Serotype prevalence of Candida albicans from blood culture isolates.

Blood culture isolates of Candida albicans were collected from 102 patients in Seattle, Wash., hospitals (n = 77) and Hong Kong (n = 25). The patients were classified by immune status into two groups. Group I patients were severely immunosppression, and group II patients had underlying risk factors for candidemia but no underlying immunosuppression. Serotyping by Hasenclever tube agglutination was done. In the Seattle area, the odds of fungemia with type B C. albicans were 3.62 times greater than the odds of type B fungemia in group II patients. Although the odds ratio could not be computed for Hong Kong patients, the direction of the relationship in this population was consistent with the data on Seattle patients. Despite the magnitude of the odds ratios, the relative prevalence of type B over type A in group I compared with group II was not significant when analyzed separately by region, probably because of relatively low numbers of isolates in group II. Accepting that the effect of immune status on serotype is equivalent across regions but presupposing that a regional effect on type B prevalence exists, the pooled odds for fungemia with serotype B in group I patients are increased 5.4-fold over those of group II patients. Logistic regression analysis controlling for region gave similar results.     

Platelet interactions with Candida albicans.

The interaction of human platelets and Candida albicans was studied. Platelet-rich plasma was obtained from freshly drawn blood or outdated platelet concentrates. From the platelet-rich plasma, a platelet extract was derived which stimulated germ tube formation by C. albicans when incubated with yeast cells at 37 degrees C. The active component(s) was heat stable, trypsin sensitive, and ribonuclease and deoxyribonuclease insensitive, and possessed cationic properties since it readily attached to carboxymethyl-Sephadex. The active component(s) seemed to bind to heparin also, since germ tube-promoting activity was eluted from a heparin-cyanogen bromide-activated Sepharose 4B column. In addition, platelet-derived growth factor (Collaborative Research, Inc.) stimulated germination when incubated with low amounts (0.4% final concentration) of bovine calf serum. The aggregation of platelets, prepared as platelet-rich plasma by C. albicans cell wall or alkali-extracted cell wall fractions, was also studied. Aggregation of platelets was observed when cell wall or cell wall fractions were incubated with platelet-poor plasma at 37 degrees C for 20 min and then added to platelet-rich plasma. The component of platelet-poor plasma which promoted aggregation of platelets by C. albicans cell wall or alkali-extracted fractions was inactivated at 56 degrees C (30 min) and by cobra venom factor, indicating a role for the alternate complement pathway in the aggregation response.     

Candida albicans group A-specific soluble antigens demonstrated by quantitative immunoelectrophoresis.

Soluble cytoplasmic extracts of Candida albicans groups A and B were prepared and compared by quantitative immunoelectrophoresis experiments performed with a commercial anti-C. albicans group A immune serum. Although crossed immunoelectrophoresis, tandem crossed immunoelectrophoresis, and line immunoelectrophoresis revealed many cross-reactions between the two groups, some components seemed to be specific to group A. However, the complexity of the extracts studied did not allow us to demonstrate specific constituents with these methods. Crossed-line immunoelectrophoresis with and without absorption of antibodies in situ was then used, and four specific antigens unique to group A cytoplasmic extract were demonstrated, one of which appeared to be quantitatively important. The value of various quantitative immunoelectrophoretic methods applied to complex antigenic preparations is discussed.     

New culture medium for the presumptive identificaion of Candida albicans and Cryptococcus neoformans.

A new medium composed of Tween 80, oxgall, caffeic acid, and Davis agar (TOC) that provides for the rapid presumptive identification of Candida albicans and Cryptococcus neoformans is described herein. C. albicans is differentiated from other yeasts by the sequential production of germ tubes and chlamydospores. In a comparison with cormeal agar control plates, there was an increase of chlamydospore-forming strains of C. albicans (97.1% versus 87.2%) and a decrease in the time required for chlamydospore formation (24 h versus 48 h). C. neoformans produced a brown pigment of TOC, which is specific for its identification, thus differentiating it from the other yeasts. A comparison of 24-h pigment production by C. neoformans on TOC with that of birdseed agar showed a dark, coffee brown color in the former cultures and a light brown color in the latter. The change in pigmentation of C. neoformans, as well as morphological changes in C. albicans, can be induced within 3 to 12 h and in not more than 24 h on the TOC medium.     

Use of fermenter-dialysis culture for the cultivation of medically relevant microbes. III. Candida albicans

For mass cultivation of Candida albicans (serotype A), a fermenter dialysis culture technique is described and compared with shaking culture and fermenter batch culture techniques. Important growth parameters such as yeast dry weight and viable cell counts demonstrate the advantage of the fermenter dialysis culture. Mannan, the major antigen from Candida albicans prepared by phenol-water extraction followed by gel chromatography was tested with the monoclonal IgM antibody H5.     

Molecular probe for typing strains of Candida albicans.

A method for separating strains of Candida albicans into nine possible groups was devised by using a cDNA probe for enolase and Southern blot analysis. Twenty-three isolates of C. albicans were found to be distributed among eight of the groups. Fifteen isolates from a single hospital segregated into four of the groups.     

Comparison of molecular typing methods for Candida albicans.

Four molecular approaches to determining the types of Candida albicans strains were compared. The strains used were those whose repeated DNA (ribosomal and mitochondrial) EcoRI restriction fragment length polymorphisms (RFLP) were determined by Stevens et al. (D. A. Stevens, F. C. Odds, and S. Scherer, Rev. Infect. Dis. 12:258-266, 1990). Scherer and Stevens (S. Scherer and D. A. Stevens, Proc. Natl. Acad. Sci. USA 85:1452-1456, 1988) used the same strains to examine the Southern blots of genomic EcoRI digests probed with the repeated sequence 27A. The results of these investigators were compared with determinations of RFLPs generated from repeated DNA by the enzyme HinfI and examination of the karyotypes of strains under two sets of conditions, one for the smaller chromosomes and one for the larger ones. Analysis of RFLPs of repeated DNA is most convenient but shows the lowest degree of resolution. Use of the repeated sequence and use of karyotype have very high resolution, but the former method is more convenient than the latter. HinfI digestion is more sensitive than EcoRI digestion but equally convenient. By using all four methods, separate types were identified for 18 of the 20 strains examined.     

Decontamination of gnotobiotic mice experimentally monoassociated with Candida albicans.

Gnotobiotic AKR mice, experimentally monoassociated with Candida albicans, were successfully decontaminated by oral treatment with amphotericin B incorporated in the drinking water. Germfree mice first were swabbed orally with viable C. albicans and then were allowed to acclimatize for 4 weeks. The log10 of number of C. albicans per gram of organ (with luminal contents) was 7.9 and 7.7 in the stomach and cecum, respectively. Direct fecal smears, as well as impresssion smears of stomach and cecum mucosal surfaces, revealed yeastphase cells, many with germ tubes, but no hyphal forms. No illness or mortality was observed over this period. The mice then were given amphotericin B DISsolved in the drinking water and offered ad libitum. At levels of 0.1 and 0.2 mg/ml, the number of fecal C. albicans was decreased but not eliminated completely. However, 0.3 mg/ml was sufficient to decontaminate the mice completely and return them to the germfree state. Residual amphotericin B was detected in the feces of the mice only while they were receiving the 0.3 mg/ml dose level. These mice remained germfree until the termination of the experiment, 10 weeks after the antibiotic had been discontinued and replaced by plain drinking water.     

The fibronectin adhesin of Candida albicans.

Candida albicans possesses on its cell surface an adhesin which binds the whole viable fungus to subendothelial extracellular matrix and matrix proteins. The adhesin is composed of 75 to 80% carbohydrate and approximately 20 to 25% protein by weight. High-performance liquid chromatography of material eluted from a fibronectin-agarose affinity column demonstrates the presence of three peaks, all of which on sodium dodecyl sulfate-polyacrylamide gel electrophoresis show the presence of one protein of approximately 60 kDa. Molecular weight sizing column chromatography, however, demonstrates that the adhesin elutes with an apparent molecular mass of 42 kDa. The N terminus of the 60-kDa glycoprotein is blocked to Edman degradation. The fibronectin adhesin of C. albicans is a glycoprotein that may be present and functional as an aggregate or multimer of a 60-kDa protein.     

Preliminary investigation of Candida albicans biovars.

A total of 126 Candida albicans strains were enzymatically evaluated by the API ZYM system. Four enzymatically based groups of C. albicans are recognized.     

New Medium for Differentiation of Candida albicans from Candida stellatoidea

A simple medium effectively differentiated Candida stellatoidea from Candida albicans on the basis of a new criterion, relative sensitivity to cycloheximide.     

Ubiquitin-like epitopes associated with Candida albicans cell surface receptors.

We have recently reported the cloning of a Candida albicans polyubiquitin gene and the presence of ubiquitin in the cell wall of this fungus. The polyubiquitin cDNA clone was isolated because of its reactivity with antibodies generated against the candidal 37-kDa laminin-binding protein. In the present study, we have further investigated the relationship between ubiquitin and cell wall components displaying receptor-like activities, including the 37-kDa laminin receptor, the 58-kDa fibrinogen-binding mannoprotein, and the candidal C3d receptor. Two-dimensional electrophoretic analysis and immunoblot experiments with antibodies against ubiquitin and the individually purified receptor-like molecules confirmed that these cell surface components are ubiquitinated. In an enzyme-linked immunosorbent assay, polyclonal antisera to each receptor reacted with ubiquitin, thus demonstrating that the purified receptor preparations used as immunogens contained ubiquitin-like epitopes. It is proposed that ubiquitin may play a role in modulating the activity of these receptors and in the interaction of C. albicans cells with host structures.     

Evaluation of bichro-latex albicans, a new method for rapid identification of Candida albicans.

A method for identification of Candida albicans within 5 min was evaluated by using 4,643 yeast isolates. Six false-positive and three false-negative reactions were observed. The specificity (99.87%) and sensitivity (99.74%) obtained indicate that the Bichro-latex albicans test is a useful method for the rapid identification of C. albicans colonies.     

Lymphocyte transformation in vitro. I. Tissue culture conditions and quantitative measurements

The quantitative evaluation of the reactivity in human PHA-stimulated lymphocyte cultures was studied. A technique for the measurement of DNA synthesis by means of thymidine-2-¹⁴C incorporation was standardized and its reproducibility determined. Using this method a number of variables in culture conditions were investigated. The results obtained are discussed.     

Comparison of the MUREX C. albicans, Albicans-Sure, and BactiCard Candida test kits with the germ tube test for presumptive identification of Candida albicans.

The MUREX C. albicans (MC)(Murex Diagnostics), Albicans-Sure (AS) (Clinical Standards Laboratories), and BactiCard Candida (BC) (Remel) test kits were compared with the germ tube (GT) test for the rapid, presumptive identification of Candida albicans. All three test kits detect the enzymes L-proline aminopeptidase and beta-galactosaminidase in yeast cells grown on culture media and are based on the principle that C. albicans produces both enzymes whereas other yeasts produce only one or neither of the enzymes. The organisms evaluated were fresh clinical isolates identified by methods routinely used in our laboratory (API 20C system and conventional methods) and included 303 C. albicans isolates, 153 Candida glabrata isolates, 70 Candida tropicalis isolates, 36 Candida parapsilosis isolates, 13 isolates of other Candida spp., 5 Cryptococcus neoformans isolates, and 3 Saccharomyces cerevisiae isolates. The MC, AS, BC, and GT tests detected 299 (98.7%), 300 (99.0%), 301 (99.3%), and 287 (94.7%) C. albicans isolates, respectively. There was one false-positive result with both the MC and BC kits and two false-positive results with the GT test. The enzymatic methods evaluated in this study provide rapid and accurate alternatives to the GT test for the presumptive identification of C. albicans.     

Purification of actin from Candida albicans and comparison with the Candida 48,000-Mr protein.

Actin was purified from Candida albicans cells by affinity chromatography by DNase-Sepharose and was recognized by immunoblotting with monoclonal antibody directed against chick muscle actin. The C. albicans 48-kilodalton protein recognized by sera from patients with invasive candidiasis was shown by DEAE chromatography and immunoblotting not to be identical with the purified C. albicans actin.     

Factors affecting filamentation in Candida albicans: changes in respiratory activity of Candida albicans during filamentation.

Glucose metabolism and respiration of Candida albicans were compared under conditions which permitted either maximal filamentous or maximal yeast growth. Changes in metabolism were monitored by comparing the quantities of ethanol produced, CO2 evolved, and oxygen consumed. Filamenting cultures produced more ethanol and less CO2 than yeasts, with oxygen consumption in the former concomitantly slower than that of the latter. Studies involving cofactors and inhibitors associated with electron transport imply that a transfer of electrons away from flavoprotein is required for maintenance of yeast morphology. Conditions consistent with a buildup of reduced flavoprotein, however, favored filament formation. These changes were expressed metabolically as a shift from an aerobic to a fermentative metabolism. The results presented are consistent with hypotheses correlating filament production with changes in carbohydrate metabolism and an interruption of electron transfer within the cell.     

Characterization of cerulenin-resistant mutants of Candida albicans.

Cerulenin, an inhibitor of fatty acid biosynthesis, has been used to study the role of the plasma membrane in germination of Candida albicans. To further elucidate this association, spontaneous, cerulenin-resistant mutants of C. albicans were isolated. Two of the mutants, 4918-2 and 4918-10, were compared biochemically with wild-type cells (4918). All strains grew equally well at 37 degrees C and synthesized fatty acids at comparable rates in the absence of the drug. In the presence of cerulenin, wild-type cells did not proceed through a logarithmic growth stage and exhibited a significantly impaired ability to incorporate [3H]acetate into newly synthesized lipid material. All strains were examined ultrastructurally. Alterations were observed in the membranous structures of cerulenin-treated wild-type cells. Such changes were not observed in cerulenin-treated mutant strains. Further examination of mutant strains revealed differences in cell wall protein and polysaccharide compositions when compared with those of wild-type cells. These apparent alterations in cell surface components may be correlated with the reduced abilities of mutant strains to adhere, in vitro, to mammalian cells.