (3) and one strain of each C. krusei and C. robusta were isolated. Otomycosis was associated with one strain of C. albicans, C. parapsilosis, C. tropicalis and C. guilliermondii. C. albicans was most frequently associated with onychomycosis, paronychia and endophthalmitis; C. parapsilosis was the second most rated yeast.     

Activity of anidulafungin against Candida biofilms

Currently, no standardized method to study the in vitro activity of antifungal agents on biofilms is available, thus, the comparison among different authors is difficult. The studies discussed in this review use the XTT reduction to measure the activity of antifungals on biofilms of 24 h of maturation. To date, biofilm anidulafungin MICs of 47 isolates of Candida spp. (25 Candida albicans, 16 Candida tropicalis, 5 Candida dubliniensis and 1 Candida parapsilosis) have been published. The geometric mean MIC of anidulafungin on biofilms of Candida spp. is of 1.18 microg/ml. Against isolates of species with great capacity of biofilm formation, the geometric mean MIC is 0.325 (C. albicans), 2 (C. parapsilosis) and 0.5 microg/ml (C. dubliniensis). No echinocandin has activity on C. tropicalis biofilms. In addition, anidulafungin can be used for lock therapy of catheters since it is the echinocandin with the least in vitro paradoxical effect.     

Contribution to the study of the enzymatic profiles of yeast organisms with medical interest

The enzymatic profiles of several yeastlike organisms were studied using 19 substrates included in the API ZYM system. The isolates evaluated were: 186 Candida albicans, 19 C. stellatoidea, 4 C. tropicalis, 2 C. parapsilosis, 2 C. pseudotropicalis, 1 C. guilliermondii, 3 C. krusei, 11 Torulopsis (Candida) glabrata, 1 Cryptococcus neoformans, 2 Saccharomyces carlsbergensis, 1 Rhodotorula rubra, and 1 R. mucilaginosa. Esterase lipase (C8), leucine arylamidase, acid phosphatase, and phosphoamidase were detected in all of the isolates while trypsin and alpha-galactosidase were not found in any of the isolates using this system. The other enzymes were produced to a variable degree. The different enzymatic profiles might prove useful in the rapid differential diagnosis of genera and species of these yeastlike organisms. To this end, more extensive studies using more isolates of each species will be required, and enzymatic activity should be verified with other techniques and substrates.     

New chromogenic agar medium for the identification of Candida spp

A new chromogenic agar medium (Candida diagnostic agar [CDA]) for differentiation of Candida spp. is described. This medium is based on Sabouraud dextrose agar (Oxoid CM41) and contains (per liter) 40.0 g of glucose, 10.0 g of mycological peptone, and 15.0 g of agar along with a novel chromogenic glucosaminidase substrate, ammonium 4-(2-[4-(2-acetamido-2-deoxy-beta-D-glucopyranosyloxy)-3-methoxyphenyl]-vinyl)-1-(propan-3-yl-oate)-quinolium bromide (0.32 g liter(-1)). The glucosaminidase substrate in CDA was hydrolyzed by Candida albicans and Candida dubliniensis, yielding white colonies with deep-red spots on a yellow transparent background after 24 to 48 h of incubation at 37 degrees C. Colonies of Candida tropicalis and Candida kefyr were uniformly pink, and colonies of other Candida spp., including Candida glabrata and Candida parapsilosis, were white. CDA was evaluated by using 115 test strains of Candida spp. and other clinically important yeasts and was compared with two commercially available chromogenic agars (Candida ID agar [bioMerieux] and CHROMagar Candida [CHROMagar Company Ltd.]). On all three agars, colonies of C. albicans were not distinguished from colonies of C. dubliniensis. However, for the group containing C. albicans plus C. dubliniensis, both the sensitivity and the specificity of detection when CDA was used were 100%, compared with values of 97.6 and 100%, respectively, with CHROMagar Candida and 100 and 96.8%, respectively, with Candida ID agar. In addition, for the group containing C. tropicalis plus C. kefyr, the sensitivity and specificity of detection when CDA was used were also 100%, compared with 72.7 and 98.1%, respectively, with CHROMagar Candida. Candida ID agar did not differentiate C. tropicalis and C. kefyr strains but did differentiate members of a broader group (C. tropicalis, C. kefyr, Candida lusitaniae plus Candida guilliermondii); the sensitivity and specificity of detection for members of this group were 94.7 and 93.8%, respectively. In addition to the increased sensitivity and/or specificity of Candida detection when CDA was used, differentiation of colony types on CDA (red spotted, pink, or no color) was unambiguous and did not require precise assessment of colony color.     

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.     

In vitro antifungal susceptibility of clinical isolates of Candida spp. obtained from patients with different predisposing factors to candidosis

The increase in the number of infections caused by Candida species and the consequent use of antifungal agents favours an increase of resistant isolates. The aim of this study was to evaluate the antifungal susceptibility of Candida spp. isolates from patients with different systemic predisposing factors to candidosis. Seventy-nine Candida spp. isolates were assayed for in vitro susceptibility to amphotericin B, fluconazole, 5-flucytosine and itraconazole using the technique proposed by the Clinical and Laboratory Standards Institute (CLSI). Four C. albicans, one C. guilliermondii, four C. parapsilosis and two C. tropicalis isolates were resistant to amphotericin B. Only two isolate was resistant to itraconazole. All the isolates tested were susceptible to fluconazole and flucytosine. It could be concluded that the most efficient drugs against the Candida isolates studied were fluconazole and flucytosine and that all of the antifungal agents used in this study were effective against the Candida spp. isolates tested.     

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.     

In-vitro activity of 5-fluorocytosine against 1,021 Spanish clinical isolates of Candida and other medically important yeasts

The aim of this study was to determine the prevalence of primary resistance to 5-fluorocytosine (5FC) among clinical isolates of yeasts in Spain where this drug is not currently available for therapy. We have tested the in vitro activity of 5FC against 1,021 recent yeast clinical isolates, including 522 Candida albicans, 140 Candida parapsilosis, 68 Candida glabrata, 41 Candida dubliniensis, 50 Candida guilliermondii, 34 Candida tropicalis, 28 Candida krusei, 20 Candida famata, 11 Cryptococcus neoformans, 5 Cryptococcus albidus, 43 Rhodotorula spp., 24 Trichosporon spp., 5 Saccharomyces cerevisiae, 9 Pichia spp., and 21 isolates from other 11 yeast species. The MICs were determined by the ATB Fungus agar microdilution test (bioMerieux, France) and the following interpretive breakpoints were used: susceptible, > 4 microg/ml; intermediate, 8 to 16 microg/ml; resistant, > 32 microg/ml. 5FC was very active against Candida spp. and other medically important yeasts as 852 (83.4%) of the studied isolates were susceptible (MIC < 4 microg/ml). The species most susceptible to 5FC were C. dubliniensis (100%of isolates; MIC90, 0.25 microg/ml), C. famata (100% of isolates; MIC90, 0.25 microg/ml), C. guilliermondii (98%of isolates; MIC90, 0.25 microg/ml), C. glabrata (95.5% of isolates; MIC90, 0.25 microg/ml), and C. neoformans (90.9% of isolates; MIC90, 2 microg/ml). Primary resistance to 5FC was very uncommon, and a MIC > 32 microg/ml, indicator of in vitro resistance, was observed in 106 isolates (10.4%): 77 C. albicans (16.5% of isolates; MIC90, > 128 microg/ml), 9 C. parapsilosis (6.4% of isolates; MIC90, 8 microg/ml), 4 C. albidus (80% of isolates, MIC50, > 128 microg/ml), 3 C. glabrata (4.4% of isolates; MIC90, 0.25 microg/ml), 3 C. tropicalis (8.8% of isolates; MIC90, 4 microg/ml), 2 C. krusei (7.1% of isolates; MIC90, 8 microg/ml), 2 Rhodotorula spp. (4.6% of isolates, MIC90, 1 microg/ml), 8 Trichosporon spp. (33.3% of isolates; MIC90, 64 microg/ml), and 1 C. lipolytica (50% of isolates). Interestingly, most C. albicans (67 out of 77 isolates) resistant to 5FC were serotype B isolates.     

Secretion of inducible proteinase by pathogenic Candida species

The ability of three isolates each of seven pathogenic Candida species to grow in a liquid medium containing bovine serum albumin (BSA) as a nitrogen source was determined. All three strains of C. albicans, two strains of C. guilliermondii and one strain of C. tropicalis grew well. At any time proteinase activity was detected in the culture filtrates of only the most virulent species--C. albicans, C. tropicalis and C. parapsilosis and this observation was related to complete hydrolysis of BSA. Serologically, cross reactions were demonstrated between anti-proteinase antiserum and C. albicans and C. tropicalis culture filtrates. These results further emphasise the role of the inducible proteinase of Candida in the pathogenesis of candidosis.     

口腔黏膜真菌鉴定方法的比较

比较常见用于黏膜真菌菌种鉴别的多种方法,探寻最佳的鉴别方法。采集230例普通人群口腔黏膜样本,分别用玉米吐温-80培养观察厚膜孢子法、糖发酵生化反应法、CHROMagar假丝酵母菌显色培养基法、ITS基因的PCR-RFLP(聚合酶链反应-限制性片段长度多态性)法、ITS测序菌种鉴定法,鉴别真菌各菌株。结果显示:有56例菌株至少通过1种方法检出真菌;玉米吐温-80分离培养假丝酵母菌37株;50例菌株ITS基因测序共鉴定出8个菌种,白假丝酵母菌(C.albicans)29株,近平滑假丝酵母菌(C.parapsilosis)10株,热带假丝酵母菌(C.tropicalis)5株,Candida metapsilosis 1株,Lodderomyces elongisporus 1株,克柔假丝酵母菌(Candida krusei)1株,乙醇假丝酵母菌(C.ethanolica)1株,季也蒙毕赤酵母菌(Pichia guilliermondii)2株;CHROMagar假丝酵母菌显色培养基法鉴定出3种菌株,分别是白假丝酵母菌、热带假丝酵母菌、近平滑假丝酵母菌;PCR-RFLP法检出5种菌株,分别是白假丝酵母菌、热带假丝酵母菌、近平滑假丝酵母菌、季也蒙毕赤酵母菌、克柔假丝酵母菌,与基因的测序鉴定一致率为91%;糖发酵生化反应法阳性标本占被检出真菌例数的46.4%(26/56)。结果表明:ITS基因的测序法可以准确鉴定真菌各个菌种;PCR-RFLP法能鉴定常见的菌种,但操作繁琐;CHROMagar假丝酵母菌显色培养基法能快速准确鉴别3种常见假丝酵母菌菌种;玉米吐温-80可以准确培养鉴别白假丝酵母菌;糖发酵生化反应法,缺乏足够的敏感度和特异性,难以准确鉴别各个菌种。     

慢性前列腺炎患者假丝酵母菌感染及耐药性分析

目的 了解慢性前列腺炎与假丝酵母菌感染的相关性及假丝酵母菌的耐药性。方法 采用常规沙堡平板分离360例慢性前列腺炎患者的前列腺液标本中的假丝酵母菌,疑似菌落用ATB Expression鉴定仪进行鉴定。采用ATB-Fungus真菌药敏板,对假丝酵母菌株进行药敏试验。结果 11．7％前列腺液标本（42／360）似丝酵母菌阳性,其中自假丝酵母菌23例（54．7％）,近平滑假丝酵母菌13例（30．9％）,其它6例（14．3％）。假丝酵母菌菌株对两性霉素B和制霉菌素敏感率均为100％,其次为酮康唑,敏感率为97．0％;对5-氟胞嘧啶耐药性最强,其耐药率为56．5％。结论 白假丝酵母菌和近平滑假丝酵母菌是慢性前列腺炎假丝酵母菌感染的优势菌种,假丝酵母菌株最敏感的药物是两性霉素B和制霉菌素。     

几种念珠菌DNA总含量的流式细胞分析

应用流式细胞术(FCM)对处于稳定生长阶段的念珠菌属(Candida)的7种8株念珠菌进行了DNA总含量的流式细胞(FCM)分析。这8株念珠菌是:白念珠菌(C.albicans)2株,热带念珠菌(C.tropicalis),克柔念珠菌(C.krusei),近平滑念珠菌(C.parapsiolosis),乳酒念珠菌(C.kefyr),白念珠菌星形变种(C.stellatoidea),即血清B型白念珠菌,季也蒙念珠菌(C.guilliermondii)各一株。应用EB一步插入法染色,用鸡红细胞(CRBC)作为内参标准进行DNA总含量测定。分析结果表明:稳定生长阶段的组方图上,大多数念珠菌细胞处于DNA合成周期的G_0/G_1期;DNA总含量有明显的种间和种内差异。     

ATB FUNGUS2法在念珠菌属和新生隐球菌抗真菌药敏试验中应用的评价

目的评价ATBFUNGUS2半固体培养基法在测定念珠菌属和新生隐球菌对4种常用抗真菌药物敏感性中的应用价值。方法利用CLSIM27．A2微量液基稀释法和ATBFUNGUS2法同时测定131株念珠菌和20株新生隐球菌对两性霉素B（AmB）、氟康唑（FLC）、氟胞嘧啶（5-Fc）和伊曲康唑（ITC）的敏感性。结果①两种方法对于AmB、5-FC、FLC和ITC的一致性分别为98％、89．4％、78．8％和78．1％;②所有受试菌株中两种方法的一致性为80％,但ATBFUNGUS2法将2／5株M27-A2法检查为FLC耐药的白念珠菌判断为敏感或剂量依赖,将8／10株M27-A2法检查为FLC剂量依赖的白念珠菌判断为敏感或耐药。③ATBFUNGUS2法中AmB的MIC值判读范围偏高,以致于实际工作中不能读出准确的值。结论ATBFUNGUS2半固体培养基法在测定念珠菌属和新生隐球菌对4种常用抗真菌药物的敏感性时不失为简单、快速而且重复性好的方法。     

Occurrence ofCandida strains in cases of paronychia

A total of 43 patients, 11 males and 32 females, with paronychia of the fingernails were examined for the presence of Candida spp. The yeast species isolated were identified using standard laboratory methods, including germ-tube production, morphology on rice agar with Tween 80, and mainly fermentation and assimilation of saccharides. In the male group, two Candida species were detected: C. albicans as the dominant species in 9 patients and C. parapsilosis in 2 cases. Similarly, C. albicans was the prevalent species also in females (n = 17); other Candida species detected were C. parapsilosis (n = 7), C. tropicalis (5) and C. krusei (3). In addition to the genus Candida, the following anaerobic and aerobic microorganisms were isolated from patients of both groups: Fusobacterium spp., Bacteroides spp., Staphylococcus aureus, alpha-hemolytic streptococci, group A beta-hemolytic streptococci, Klebsiella pneumoniae, Neisseria spp. and Pseudomonas aeruginosa.     

Differential adhesion of pathogenic Candida species to epithelial and inert surfaces

Abstract Growth in medium containing 500 mM galactose is known to promote the adhesion of Candida albicans to buccal epithelial cells or to acrylic in vitro. Of 5 other Candida species tested, only C. tropicalis (one strain) showed substantially increased adhesion to buccal cells (but not to acrylic) after growth under these conditions. A second strain of C. tropicalis as well as C. stellatoidea, C. parapsilosis, C. pseudotropicalis, C. guilliermondii and Saccharomyces cerevisiae showed little or no increased adhesion to either surface. However, after growth in medium containing 50 mM glucose, C. tropicalis and C. parapsilosis were significantly more adherent to acrylic than glucose-grown yeasts of the other species, including C. albicans . These results are discussed in relation to the colonization and infection potential of the pathogenic Candida species.     

A comparison of experimental pathogenicity of Candida species in cyclophosphamide-immunodepressed mice

The experimental pathogenicity of Candida albicans, C. krusei, C. guilliermondii, C. parapsilosis, C. tropicalis and C. viswanathii was tested in normal and in cyclophosphamide-(Cy) immunodepressed mice. In unpretreated CD1 mice only C. albicans, C. tropicalis and C. viswanathii were pathogenic on intravenous challenge, with LD50 of 1.0 X 10(6), 4.8 X 10(6), 7.2 X 10(8) cells, respectively, per kg. Three days after a single intraperitoneal injection of Cy (150 mg kg-1) mice had a marked decrease in spleen weight and cellularity as well as reduced numbers of circulating leukocytes. Under these conditions, there was a significant, proportional increase in pathogenicity of C. albicans, C. tropicalis and C. viswanathii but the animals were still resistant to challenge with C. krusei, C. guilliermondii and C. parapsilosis. This pattern of susceptibility was not influenced by higher doses of Cy. Only C. albicans and C. tropicalis were capable of rapid and extensive multiplication in target organs such as kidney and brain in normal and Cy-treated mice and for both these species of Candida, there was a 'rebound' effect of increased resistance to experimental infection after 12 days from Cy administration. This study shows that the strong immunodepression provoked by Cy does not modify significantly the susceptibility of the animal to those species of Candida which were endowed with low or no pathogenicity for normal mice, but it greatly increases the susceptibility to those species of Candida that are already pathogenic for unmodified host.     

Comparison of secretory acid proteinases from Candida tropicalis, C. parapsilosis and C. albicans.

Acid proteinases secreted by Candida tropicalis and C. parapsilosis were newly isolated. Their physico-chemical and enzymatic properties of molecular weight, pH stability, isoelectric points, specific activity, and N-terminal amino acid sequences were determined and compared with those of a C. albicans acid proteinase. The two acid proteinases secreted by C. parapsilosis were found to be new enzymes in their molecular weights. The acid proteinases from C. tropicalis and C. parapsilosis showed lower activity at neutral pH, less resistance to neutral and alkaline pH than that from C. albicans, and a half or a third of the specific activity of the C. albicans enzyme. These differences seemed to be associated with the difference of pathogenesis between Candida species. Of the 31 N-terminal amino acids, the enzymes of these three Candida species revealed 12 homologous amino acids.     

Preliminary characterization and grouping of Candida species by numerical analysis of protein profiles obtained by polyacrylamide gel electrophoresis

Whole-cell proteins from isolates of five Candida species (Candida albicans, Candida krusei, Candida parapsilosis, Candida tropicalis and Candida guilliermondii) were separated by SDS-PAGE and the profiles obtained were converted into a binary data matrix that produced a cophenetic correlation phenogram. The analysis of the phenogram allowed detection of the cophenetic correlation levels existing among these species.     

Amphotericin B-metronidazole combination against Candida spp

Oropharyngeal candidiasis (OPC) remains a common opportunistic infection in HIV-infected patients. Candida albicans is the most frequent causative agent of OPC. However, non-albicans spp. are being increasingly isolated. Candidal cell wall proteins and mannoproteins play important roles in the biology and patogenesis of candidiasis. In the present study, we have analyzed the proteinaceous components associated with cell wall extracts from C. albicans, Candida tropicalis, Candida pseudotropicalis, Candida krusei, Candida glabrata, Candida parapsilosis, Candida guilliermondii and Candida rugosa obtained from HIV-infected patients with recurrent OPC. Cell wall proteinaceous components were extracted with beta-mercaptoethanol and analyzed using electrophoresis, immunoblotting (with antisera generated against C. albicans cell wall components, and with serum samples and oral saline rinses from patients with OPC), and lectin-blotting (concanavalin A) techniques. Numerous molecular species were solubilized from the various isolates. Major qualitative and quantitative differences in the polypeptidic and antigenic profiles associated with the cell wall extracts from the different Candida spp. were discernible. Some of the antibody preparations generated against C. albicans cell wall components were able to recognize homologous materials present in the extracts from non-albicans spp. Information on cell wall antigens of Candida species may be important in the therapy and prevention of HIV-related OPC.