An antibody reactivity-based assay for diagnosis of invasive candidiasis using protein array.

The increased incidence of invasive candidiasis and of patients at risk requires early diagnosis and treatment to improve prognosis and survival. The aim of this study was to set up a ten-protein array-based immunoassay to assess the IgG antibody responses against ten well-known immunogenic C. albicans proteins (Bgl2, Eno1, Pgk1, Pdc11, Fba1, Adh1, Als3, Hwp1, Hsp90 and Grp2) in 51 patients with invasive candidiasis (IC) and in 38 culture-negative controls (non-IC). Antibody levels were higher against Bgl2, Eno1, Pgk1, Als3, Hwp1 and Grp2, than against Adh1, Pdc11, Fba1 and Hsp90, irrespectively of the patient group considered. Moreover, the IgG levels against Bgl2, Eno1, Pgk1 and Grp2 were significantly higher in IC than in non-IC patients. Furthermore, the ROC curves generated by the analysis of the antibody responses against Bgl2, Grp2 and Pgk1 displayed AUC values above 0.7, thus discriminating IC and non-IC patients. According to these results, the employment of the microarray immunoassay (a rapid, sensitive and multiparametric system), in parallel with conventional diagnostics, can help to spot IC patients. This ultimately will allow to initiate an early, focused and optimized antifungal therapy.

Yeast associated with spontaneous fermentations of white wines from the "Txakoli de Bizkaia" region (Basque Country, North Spain).

The microbiota of eight spontaneous fermentation of white wine from different grape varieties and different wineries from the "Txakoli de Bizkaia" region (Basque country, North Spain), in 1996 and 1997 campaigns was studied. The yeast population was higher in grapes harvested in 1997, in which late summer and early autumn was warmer and drier. Eight species belonging to five genera were identified in total. The most frequent genera in grapes were Rhodotorula in 1996 and Kloeckera in 1997. Saccharomyces bayanus was the most frequent species during vigorous and final fermentation, and it was occasionally isolated from grapes and must. Only another Saccharomyces spp., i.e., S. kluyvery, was identified in some samples from 1997.

Differences in the Candida albicans antigenic expression after heat shock and infection.

Heat-shock and infection induce changes in protein expression in C. albicans. To investigate if these alterations induce changes in antigenicity, we have compared the reactivity mediated by IgA antibodies of protein extracts from a strain of C. albicans and the same strain recovered from an infected animal, both at 24 degrees C and 37 degrees C. The antigenic variability was detected mainly in antigens recognized by salivary IgA. Antigens of 223, 205, 180 and 140 kDa were over-expressed in both strains at 37 degrees C, indicating that variations due to heat shock were present before and after infection. The antigens were characterized as mannoproteins located at the outer side of the cell wall. An antigen of 61 kDa was also detected in which the expression decreased significantly after infection This was independent of heat shock.

Characterization of Candida albicans antigenic determinants by two-dimensional polyacrylamide gel electrophoresis and enhanced chemiluminescence.

The use of a two-dimensional polyacrylamide gel electrophoresis joined with Western blotting allowed us to investigate the reactivities of antibodies present in sera from mice and humans to antigens of Candida albicans blastoconidia. The analysis of the antibody response in the two models studied and the comparison between the antibody response in infected and noninfected individuals showed that the infection by C. albicans produces changes in the antibody response which may be of relevance in the serodiagnosis of invasive candidiasis. These changes include the induction of antibodies against new antigens, the disappearance of antibodies against a group of antigens and variations in the reactivity of antibodies directed to a different group of antigens. The technique used resolved the isoforms of several antigens including enolase. It is concluded that the antibody response in humans and mice with candidiasis is not homogeneously directed to all the isoforms of an antigen.

Ecology of inoculated and spontaneous fermentations in Rioja (Spain) musts, examined by mitochondrial DNA restriction analysis.

Analysis of mitochondrial DNA restriction patterns was used to study the introduction of a selected strain of Saccharomyces cerevisiae for fermentation of non-sterile musts of La Rioja (Spain). All of the isolates from the inoculated musts showed the restriction pattern of the selected strain. The same technique was used to study the spontaneous fermentation of musts, showing that a few strains were responsible for the fermentations. One of the strains identified from the spontaneous fermentations had been identified in a previous vintage.

Resistance to candidiasis and macrophage activity in chitin-treated mice.

The effect of chitin, a polysaccharide of the cell wall of Candida albicans, on both the survival of C. albicans infected mice and the activity of the murine peritoneal macrophages has been studied. Pretreatment of mice with 30 mg kg(-1) C. albicans chitin enhanced the survival of the infected animals. The protective effect was concomitant with an enhancement of both phagocytic and candidacidal activities of the peritoneal macrophages. Chitin by itself did not induce the nitric oxide (NO) synthase in the macrophages, which remained at a level similar to that shown by the macrophages from untreated animals. The administration of 10 mg kg(-1) C. albicans chitin diminished the long term survival of the infected animals. This effect was coincident with a lower candidacidal activity and NO production by the macrophages of the chitin treated and infected animals, compared to the untreated infected animals.

Re-expression by Candida albicans germ tubes of antigens lost during subculture of blastospores.

The effect of germ tube induction on the antigenic variability in C.albicans was studied in strains from blood cultures (Group I) and superficial candidiasis (Group II). When compared by immunoblotting with a rabbit antiserum, antigenic extracts from Group I strains grown as blastospores showed a higher reactivity than that of Group II strains. Major bands in Group I strains (45-47, 33, 30 kDa) were continuously expressed through the subcultures in vitro but, with the exception of the 45 kDa band, the reactivity of all of them decreased or disappeared after the tenth subculture in Group II strains. The induction of the germ tubes produced the re-expression of the antigens lost during subculture in the yeast form, the effect being very clear in Group II strains. The re-expression by C. albicans germ tubes of antigens lost during subculture of blastospores in vitro and the higher reactivity shown by Group I strains grown in mycelial phase should be taken into consideration when a test to detect anti-C. albicans antibodies is to be developed.

Nitric oxide-dependent killing of Candida albicans by murine peritoneal cells during an experimental infection.

The phagocytic and candidacidal activities of the peritoneal cells of Candida albicans-infected mice were studied 20 days following experimental infection. Both activities were enhanced during infection. The production of nitric oxide (NO) by the peritoneal cells of infected mice was determined, and an increase in the nitrite concentration in supernatants of peritoneal cell cultures was detected. The period of NO production by the peritoneal cells coincided partially with the period of enhanced C. albicans killing. The inhibition of NO synthesis by N-monomethyl-L-arginine was concomitant with inhibition of candidacidal activity. We conclude that NO synthesis is the primary candidacidal mechanism of the murine peritoneal cells activated by C. albicans infection.

Chitin assay to estimate the growth of Candida albicans in organs of infected mice.

Two methods have been used to estimate chitin. In both, the chitin was first converted into chitosan. The insoluble chitosan was then either depolymerized and deaminated with HNO2 and the product colorimetrically determined with 3-methylbenzo-2-thiazolone hydrazone and Fe3+ (method A), or hydrolysed in HCl and the glucosamine determined with p-dimethylaminobenzaldehyde (method B). Method B showed a better correlation between chitin concentration and absorbance, and its reliability was higher. This method was equally convenient to estimate the chitin content of germ tubes of Candida albicans grown in vitro, showing good correlation between mycelial growth and chitin content. Finally, method B was used to measure the growth of C. albicans in organs of animals infected experimentally.

Growth and macromolecular content of the dimorphic fungus Aureobasidium pullulans and the effect of hydroxyurea and other inhibitors.

The growth kinetics and the macromolecular content of the yeast and ethanol-induced hyphal forms of Aureobasidium pullulans were studied. During the morphological transition from yeasts to hyphae, both the protein and RNA content decreased significantly, the mycelial form containing only 76% of the amount of protein in the yeasts, and 38% of the RNA. The DNA was the only component tested whose level increased during the transition. Among several compounds inhibiting macromolecular synthesis, only hydroxyurea showed a remarkable effect on the morphology of A. pullulans, inducing the mycelial morphology. The macromolecular composition of hydroxyurea-treated cultures changed with time in a way similar to that of the ethanol-Tween 80-ammonia medium, and to that of carbon-starved cultures, without ethanol or glucose.

[Evolution of several enzyme activities of Aureobasidium pullulans during the transition from yeast to mycelium induced by ethanol]. / Evolución de algunas actividades enzimáticas de Aureobasidium pullulans durante la transición de levadura a micelio inducida por etanol.

Some key enzyme activities from the energy metabolism of A. pullulans have been studied during the ethanol-induced yeast-to-mycelium transition. Both the mycelial and yeast-like forms showed greater glucose-6-phosphate dehydrogenase activity than phosphofructokinase. During the morphological transition, the most outstanding variations occurred in large cells (3 days), especially for citrate synthase, malate dehydrogenase and isocitrate lyase activities. However, similar variations were detected in cultures without glucose or ethanol, which showed no morphological transition. Therefore, the observed changes in the enzymatic activities may be attributed to the absence of glucose. As this is not sufficient to induce the morphological transition, we conclude that there is no evidence of a clear-cut relationship between morphology and the activity of the enzymes studied.

Development of Candida albicans hyphae in different growth media--variations in growth rates, cell dimensions and timing of morphogenetic events.

In six liquid culture media, all of which stimulated Candida albicans to grow in the hyphal form, the rates of hyphal extension and increase in cellular ATP concentration, hyphal diameters, times of evagination of hyphae, times of septum formation and positions of septa in the hyphae appeared to vary independently. There were no discernible associations between properties such as length or volume of hyphal compartments at the time of septation and temporal parameters of hyphal growth. The results suggest that growth environment influences many of the processes contributing to hyphal development, but that these processes are not necessarily interrelated.

Consistency of protein patterns in Candida albicans during hyphal septum and branch formation.

Candida albicans was grown in its hyphal form in five different media and in the presence of two concentrations of ketoconazole, and in its yeast form in one medium. Polyacrylamide gel electrophoresis patterns of proteins obtained by disruption of the fungal cells at different times around the stages of septum formation and branch initiation revealed no differences in intracellular protein composition related to growth conditions or time. Autoradiography of proteins labelled with [35S1]-methionine similarly revealed no differences in protein composition related to growth conditions or time. These findings suggest either that septum formation and branch initiation proceed independently of synthesis of new proteins or that proteins related to these processes are synthesized too transiently or in amounts too small to be detected under the conditions of the experiments.