Rapid, colorimetric identification of Candida albicans.

A total of 706 yeast isolates were evaluated in parallel by the Candida albicans Screen (CAS; Carr-Scarborough Microbiologicals, Inc., Stone Mountain, Ga.) test and the germ tube (GT) test in comparison with the API 20C Yeast Identification System. The CAS and GT tests correctly identified 419 of the 422 isolates of C. albicans (99.3%). Two of the false-negative reactions occurring with the CAS were with GT-negative strains of C. albicans. There were two false-positive CAS reactions involving a single strain each of C. parapsilosis and C. tropicalis. Sensitivity and specificity for both tests exceeded 99%, with positive and negative predictive values of 99 and 98%, respectively.     

Serum tube identification of Candida albicans

The production of filaments (germ tubes) by cells of Candida albicans in serum tubes permits presumptive identification to be made within two to three hours. The proportion of yeast-like cells forming filaments is progressively decreased with increasing cell concentration. The test is effective over a comparatively wide range of temperatures and using different types and concentrations of sera.     

Rapid identification of Candida albicans: evaluation of "Rapidec albicans". Study of 444 yeast strains]

Rapid identification of Candida albicans is of great importance as it is the most frequently isolated yeast pathogen. Rapidec albicans, a new 2-h micromethod, performs two fluorescent enzymatic activities: hexosaminidase and proline arylamidase. A total of 444 yeast strains (334 from type culture collections and 110 from recent clinical isolates) were tested. The sensitivity was 98.5% and the specificity 95.8%. When only considering the clinical strains, 47/47 Candida albicans were identified by Rapidec albicans (sensitivity 100%) but only 43/47 by the germ tube test (sensitivity 91.5%). The specificities of the two tests were respectively 98.2% and 100%. This new system is therefore very efficient for the routine diagnosis of Candida albicans in the clinical field. It is easier and quicker than the germ tube test.     

Direct identification and recognition of yeast species from clinical material by using albicans ID and CHROMagar Candida plates.

Two chromogenic media, Albicans ID and CHROMagar Candida agar plates, were compared with a reference medium, Sabouraud-chloramphenicol agar, and standard methods for the identification of yeast species. This study involved 951 clinical specimens. The detection rates for the two chromogenic media for polymicrobial specimens were 20% higher than that for the Sabouraud-chloramphenicol agar plates. The rates of identification of Candida albicans for Albicans ID and CHROMagar Candida agar plates were, respectively, 37.0 and 6.0% after 24 h of incubation and 93.6 and 92.2% after 72 h of incubation, with specificities of 99.8 and 100%. Furthermore, CHROMagar Candida plates identified 13 of 14 Candida tropicalis and 9 of 12 Candida krusei strains after 48 h of incubation.     

Rapid killing of monocytes in vitro by Candida albicans yeast cells.

To study the interaction between Candida albicans blastoconidia and human phagocytes, we incubated peripheral leukocytes with fungi for 1 h at 37 degrees C and stained the cells with fluorescent vital stains ethidium bromide (EB) and fluorescein diacetate. Fungi that had been phagocytosed showed little staining; however, some leukocytes containing blastoconidia exhibited nuclear staining with EB, even though their cell membranes showed no signs of penetration by fungi. The number of EB-positive leukocytes was related to viability of the yeast cells and the temperature at which they were maintained before use. Because efforts to quantitate EB-positive leukocytes microscopically were frustrated by cell aggregation, we labeled the leukocytes with 51Cr and measured isotope release. We determined that leukocytes incubated with viable fungi released significantly more isotope than cells incubated alone or with killed blastoconidia. Furthermore, 51Cr release correlated directly with concentration of fungi in the assay, time of incubation, and temperature at which fungi were maintained before use. Using a number of isolates of C. albicans and several other species of Candida, we found that all exhibited cytotoxic activity against leukocytes, but the level of activity varied among organisms. Finally, we depleted or enriched peripheral leukocytes for specific cell populations and determined that only monocytes released more 51Cr after incubation with viable blastoconidia. Blastoconidia can lyse phagocytic cells through germination and penetration of cell membranes within 1 to 2 h, but the cytotoxic phenomenon we describe occurs within 15 to 30 min after yeast cells have been phagocytosed. Therefore, this capacity may represent a more immediate response by blastoconidia against phagocytosis and killing by monocytes.     

Isolation and characterization of an avirulent Candida albicans yeast monomorphic mutant.

Mutagenesis of Candida albicans strain ATCC 26555 with N-methyl-nitro-N-nitrosoguanidine followed by plating on solid yeast nitrogen base-N-acetylglucosamine medium at 37 degrees C yielded colony morphology variants that were characterized as forming smooth colonies, in contrast to the rough colonies formed by the parental strain. One yeast monomorphic mutant, CAL4, was studied in detail. Strain CAL4 is defective in filamentous growth, unable to form hyphae or pseudohyphae in vivo and in vitro. These filamentous structures are not elicited by commonly used external stimuli such as serum. The mutant had no obvious alterations in its mannan, glucan or chitin content. The total quantity of non-covalently linked wall proteins was reduced in the mutant strain, but the electrophoretic pattern shown by these proteins was identical to that of proteins from the parental strain. CAL4 showed major differences from the parental strain in its formation of covalently linked wall proteins. An important aspect of these differences lay in the practical absence of proteins recognized by two monoclonal antibodies, 1B12 and 3H8, which are considered valuable tools in the diagnosis of candidiasis in part because they normally react strongly with all strains. The C. albicans mutant, blocked in yeast-mycelium transition, was avirulent in a mouse model, although it was able to grow in animal tissues.     

Umbelliferyl-labeled galactosaminide as an aid in identification of Candida albicans.

The initial evaluation of a fluorescence, nonmicroscopic method of rapid identification of Candida albicans is described. A total of 524 yeast isolates were evaluated in parallel by the umbelliferyl-conjugated N-acetyl-beta-D-galactosaminide (UAG) test and the germ tube (GT) test in comparison with the API 20C Yeast Identification System. The UAG test correctly identified 333 of the 334 isolates of C. albicans (99.7%), and the GT test identified 328 (98%). There were three false-positive GT reactions and five false-positive UAG reactions with 69 isolates of C. tropicalis. The sensitivity and specificity were 99 and 97%, respectively, for the UAG test and were both 98% for the GT test. The UAG test requires less than 2 h for test completion compared with 2 to 4 h for the GT test, is similar in cost, has a nonmicroscopic visual endpoint, and eliminates the health hazards of handling pooled human sera.     

Multiple LTR-retrotransposon families in the asexual yeast Candida albicans

We have begun a characterization of the long terminal repeat (LTR) retrotransposons in the asexual yeast Candida albicans. A database of assembled C. albicans genomic sequence at Stanford University, which represents 14.9 Mb of the 16-Mb haploid genome, was screened and >350 distinct retrotransposon insertions were identified. The majority of these insertions represent previously unrecognized retrotransposons. The various elements were classified into 34 distinct families, each family being similar, in terms of the range of sequences that it represents, to a typical Ty element family of the related yeast Saccharomyces cerevisiae. These C. albicans retrotransposon families are generally of low copy number and vary widely in coding capacity. For only three families, was a full-length and apparently intact retrotransposon identified. For many families, only solo LTRs and LTR fragments remain. Several families of highly degenerate elements appear to be still capable of transposition, presumably via trans-activation. The overall structure of the retrotransposon population in C. albicans differs considerably from that of S. cerevisiae. In that species, retrotransposon insertions can be assigned to just five families. Most of these families still retain functional examples, and they generally appear at higher copy numbers than the C. albicans families. The possibility that these differences between the two species are attributable to the nonstandard genetic code of C. albicans or the asexual nature of its genome is discussed. A region rich in retrotransposon fragments, that lies adjacent to many of the CARE-2/Rel-2 sub-telomeric repeats, and which appears to have arisen through multiple rounds of duplication and recombination, is also described.     

Zinc and regulation of growth and phenotype in the infectious yeast Candida albicans.

When Candida albicans is grown at 25 degrees C in suspension in defined medium, cells accumulate at stationary phase as singlets in G1 of the deoxyribonucleic acid replication cycle and acquire the capacity to form mycelia. When cells were removed from a stationary-phase culture and a low concentration of fresh cells was inoculated into the cell-free, stationary-phase medium, the fresh cells grew to approximately the same cell density as the original culture. We demonstrated that in the accompanying decrease in pH, nor due to a depletion of O2, an accumulation of CO2, a physical crowding effect, or accumulation of the putative autoinhibitors tryptophol and 2-phenylethyl alcohol. Rather, cells stop multiplying at stationary phase due to the depletion of zinc from the culture medium. The manipulation of cultures with glassware to remove stationary-phase cells and to add fresh cells led to the addition of zinc to the medium and hence a new round of culture growth. The same manipulations with plasticware did not result in zinc supplementation and hence in now new round of culture growth. When cells enter stationary phase in excess zinc, they do not accumulate as singlets; rather, they accumulate as budded cells. When these cells were induced to form mycelia, they did so in half the time it took zinc-starved cells. The usefulness of employing zinc starvation as a method for obtaining a uniform stationary-phase phenotype and for synchronizing induced mycelium or bud formation is discussed.     

Rapid identification of Candida dubliniensis with commercial yeast identification systems.

Candida dubliniensis is a newly described species that is closely related phylogenetically to Candida albicans and that is commonly associated with oral candidiasis in human immunodeficiency virus-positive patients. Several recent studies have attempted to elucidate phenotypic and genotypic characteristics of use in separating the two species. However, results obtained with simple phenotypic tests were too variable and tests that provided more definitive data were too complex for routine use in the clinical laboratory setting. The objective of this study was to determine if reproducible identification of C. dubliniensis could be obtained with commercial identification kits. The substrate reactivity profiles of 80 C. dubliniensis isolates were obtained by using the API 20C AUX, ID 32 C, RapID Yeast Plus, VITEK YBC, and VITEK 2 ID-YST systems. The percentages of C. dubliniensis isolates capable of assimilating or hydrolyzing each substrate were compared with the percentages from the C. albicans profiles in each kit's database, and the results were expressed as percent C. dubliniensis and percent C. albicans. Any substrate that showed >50% difference in reactivity was considered useful in differentiating the species. In addition, assimilation of methyl-alpha-D-glucoside (MDG), D-trehalose (TRE), and D-xylose (XYL) by the same isolates was investigated by the traditional procedure of Wickerham and Burton (L. J. Wickerham and K. A. Burton, J. Bacteriol. 56:363-371, 1948). At 48 h (the time recommended by the manufacturer for its new database), we found that the assimilation of four carbohydrates in the API 20C AUX system could be used to distinguish the species, i.e., glycerol (GLY; 88 and 14%), XYL (0 and 88%), MDG (0 and 85%), and TRE (15 and 97%). Similarly, results with the ID 32 C system at 48 h showed that XYL (0 and 98%), MDG (0 and 98%), lactate (LAT; 0 and 96%), and TRE (30 and 96%) could be used to separate the two species. Phosphatase (PHS; 9 and 76%) and alpha-D-glucosidase (23 and 94%) proved to be the most useful for separation of the species in the RapID Yeast Plus system. While at 24 h the profiles obtained with the VITEK YBC system showed that MDG (10 and 95%), XYL (0 and 95%), and GLY (26 and 80%) could be used to separate the two species, at 48 h only XYL (6 and 95%) could be used to separate the two species. The most useful substrates in the VITEK 2 ID-YST system were TRE (1 and 89%), MDG (1 and 99%), LAT (4 and 98%), and PHS (83 and 1%). While the latter kit was not yet commercially available at the time of the study, it would appear to be the most valuable for the identification of C. dubliniensis. Although assimilation of MDG, TRE, and XYL proved to be the most useful for species differentiation by the majority of commercial systems, the results with these carbohydrates by the Wickerham and Burton procedure were essentially the same for both species, albeit following protracted incubation. Thus, it is the rapidity of the assimilation achieved with the commercial systems that allows the differentiation of C. dubliniensis from C. albicans.     

Role of yeast cell growth temperature on Candida albicans virulence in mice.

Previous studies have suggested that yeast cell growth temperature may influence the relative virulence of the opportunistic dimorphic fungus Candida albicans. To test this possibility, mice were challenged with C. albicans yeast cells which were grown at either room temperature or 37 degrees C, and their survival was monitored daily. Mice which received room temperature-grown cells died faster. The interaction of glycogen-elicited polymorphonucleated neutrophils (PMNs) with C. albicans yeast cells grown at the two temperatures was examined, because PMNs have been shown to have a critical role in preventing development of candidiasis in normal individuals. In the absence of serum (i.e., nonopsonic conditions), more PMNs conjugated and engulfed C. albicans cells grown at room temperature than those grown at 37 degrees C. However, PMNs were less able to kill cells grown at room temperature than cells grown at 37 degrees C. Cells grown at room temperature also produced abundant germ tubes after engulfment and were thus more likely to escape killing by phagocytes. These results suggest that cells grown at room temperature are more virulent because they are less likely to be killed by phagocytes and are more likely to disseminate. The possibility that expression of cell surface hydrophobicity is involved in these events is discussed.     

Phenotypic identification of Candida albicans by growth on chocolate agar.

In this study, we describe a simple method for the identification of Candida albicans in clinical samples. A total of 383 clinical isolates of Candida species were streaked onto chocolate agar and incubated for 48 h at 37 degrees C in the presence of an atmosphere of 6% CO2. All 208 of the C. albicans isolates tested, developed an easy to identify filamentous colony morphology. Of 175 other Candida species tested, 172 (98.3%) were distinguishable from C. albicans by their smooth colony morphology. Three isolates (1.7%) exhibited weak filamentation after prolonged incubation. Although not a routine medium in medical mycology a significant advantage of using chocolate agar lies in its use in clinical bacteriology laboratories for the isolation of fastidious bacteria. Implementation of the proposed method is applicable across a range of specimen types, thus allowing the direct identification of C. albicans in clinical samples. This simple method may allow a quicker entry into directed treatment.     

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.     

Antibodies to germinating and yeast cells of Candida albicans in human and rabbit sera.

Two major antigenic components, I and II, were detected by double immunodiffusion in sonic extracts of the germinating (G) or yeast (Y) cells of the dimorphis organism, Candida albicans group A. Component I may be a heterogeneous mixture of antigens which are stable to heating and phenol. Component II is more homogeneous but is labile to heat and phenol. Rabbit antisera, showing only precipitin to component II or certain human sera at high dilution, were found to react with G cells to give an immunofluorescence which was confined to the germ tubes. This suggested that component II is localised on the germ tubes, whereas no immunofluorescent reaction against the yeast cells could be detected under the same conditions although component II was as readily extracted from these cells as from G cells. This suggested that component II might exist in a cryptic state in the Y cells. In support of the latter contention it was shown that live Y cells did not absorb precipitin to component II nor were they capable of providing these antibodies in rabbits. Using both human and rabbit sera, it was shown that the antigenic specificity of the immunofluorescence assay where Y cells were used was related to component I and that where G cells were used it was related to both components I and II.     

Rapid identification of Candida albicans by dot-enzyme immunoassay

A highly sensitive and specific dot-enzyme immunoassay for the rapid identification of Candida albicans was developed using a murine monoclonal antibody (Mab), which adsorbed to cell surface-exposed determinants. This Mab reacted with 28 of the 28 C. albicans strains tested including the serotypes A and B and 2 C. stellatoidea. It did not react with 32 other isolates representing eight other Candida species commonly encountered in human materials. All the test could be performed in four steps in less than an hour. The yeasts were directly spotted on a strip of immunodyne membrane. Then the strip was incubated for 5 min. with the Mab, for 15 min. with a peroxidase-conjugate and for 30 min. with the enzyme substrate and 4-chloro 1 naphthol. This test proved useful for rapid and easy identification of C. albicans.     

Candida albicans colony identification in 5 minutes in a general microbiology laboratory.

A total of 381 fully identified yeast isolates were tested by the germ tube (GT) and Albistrip (Lab M Ltd., Bury, United Kingdom) methods, and the results were compared. As a test system for the identification of Candida albicans, the Albistrip showed two false-positive and two false-negative results, whereas the GT showed seven false-negative and no false-positive results. With the same methods, 736 yeast isolates from clinical samples were tested in a laboratory that did not specialize in mycology. In this second experiment, when the results of the tests disagreed, further identification was carried out with the API 20C Yeast Identification System (API-Biomerieux Ltd., Vercieu, France). When the statistics of the first experiment were used to justify the results, this second experiment showed the Albistrip to be 98% sensitive and 98% specific, whereas the GT was 98% sensitive and 95% specific.     

Differential adherence of hydrophobic and hydrophilic Candida albicans yeast cells to mouse tissues.

Using an ex vivo binding assay, we previously demonstrated that yeast cells grown at 37 degrees C display binding specificity in mouse spleen, lymph node, and kidney tissues. In spleen and lymph node tissues, binding was predominantly in regions rich in macrophages. Here, we tested the possibility that hydrophobic and hydrophilic cells bind differentially to host tissues. Hydrophobic and hydrophilic yeast cells of four Candida albicans strains were incubated for 15 min at 4 degrees C with cryostat sections of organs that had been rapidly frozen after removal from BALB/cByJ mice. Unattached cells were removed by washing, and the sections were examined. Hydrophobic cells bound diffusely and abundantly to all tissues, while hydrophilic cell attachment was restricted to specific sites. For example, hydrophobic cells bound to the white and red pulp and the marginal zones in spleens, whereas hydrophilic cells attached primarily to the marginal zones. Hydrophobic yeast cells attached throughout lymph node tissue including paracortical areas, but hydrophilic cell attachment occurred primarily at the subcapsular and trabecular sinuses, EDTA inhibited the adherence of hydrophilic cells but not hydrophobic cells to mouse tissues. Hydrophobic C. albicans strains displaying similar levels of hydrophobicity differed quantitatively in their levels of attachment to kidney and spleen tissues, confirming our earlier observation that surface hydrophobicity is not the sole determinant in adherence to host cells. Other studies have shown that hydrophobic and hydrophilic cells display different virulence characteristics related to their surface properties and that hydrophobic cells are more virulent than hydrophilic cells. Taken together, the present results suggest that the enhanced virulence of hydrophobic cells over hydrophilic cells may be due, in part, to the potential of hydrophobic cells to bind throughout various organs following clearance from the bloodstream.     

Cytoplasmic antigens unique to the mycelial or yeast phase of Candida albicans.

Crossed immunoelectrophoresis with absorption in situ was used to distinguish the cytoplasmic antigens unique to the mycelial or yeast phase of Candida albicans from cytoplasmic antigens shared by both phases. The soluble cytoplasmic extracts of each growth phase had at least six distinct antigenic constituents not shared by the other phase. This technique is recommended for the analysis of closely related antigenic complexes.     

New germ tube induction medium for the identification of Candida albicans.

A new germ tube induction medium, composed of three parts Rabbit Coagulase Plasma with EDTA (BBL Microbiology Systems) and two parts Tryp-Soy broth (Scott Laboratories, Inc.), was effective for the presumptive identification of Candida albicans. This medium was safer to use, more accurate, and less expensive than other commercial germ tube induction media.