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Severe infection often causes a septic cytokine storm followed by immune exhaustion/paralysis. Not surprisingly, many pathogens are equipped with various anti-inflammatory mechanisms. Such mechanisms might be leveraged clinically to control septic cytokine storms. Here we show that N-glycan from pathogenic C. albicans ameliorates mouse sepsis through immunosuppressive cytokine IL-10. In a sepsis model using lipopolysaccharide (LPS), injection of the N-glycan upregulated serum IL-10, and suppressed pro-inflammatory IL-1β, TNF-α and IFN-γ. The N-glycan also improved the survival of mice challenged by LPS. Analyses of structurally defined N-glycans from several yeast strains revealed that the mannose core is key to the upregulation of IL-10. Knocking out the C-type lectin Dectin-2 abrogated the N-glycan-mediated IL-10 augmentation. Furthermore, C. albicans N-glycan ameliorated immune exhaustion/immune paralysis after acute inflammation. Our results suggest a strategy where the immunosuppressive mechanism of one pathogen can be applied to attenuate a severe inflammation/cytokine storm caused by another pathogen.

Candida albicans is a commensal fungus in human mucosal surfaces, including the oral cavity. Lactoferrin (LF) and the lactoperoxidase (LPO) system, which are host protection components in exocrine secretions, each exhibit weak anti-candida activity. We herein examined the effects of the combination of LF and the LPO system on C. albicans. Morphological observations indicated that the combination of LF and the LPO system reduced the mycelial volume of C. albicans and changed the size and shape of cells more than each agent alone. The combination of LF and the LPO system also exerted strong inhibitory effects on the cellular metabolic activity and adhesive hyphal form of C. albicans. A checkerboard analysis revealed that the anti-candida activity of LF and the LPO system was synergistic. These results suggest that the combination of LF and the LPO system is useful for preventing candidiasis.

Protamine peptide (PP) derived from salmon is a 14-mer with 10 arginine residues. We investigated the in vitro and in vivo antifungal activity of PP against Candida albicans PP showed a concentration-dependent dual mode of action, with fungicidal activity and inhibitory activity for hyphal development in vitro. At lethal concentrations of PP, intracellular accumulation of PP was energy-dependent but independent of endocytosis, and resulted in ATP efflux and the generation of reactive oxygen species in the cells. PP at sublethal concentrations inhibited hyphal development in C. albicans by binding to the cell surface. Though antifungal activity of PP was inactivated by high concentrations of NaCl, the antifungal activity of the synthetic cyclic (via a disulfide bond) form of PP (cyclic PP) was not. Cyclic PP also showed the concentration-dependent dual mode of action, and had five-fold greater antifungal activity than PP. The advantage of antifungal activity of cyclic PP compared with PP in vitro resulted in a high in vivo efficacy in a murine oral candidiasis model. Oral treatment with cyclic PP inhibited hyphal development of C. albicans on mouse tongues and protected against the development of severe candidiasis. This study shows the therapeutic potential of cyclic PP as an antifungal peptide against C. albicans., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The binding of Candida albicans cells to chitin was examined in a cell-binding assay. Microscopic observations indicated that both living and heat-killed Candida cells bound to chitin-coated substrates. C. albicans preferentially bound to chitin-coated plastic plates over chitosan-coated and uncoated plates. We prepared 125 I-labeled Candida cells for quantitative analysis of their binding to chitin. Heat-killed 125 I-labeled Candida cells bound to chitin-coated plates in a time-dependent manner until 1.5 hours after start of incubation at 4℃. The binding of 125 I-labeled Candida cells to chitin-coated plates was inhibited by adding unlabeled living or unlabeled heat-killed Candida cells. The binding of Candida to chitin was also reduced by addition of 25 mg/ml chitin or chitosan up to 10%. N-acetylglucosamine (GlcNAc), which is a constituent of chitin, inhibited binding of Candida to chitin in a dose-dependent manner between 12.5 and 200 mM. Glucosamine, which is a constituent of chitosan, showed no such inhibitory effect. These findings suggest that the binding of Candida to chitin may be mediated by recognition of GlcNAc.

A lysozyme-chitosan conjugate preparation (LYZOX), produced from egg white lysozyme and chitosan by Maillard reaction, is a commercial product developed as a cosmetic ingredient or food additive. Effects of LYZOX on in vitro growth of Candida albicans were examined. C. albicans cells were treated with LYZOX for 3 hrs, and then washed and cultured for an additional 16 hrs in modified RPMI1640 medium. Mycelial growth of C. albicans was clearly inhibited by more than 100 μg/ml of LYZOX in a concentration-dependent manner. On the other hand, corresponding concentration of chitosan or lysozyme or their mixture only scarcely showed clear inhibitory effect. Similarly, anti-Candida activity of the combination of LYZOX and decanoic acid, a middle-chain fatty acid, was also examined. Inhibitory activity of this combination against mycelial growth of C. albicans was very potent and appeared synergistic, since fractionated inhibitory concentration (FIC) index for 70% growth inhibition was calculated to be 0.20. Oral application of this combination improved the symptoms of Candida-infected-tongue in an experimental murine candidiasis model. On the basis of these results, the possible application of LYZOX as a new functional product with anti-candida activity was discussed.

We previously showed a prophylactic effect of Lactobacillus pentosus strain S-PT84 against oral candidiasis in mice. In the present study, we evaluated the protective effect of S-PT84 against Candida infection of the gastrointestinal tract. As the first step, we used an in vitro assay to compare the inhibitory effects of several lactobacilli (S-PT84 and Lactobacillus pentosus type strain JCM1558 T , Lactobacillus gasseri type strain JCM1131 T and Lactobacillus casei type strain JCM1134 T ) on mycelial growth of Candida albicans. S-PT84 directly adhered to Candida cells and showed the strongest growth-inhibitory activity among the tested Lactobacillus strains. In the second experiment, we used an in vivo assay to evaluate the effect of S-PT84 ingestion on severity score of stomach lesion and gastric inflammation in a mouse model of gastrointestinal candidiasis. The severity scores were significantly improved by oral administration of S-PT84 (6 mg/ 200 μL), consistent with decreased coverage of stomach lesions by patchy whitish plaques. The attenuation of stomach lesion severity by S-PT84 was more pronounced than that obtained with L. gasseri type strain JCM1131 T , consistent with the results of the above in vitro study. Histological analysis also indicated that S-PT84 prevented the adhesion of C. albicans to the stomach surface and suppressed stomach inflammation caused by neutrophil infiltration. Furthermore, S-PT84 also suppressed the vascular permeability observed in Candida-infected stomach. These results suggest that oral administration of S-PT84 might be effective not only in inhibiting Candida infection but also in preventing gastric inflammation induced by Candida infection.

We previously showed a prophylactic effect of Lactobacillus pentosus strain S-PT84 against oral candidiasis in mice. In the present study, we evaluated the protective effect of S-PT84 against Candida infection of the gastrointestinal tract. As the first step, we used an in vitro assay to compare the inhibitory effects of several lactobacilli (S-PT84 and Lactobacillus pentosus type strain JCM1558 T , Lactobacillus gasseri type strain JCM1131 T and Lactobacillus casei type strain JCM1134 T ) on mycelial growth of Candida albicans. S-PT84 directly adhered to Candida cells and showed the strongest growth-inhibitory activity among the tested Lactobacillus strains. In the second experiment, we used an in vivo assay to evaluate the effect of S-PT84 ingestion on severity score of stomach lesion and gastric inflammation in a mouse model of gastrointestinal candidiasis. The severity scores were significantly improved by oral administration of S-PT84 (6 mg/ 200 μL), consistent with decreased coverage of stomach lesions by patchy whitish plaques. The attenuation of stomach lesion severity by S-PT84 was more pronounced than that obtained with L. gasseri type strain JCM1131 T , consistent with the results of the above in vitro study. Histological analysis also indicated that S-PT84 prevented the adhesion of C. albicans to the stomach surface and suppressed stomach inflammation caused by neutrophil infiltration. Furthermore, S-PT84 also suppressed the vascular permeability observed in Candida-infected stomach. These results suggest that oral administration of S-PT84 might be effective not only in inhibiting Candida infection but also in preventing gastric inflammation induced by Candida infection.

We examined the effect of 5 saturated fatty acids and their related alcohols on the growth of Candida albicans. The inhibitory effects of these compounds against the yeast and hyphal growth forms of C. albicans were examined using the modified NCCLS method and crystal violet staining, respectively. Among these compounds, capric acid inhibited both types of growth at the lowest concentration. The IC(80), i.e., the concentration at which the compounds reduced the growth of C. albicans by 80% in comparison with the growth of control cells, of capric acid for the hyphal growth of this fungus, which is indispensable for its mucosal invasion, was 16.7 μM. These fatty acids, including capric acid, have an unpleasant smell, which may limit their therapeutic use. To test them at reduced concentrations, the combined effect of these fatty acids and oligonol, a depolymerized polyphenol, was evaluated in vitro. These combinations showed potent synergistic inhibition of hyphal growth [fractional inhibitory concentration (FIC) index = 0.319]. Our results demonstrated that capric acid combined with oligonol could be used as an effective anti-Candida compound. It may be a candidate prophylactic or therapeutic tool against mucosal Candida infection.

In a preceding paper, we showed that aroma candy containing oligonol, capric acid, and cinnamon (cassia) powder had potent inhibitory activity against mycelial growth of Candida albicans in vitro and protective activity against murine oral candidiasis. In order to assess the effects of this candy (the test candy) on oral C. albicans colony-forming units (CFU) and oral hygiene states, a placebo-controlled double-blind crossover comparative study was performed. Twenty subjects were divided into two groups. One group ingested the test candy in the first 7 days followed by 2 weeks washing-off period, then ingested the placebo candy (control candy) for 7 days. The other group was vice versa. C. albicans CFU in all oral rinse samples from the subjects before and after 7 days ingestion of candy was measured. The degree of oral malodor in all subjects was monitored using a portable measuring instrument. The results showed no statistically significant difference between test-candy group and placebo group for C. albicans CFU. However, C. albicans CFU in test-candy group with>4,000 CFUs was significantly decreased after 7 days ingestion of test-candy (p<0.05). Scores of oral malodor in the test-candy group was significantly decreased after 7 days ingestion of test-candy (p<0.05). A questionnaire survey of oral hygiene states indicated that in the test-candy group, oral malodor, glutinous feeling, and refreshing feeling significantly improved in comparison with control-candy group (p<0.05). Our study suggests that the aroma candy is effective in oral health care of elderly carrying C. albicans.

To develop a new therapy against oral candidiasis, a commensal microorganism, Enterococcus faecalis was tested for its ability to modulate Candida growth in vitro and its therapeutic activities against a murine model in vivo. Addition of heat-killed E. faecalis strain EF2001 (EF2001) isolated from healthy human feces to the culture of C. albicans strain TIMM1768 inhibited adherence of the latter to a microtiter plate in a dose dependent manner and Candida cells surrounded by EF2001 were increased. To examine the protective activities of EF2001 in vivo, heat-killed EF2001 was applied orally before and after inoculation of Candida to the tongue of mice previously immunosuppressed. Two days after inoculation this inoculation, both the symptom score and CFU from swabbed-tongue were significantly reduced in the EF2001-treated animals. Histological analysis indicated that EF2001 may potentiate the accumulation of polymorphnuclear cells near a Candida-infected region. These results suggest that oral administration of EF2001 has protective activity against oral candidiasis and that the in vivo activity may be reflected by direct interaction between EF2001 and Candida cells in vitro and the potentiation of an immunostimulatory effect of EF2001.

The effect of S-PT84, a heat-killed preparation of Lactobacillus pentosus on growth of Candida albicans was examined in vitro and in vivo. The mycelial growth was effectively inhibited by S-PT84 and seemed to bind to the hyphae. We assessed the potential of S-PT84 for treatment of oral and gastric candidiasis using a murine model. When 2 mg of S-PT84 was administered three times into the oral cavity of orally Candida infected mice, the score of lesions on the tongue was improved on day 2. When 50 μl and 200 μl of S-PT84 (10 mg/ml) were administered three times into the oral cavity (0.5 mg × 3) and the stomach (2 mg × 3) of the same mouse model, the number of viable Candida cells in the stomach was reduced significantly on day 2. These findings suggest the possibility that S-PT84 has potential as a food ingredient supporting anti-Candida treatment, especially for Candida infection in the gastrointestinal tract.

We examined the viability and morphology of Candida albicans under experimental conditions after treatment with varying concentrations of cinnamaldehyde, the major component of cassia (Cinnamomum cassia), using XTT assay, fluorescent microscopy, scanning electron microscopy, and thin-section electron microscopy. At 10 μg/ml level, cinnamaldehyde inhibited mycelial growth, but did not affect the growth of yeast cells, metabolic activity, cell shape, or the ultrastructure of the cells. At 40 μg/ml level, cinnamaldehyde showed fungicidal activity accompanied by alteration of the membrane and interior of Candida cells. These findings indicate that cinnamaldehyde has both fungistatic and fungicidal activities against C. albicans and affects the structure of the cells.

The combined effect of terpinen-4-ol, the main component of tea tree oil, and capric acid against mycelial growth of Candida albicans and murine oral candidiasis was evaluated in vitro and in vivo. Mycelial growth of C. albicans was estimated by the Cristal violet method. Combination of these compounds revealed a potent synergistic inhibition of growth. Therapeutic efficacy of the combination was evaluated microbiologically in murine oral candidiasis, and its application of the compounds clearly demonstrated therapeutic activity. Based on these results, the combined agent of terpinen-4-ol and capric acid was discussed as a possible candidate for oral candidiasis therapy.

Colony forming units (CFU) of Candida albicans in cell suspensions or homogenates prepared from C. albicans-infected tissues are not always accurate indicators of the severity of infection in mucosal tissues. In order to improve the reliability of CFU counts in the murine oral candidiasis model, we developed a new cell preparation method in which a dispersal process involving trypsin digestion was included in the processing of Candida albicans-infected tongue tissues. Trypsin digestion, which was added to the conventional method for preparing Candida suspension from tongue homogenates, improved the recovery yield as evidenced by an increase in Candida CFU's. This method also increased the number of planktonic Candida cells which could pass through a mesh filter, perhaps because the trypsin contributed to the break up of the complex mass of tissue-debris and C.albicans cells. Using this trypsin digestion technique, we confirmed the protective action of farnesol by a relative decrease in the number of viable Candida cells in homogenates of infected tongues, which was correlated with improvement of the symptoms of oral candidiasis. These results indicate that our new method of trypsin digestion is valuable in that it reflects the protective activity of some bioactive substances against mucosal candidiasis.

Oral candidiasis is often accompanied by severe inflammation, resulting in a decline in the quality of life of immunosuppressed individuals and elderly people. To develop a new oral therapeutic option for candidiasis, a nonpathogenic commensal oral probiotic microorganism, Streptococcus salivarius K12, was evaluated for its ability to modulate Candida albicans growth in vitro, and its therapeutic activity in an experimental oral candidiasis model was tested. In vitro inhibition of mycelial growth of C. albicans was determined by plate assay and fluorescence microscopy. Addition of S. salivarius K12 to modified RPMI 1640 culture medium inhibited the adherence of C. albicans to the plastic petri dish in a dose-dependent manner. Preculture of S. salivarius K12 potentiated its inhibitory activity for adherence of C. albicans. Interestingly, S. salivarius K12 was not directly fungicidal but appeared to inhibit Candida adhesion to the substratum by preferentially binding to hyphae rather than yeast. To determine the potentially anti-infective attributes of S. salivarius K12 in oral candidiasis, the probiotic was administered to mice with orally induced candidiasis. Oral treatment with S. salivarius K12 significantly protected the mice from severe candidiasis. These findings suggest that S. salivarius K12 may inhibit the process of invasion of C. albicans into mucous surfaces or its adhesion to denture acrylic resins by mechanisms not associated with the antimicrobial activity of the bacteriocin. S. salivarius K12 may be useful as a probiotic as a protective tool for oral care, especially with regard to candidiasis.

The amino sugar N-acetylglucosamine (GlcNAc) is an in vitro inducer of the hyphal mode of growth of the opportunistic pathogen Candida albicans. The development of hyphae by C. albicans is considered to contribute to the pathogenesis of mucosal oral candidiasis. GlcNAc is also a commonly used nutritional supplement for the self-treatment of conditions such as arthritis. To date, no study has investigated whether ingestion of GlcNAc has an effect on the in vivo growth of C. albicans or the pathogenesis of a C. albicans infection. Using a murine model of oral candidiasis, we have found that administration of GlcNAc, but not glucose, increased oral symptoms of candidiasis and fungal burden. Groups of mice were given GlcNAc in either water or in a viscous carrier, i.e., 1% methylcellulose. There was a dose-dependent relationship between GlcNAc concentration and the severity of oral symptoms. Mice given the highest dose of GlcNAc, 45.2 mM, also showed a significant increase in fungal burden, and increased histological evidence of infection compared to controls given water alone. We propose that ingestion of GlcNAc, as a nutritional supplement, may have an impact on oral health in people susceptible to oral candidiasis.

Clinical management of patients undergoing treatment of oropharyngeal candidiasis with azole antifungals can be impaired by azole resistance. High-level azole resistance is often caused by the overexpression of Candida albicans efflux pump Cdr1p. Inhibition of this pump therefore represents a target for combination therapies that reverse azole resistance. We assessed the therapeutic potential of the D-octapeptide derivative RC21v3, a Cdr1p inhibitor, in the treatment of murine oral candidiasis caused by either the azole-resistant C. albicans clinical isolate MML611 or its azole-susceptible parental strain MML610. RC21v3, fluconazole (FLC), or a combination of both drugs were administered orally to immunosuppressed ICR mice at 3, 24, and 27 h after oral inoculation with C. albicans. FLC protected the mice inoculated with MML610 from oral candidiasis, but was only partially effective in MML611-infected mice. The co-application of RC21v3 (0.02 μmol per dose) potentiated the therapeutic performance of FLC for mice infected with either strain. It caused a statistically significant decrease in C. albicans cfu isolated from the oral cavity of the infected mice and reduced oral lesions. RC21v3 also enhanced the therapeutic activity of itraconazole against MML611 infection. These results indicate that RC21v3 in combination with azoles has potential as a therapy against azole-resistant oral candidiasis., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Leaf hydrosols prepared from 18 weakly aromatic Japanese herbs used traditionally were tested on the filamentation-inhibitory activity of Candida albicans. These hydrosols were divided into two classes, A and B. The inhibitory activity of 13 hydrosols belonging to class A was markedly altered depending on the drying process of the parent herbs. On the other hand, the remaining 5 hydrosols belonging to class B showed no significant change on the composition and inhibitory activity upon drying. The change of the bioactivity was correlated with the change and concentration of the respective major constituents. Especially strong bioactivity shown by hydrosols of dried Houttuynia cordata and fresh Prunus pendula was ascribed to n-capric acid and cyanide, respectively. Eight hydrosols exhibited weak or moderate activity against the growth of C. albicans.

This study investigated the effects of cinnamaldehyde in combatting the hyphal growth of Candida albicans under varying concentrations, treatment times, and temperatures to determine the potential benefits of applying this substance to anti-Candida foods or gargles. From the results of pretreatment with cinnamaldehyde against Candida hyphae, we found that its inhibitory activity seemed to be strengthened in parallel with prolonged pretreatment time and a rise in temperature, and that pretreatment of 2,000 μg/ml for only 1 minute significantly inhibited the hyphal growth of C. albicans. We also demonstrated by XTT assay that pretreatment with cinnamaldehyde affected the metabolic activity of Candida hyphal cells. These findings suggest that a warm drink or mouthwash containing cinnamaldehyde might be a candidate as a prophylactic or therapeutic tool against oral Candida infection.

We assessed anti-C. albicans activities of the 4 fatty acids : caproic acid, caprylic acid, capric acid and lauric acid in vitro. All four inhibited not only the mycelial but also the yeast-form growth of Candida albicans. In particular, capric acid and caprylic acid inhibited Candida mycelia growth at very low concentrations. The effects of treatment of these two fatty acids on oral candidiasis were examined using a murine model. When 50 µl of capric acid (more than 48.8 µM) was administered three times into the oral cavity of Candida-infected mice, symptom scores of tongues of the mice were significantly improved. Histological studies of the capric acid-treated animals indicated that the fatty acid suppressed mycelial growth of the fungus on the tongue surface. These results suggest that all four fatty acids, and especially capric acid, have potential as substances supporting anti-Candida treatment.

Farnesol is well known as a quorum-sensing molecule of Candida albicans. To assess the pathological function of farnesol, its effects on macrophage viability and functions including growth inhibitory activities against C. albicans were examined in vitro. Murine macrophages, when cultured in the presence of 56-112 microM of farnesol for 1-2 hr, decreased their activity inhibiting the mycelial growth of C. albicans and lost their viability. This suppression of macrophage function by farnesol was neutralized by the coexistence of the anti-oxidants probucol and trolox. Macrophages cultured in the presence of farnesol for 2 hr displayed morphological change of nuclei and DNA fragmentation, which suggested apoptosis of the cells. Intracellular production of ROS in the farnesol-treated macrophages was shown by fluorescence of DCFH-DA and increase of peroxidized materials. These effects of farnesol were blocked by probucol or trolox. These results indicate that farnesol lowered viability of the murine macrophages and suppressed their anti-Candida activity, perhaps through induction of ROS.

The antifungal activity of 43 hydrosols, 7 herbal teas and 12 essential oils was determined using Candida albicans as a test organism. All of the hydrosols examined showed more potent inhibition against the filamentous form than the yeast form of C. albicans. In particular, the filamentous form was markedly inhibited by seven hydrosols, of which monarda, santolina and clove water also inhibited the growth of the yeast form. Most of the inhibitory activity of the hydrosols was correlated with that of their respective major components. Poor correlation was observed between the inhibition of filament formation and the growth inhibition of the yeast form among the hydrosols examined, among essential oils and among the major components of hydrosols and essential oils. Seven herbal teas showed moderate or weak activity against the filament formation of C. albicans, but no inhibition against the yeast form.

We analyzed the morphologic and microbiologic aspects of the process of adhesion and invasion in the early stages of Candida albicans oral infection in a murine system. ICR mice were anesthetized by intramuscular injection with chlorpromazine chloride and then orally inoculated by swabbing with the C. albicans yeast cells. Their tongues were resected 1-3h after inoculation, washed sequentially with a physiological saline and 0.25% trypsin-solution and then homogenized. The number of viable C. albicans cells on the tongue surface was counted and fround to increase from 1-3h after inoculation. Most of the Candida cells attached to the tongue surface were present in clusters, mainly located in the gaps between lingual papillae and were covered with a mucoidal substance. By 3h after inoculation, these clusters frequently formed mycelia and could not be easily detached from the tongue surface by trypsin treatment. Observation of SEM and histological sections stained by Fungiflora Y revealed that the Candida hyphae at 3h stretched out of the cluster and entered the tongues through the surface. These results indicate that Candida hyphae begin to invade the tongue surface within 3h after inoculation and suggest that the mucus-like substance covering these cells may have an important early role in the interaction between the Candida cells and the tongue mucosal epithelium.

Farnesol is known as a quorum-sensing molecule for Candida albicans and is recognized to play pathogenic roles in Candida infection. To assess the possible role of farnesol in mucosal C. albicans infection, the effects of farnesol treatment against experimental oral candidiasis in mice were examined. Prednisolone-pretreated ICR mice were orally infected with C. albicans and 3, 24 and 30 hr later the animals were orally given farnesol. Forty-eight hr later they were killed for observation. Farnesol treatment in a dose ranging between 1.125 and 9 micromol/mouse showed a protective effect against oral candidiasis in a dose-dependent manner, at least as estimated by symptom scores of tongues. At 9 micromol/mouse it decreased bodyweight loss. Histological studies of 2.25 micromol/mouse farnesol-treated animals indicated that farnesol suppressed mycelial growth of C. albicans on the surface of tongues, but microbiological study did not prevent the change of CFU of C. albicans cells not only on tongues but also in feces, kidneys and livers. These results suggest that farnesol has very characteristic roles in protection against mucosal candidiasis.

Objective: The therapeutic activities of cyclodextrin-associated itraconazole oral solution (itraconazole OS) by two administration routes in experimental oral and oesophageal candidiasis in mice were examined and compared., Methods: Using experimental oral and oesophageal candidiasis models in ICR mice, we investigated the efficacy of oral and intragastric administration of itraconazole OS and checked the concentration of itraconazole and its metabolite hydroxyitraconazole (OH-itraconazole) in tongues or oesophagus tissue after administration of itraconazole OS., Results: Oral administration of itraconazole OS at doses of 0.8, 4.0 or 20 mg/kg/day clearly decreased the number of viable Candida albicans cells in the oral cavity of mice with oral candidiasis in a dose-dependent manner at 3 days after infection. Intragastric administration of itraconazole OS at doses of 4 and 20 mg/kg/day once a day were also effective but to a lesser degree than that of oral administration. In the oesophageal candidiasis model, oral administration of itraconazole OS displayed superior therapeutic efficacy to the intragastric route. In coincidence with the greater efficacy, itraconazole was detected in lesional tissues after oral administration of itraconazole OS., Conclusions: Oral administration of itraconazole OS displayed therapeutic efficacy against murine oral and oesophageal candidiasis superior to that achieved by intragastric administration. This can be explained by there being higher concentrations of itraconazole in tongues or oesophagus tissues after administration of the suspension by the oral route.

A simple method to establish a murine esophageal candidiasis model that displayed characteristic symptoms of the condition was developed using the sedative agent, chlorpromazine. Mice were immunosuppressed with prednisolone and were given tetracycline hydrochloride. One day later, the mice received chlorpromazine to keep them in a sedated state for about 3 hr. Under the sedated condition, they were infected with 4 x 10(7) viable cells of Candida albicans by intra-esophageal injection with a round-head needle on syringe. From day 3 to day 6 post inoculation, 10(5)-10(6) colony forming units of C. albicans were recovered from the esophageal tube of each mouse and whitish, curd-like patches were observed on most of the inner surface of the tube. Histological examination showed that C. albicans in esophageal lesions grew mainly in mycelial form. In this experimental model, intragastric administration of an itraconazole oral solution (20 mg/kg/day) was clearly effective. This model would provide a useful tool to investigate the pathogenesis of C. albicans esophageal infection and the efficacy of various antifungal agents microbiologically and symptomatically.

Bone marrow cells (BMC) obtained from normal and cyclophosphamide (CY)-treated mice were cultured in the presence of recombinant human granulocyte-colony stimulating factor (rhG-CSF) and their effector cell activities inhibiting growth of C. albicans were examined. When BMC from CY-treated mice were preincubated with 0.05 ng/ml of rhG-CSF, effector cells with enhanced anti-C. albicans activity were recovered in the adherent cell population, whereas anti-C. albicans activity of BMC from normal mice was found in the non-adherent cell population. During culture without the presence of rhG-CSF, nonadherent BMC, seemingly granulocytes, from normal mice showed apoptotic change, but addition of rhGCSF clearly inhibited this change. On the other hand, when BMC from CY-treated mice were cultured with rhG-CSF, adherent cells as the main effector had the appearance of monocytes. These differences between the effectors with anti-C. albicans activity obtained from normal and CY-treated mice are discussed.

The protective effects of human saliva, known to contain various proteinaceous factors that have anti- Candida activity in vitro on oral candidiasis in the mouse model, were examined in vivo. Oral candidiasis was established by oral inoculation of viable Candida albicans (C. albicans) cells to ICR mice, 24 h after administration of predonisolone. These mice received orally 0.1 ml human saliva or sterile distilled water into the oral cavity a total of five times at specific intervals: 3, 6, 12, 24, and 36 h after inoculation. Seventy-two hours after inoculation with C. albicans, viable C. albicans cells in the oral cavity of the mice were counted and a subjective score for the extent of white patches on the tongue surface determined. The results showed that viable counts were significantly lower in the human saliva group than in the distilled water group (P < 0.05). Scores for white patches on the tongue were also significantly lower in the saliva group than in the distilled water group (P < 0.05). These results suggest that administration of human saliva may inhibit the colonization of the oral cavity by C. albicans in mice and the subsequent onset of oral candidiasis.

The effects of picolinic acid (PLA), a product of tryptophan catabolism, on anti- Candida activity of neutrophils were studied. Casein-induced peritoneal neutrophils of C3H/He mice partially inhibited mycelial growth of Candida albicans when cultured with C. albicans for 16 h in vitro. The growth inhibition of Candida was augmented by a combination of neutrophils and more than 4 mM picolinic acid. Especially in the presence of 200 U/ml murine interferon-gamma (IFN-gamma), 2 mM picolinic acid augmented the anti- Candida activity of neutrophils. The physiological significance of the augmenting effects of picolinic acid is discussed.

Therapeutic activity against oral candidiasis of orally administered bovine lactoferrin (LF), a multifunctional milk protein, was shown in a previous report using an immunosuppressed murine model. In the present study, the influence of orally administered LF on immune responses relevant to this therapeutic effect was examined. Because mice were immunosuppressed with prednisolone 1 day before and 3 days after the infection with Candida, the numbers of peripheral blood leukocytes (PBL) and cervical lymph node (CLN) cells were reduced. LF feeding prevented the reduction in the numbers of PBL on day 1 and CLN cells on days 1, 5 and 6 in the Candida-infected mice. The number of CLN cells of individual mice on days 5 and 6 was inversely correlated with the Candida c.f.u. in the oral cavity. Increased production of IFN-gamma and TNF-alpha by CLN cells stimulated with heat-killed Candida albicans on day 6 was observed in LF-treated mice compared with non-treated mice. Concanavalin A (ConA)-stimulated CLN cells from LF-treated mice also showed a significant increase in the production of IFN-gamma and IL12 on day 5 and a tendency for increased production of IFN-gamma and TNF-alpha on day 6. The levels of cytokine production by ConA-stimulated CLN cells on day 6 were inversely correlated with the Candida c.f.u. in the oral cavity. In conclusion, the alleviation of oral candidiasis by LF feeding in this model may correlate with the enhancement of the number of leukocytes and their cytokine responses in regional lymph nodes against Candida infection.

Background: Production of antibodies that are specific for allergens is an important pathological process in inflammatory allergic diseases. These contain the antibodies against antigens of Candida albicans, one of the normal microbial flora in an intestinal tract. We studied the effects of the prednisolone administration on the production of anti-Candida antibodies in the gastrointestinally C. albicans-colonized mice., Methods and Materials: BALB/c mice, treated with antibacterial antibiotics to decontaminate indigenous intestinal bacterial flora, were inoculated intragastrically with C. albicans. The mice, in which C. albicans grows intestinally, were administered prednisolone to induce temporary immunosuppression. The Candida growth in their intestinal tract and their antibody response to Candida were examined., Results: Antibiotic treatment allowed establishment of C. albicans gastrointestinal colonization, but did not cause subsequent systemic dissemination of C. albicans in all the animals. When these animals received an additional treatment with prednisolone, they showed a significantly higher population of C. albicans in their feces than those of animals treated with antibiotics alone, and the organisms were recovered even from their kidney. This systemic dissemination by C. albicans appeared to be temporal, because all the mice survived without any symptoms for more than 2 months. Examination of the serum titers of total immunoglobulin (Ig)E antibodies and specific IgE and IgG antibodies against Candida antigens demonstrated that titers of total IgE increased, partially by day 14 and clearly at day 27, in prednisolone-treated Candida-colonized mice. Without prednisolone treatment, an increment of the serum titer was scarcely observed. By day 27, corresponding to the increase of total IgE, the anti-Candida IgE and IgG titer increased in mice of the prednisolone-treated group., Conclusion: Administration of prednisolone to Candida-colonized mice can induce production of the IgG, IgE antibodies against Candida antigens, perhaps through temporal systemic dissemination of Candida from the intestinal tract.

Saliva in healthy human subjects contains protenaceous factors that detach Candida albicans cells from plastic plates. To investigate the physiological role of the detachment activity of human saliva, we compared the activity of salivary specimens collected from the elderly (who are at greater risk of oral candidiasis) to the activity of specimens collected from children and nonelderly adults. C. albicans cells were cultured and attached to the bottoms of the wells of a 96-well plate, and saliva preparations were then added to each well. After 3 h of incubation, Candida cells attached to the bottoms of the wells were counted, using the crystal violet staining method. The results showed that the activity of salivary detachment factors was significantly lower among the elderly than among nonelderly adults and children. Furthermore, the activity of salivary detachment factors was reduced in 12 of the 43 elderly subjects (27.9%), a significantly higher proportion relative to that in the children and nonelderly adults. However, there was no significant difference in the level of salivary proteins between the samples collected from elderly subjects displaying low and high salivary detachment activity. These results suggest that the increased number of people with lower salivary detachment activity among the elderly may be one of the reasons that oral candidiasis is more common among the elderly than in nonelderly adults and children.

The effects of human saliva on Candida albicans attached to plastic plates were investigated. C. albicans cells were cultured for attachment to the bottoms of the wells of a 96-well plate, and saliva preparations collected from healthy adults were then added to each well. After various periods of incubation, the number of Candida cells attached to the bottom of each well was measured by the crystal violet staining method. The results showed that, when incubated with human saliva (final concentration, 10% or higher) for 3 h, Candida cells that were attached to the plates became detached from the wells. For characterization of the detachment factor(s), the saliva was divided into three fractions by ultrafiltration. The detachment activity recovered in fractions with molecular weights of 50 K or higher. When the saliva was heated at 100 degrees C for 20 min, its ability to detach Candida cells completely disappeared. When the saliva was treated with 0.025% trypsin, the detachment activity decreased by 57%. These findings suggested that human saliva contains factors for detachment of Candida cells, which are considered to be proteins with molecular weights of 50 K or higher.

The effects of 12 essential oils, popularly used as antifungal treatments in aromatherapy, on growth of Candida albicans were investigated. Mycelial growth of C. albicans, which is known to give the fungus the capacity to invade mucosal tissues, was inhibited in the medium containing 100 micro g/ml of the oils: lemongrass (Cymbopogon citratus), thyme (Thymus vulgaris), patchouli (Pogostemon cablin) and cedarwood (Cedrus atlantica). Not only lemongrass oil but also citral, a major component of lemongrass oil (80%), in the range of 25 and 200 micro g/ml inhibited the mycelial growth but allowed yeast-form growth. More than 200 micro g/ml of citral clearly inhibited both mycelial and yeast-form growth of C. albicans. These results provide experimental evidence suggesting the potential value of lemongrass oil for the treatment of oral or vaginal candidiasis.

A conventional and easy method to establish a murine oral candidiasis model, which has not only a stable yeast population in the oral cavity but also symptoms characteristic of oral thrush, was developed by using a sedative agent. Mice were immunosuppressed with prednisolone and were given tetracycline hydrochloride. They were orally infected with 10(6) viable cells of Candida albicans by means of a cotton swab and enough chlorpromazine chloride had been injected to keep them in a sedative state about for 3 hr after inoculation. From day 3 to day 7 post inoculation, 10(5)-10(6) colony forming units of Candida were recovered from the oral cavity of each mouse and whitish, curd-like patches were observed on most parts of tongue. Microscopically, germ tubes had appeared on the tongue surface. This model would be a useful experimental oral candidiasis for investigating the pathogenesis of C. albicans oral infection and the efficacy of various antifungal agents microbiologically and symptomatically.

It is important to develop a sensitive assay method for evaluation of immunomodulating candidates including granulocyte-colony stimulating factor (G-CSF) which may augment host defense function against opportunistic infections. Effects of CSFs on anti-Candida activity of bone marrow cells (BMC) obtained from normal or cyclophosphamide-treated immunocompromised mice (CY-mice) were investigated. When BMC from CY-mice were preincubated for 24 hours in the presense of more than 0.005 ng/ml of G-CSF, anti-Candida activity was enhanced at 160 of effector (BMC) to target (C. albicans) ratio, whereas anti-Candida activity of BMC from CY-untreated mice was not changed at concentrations less than only by more than 0.5 ng/ml of G-CSF. Similar enhancement of activity was also observed in the presence of more than 0.05 ng/ml of granulocyte-macrophage colony stimulating factor (GM-CSF) in this assay using BMC from CY-mice. These findings suggest that this in vitro assay method can evaluate the activity of G-CSF and GM-CSF to augment leukocyte's anti-Candida activity.

This study investigated the influences of lansoprazole (AG1749), a proton pump inhibitor (PPI), and its active derivative (AG2000) on Candida albicans growth and the anti-Candida activity of macrophages. Under concentration of 100 microM, AG1749 and AG2000 had no effect on Candida growth. Murine peritoneal macrophages inhibited the growth of C. albicans in vitro. AG2000 suppressed the anti-Candida activity of macrophages dose-dependently, but AG1749 didn't. The suppressing activity of AG2000 for macrophages was neutralized by adding a SH-compound (L-cysteine) in the medium. This suggests that AG2000 may suppress macrophage function in a similar manner with inhibition of proton pump through binding to SH-molecules. When macrophages were preincubated with AG2000 for 1 hr and washed, their anti-Candida activity remained to be partially inhibited for 14 hrs. These results were discussed in relation to the pathogenesis of esophageal candidiasis.

Synergism of lysozyme with the imidazole antifungal lanoconazole (LCZ) has been demonstrated in vitro against Candida albicans. To determine the mechanism of the synergistic action, the effects of subinhibitory concentrations of lysozyme (50 microg ml(-1)) and LCZ (1.25 microg ml(-1)), used alone and in combination, on the morphology and ultrastructure of C. albicans cells were studied by fluorescence microscopy using Fungiflora Y, as well as by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In the presence of lysozyme, the separation of apparently mature cells from each other was inhibited, and cells in which cell wall-like material had markedly accumulated in the periplasmic space were frequently observed. Such intracellular ultrastructural changes were not seen in LCZ-treated cells, but normal septal wall biogenesis was inhibited with the resultant formation of long chains of interconnecting cells. When the cultures were treated with the combination of lysozyme and LCZ, intracellular accumulation of cell wall-like materials increased in extent, and the peripheral wall structure was severely damaged. These morphological and ultrastructural findings lead us to the following conclusions: (1) lysozyme and LCZ inhibit the process of cell division in different manners; (2) lysozyme appears to affect cell wall formation more directly and drastically by interrupting the normal assembly and integration of cell wall components; and (3) these effects of lysozyme are noticeably enhanced when combined with LCZ.

The effect of leukocytes on the anti-Candida activity of neutrophils was examined. Murine neutrophils which were purified from casein-induced peritoneal cells inhibited the mycelial growth of Candida albicans. This anti-Candida activity of neutrophils was augmented by the addition of spleen cells prepared from mice pretreated with bacterial lipopolysaccharide 3 hr before, but not from non-treated mice. The population in the spleen cells, which enhanced the anti-Candida activity of neutrophils, was plastic-plate adherent, nylon-fiber columns adherent and anti-Mac-1 antigen-positive. These immunological profiles suggested that the enhancing cells are classified to splenic macrophages. Peritoneal-exudated macrophages from mice treated with lipopolysaccharide also augmented the anti-Candida activity of neutrophils. These results suggest that the anti-Candida activity of neutrophils may be upregulated by activated macrophages.

The effects of ascites fluids and sera of tumor-bearing mice on the mycelial growth of Candida albicans were examined. When the ascites fluids or the sera obtained from mice inoculated with MM46 mammary carcinoma were added to the culture medium, mycelial growth of C. albicans was strongly inhibited. The molecular size of the growth inhibitory factor in the ascites fluids was estimated to be approximately 80 K dalton by gel-filtration chromatography. Ferric chloride (6 microM) neutralized the anti-Candida activity. On the basis of these results including morphological observation, a possible role of a transferrin-like molecule was discussed.

The effects of bovine lactoferrin (LF) or the LF-derived antimicrobial peptide lactoferricin B (LFcin B) on the growth of Candida albicans hyphae, including those of three azole-resistant strains, were investigated by a crystal violet staining method. The hyphae of two highly azole-resistant strains were more susceptible to inhibition by LF or LFcin B than the azole-susceptible strains tested. One moderately azole-resistant strain was defective in the formation of hyphae and showed a susceptibility to LF greater than that of the susceptible strains but a susceptibility to LFcin B similar to that of the susceptible strains. The highly azole-resistant strain TIMM3317 showed trailing growth in the presence of fluconazole or itraconazole, while the extent of growth was reduced by the addition of LF or LFcin B at a sub-MIC. Thus, the addition of LF or LFcin B at a sub-MIC resulted in a substantial decrease in the MICs of fluconazole and itraconazole for two highly azole-resistant strains; e.g., the MIC of fluconazole for TIMM3317 was shifted from > 256 to 0.25 micrograms/ml by LF, but the MICs were not decreased for the susceptible strains. The combination effects observed with triazoles and LF-related compounds in the case of the two highly azole-resistant strains were confirmed to be synergistic by the fractional inhibitory concentration index. These results demonstrate that for some azole-resistant C. albicans strains, LF-related compounds combined with triazoles can inhibit the growth of hyphae, an important form of this organism in pathogenesis.

The combined effects of lysozyme and various antifungal agents were investigated by microbroth dilution method against Candida albicans in vitro. Synergistic anti-Candida activity was observed between egg white lysozyme and itraconazole, clotrimazole, miconazole and lanoconazole. Similar, although not as clear, combination anti-Candida activities were seen in the cases of ketoconazole, bifonazole, amphotericin B and nystatin. Anti-Candida activity of fluconazole was not affected by the addition of lysozyme, however. Physiological roles of this combination effect in anti-Candida therapy by azole antifungals were discussed.

We investigated the effects of lactoferrin (Lf)-related compounds on growth inhibition of Candida albicans by neutrophils or antifungal agents in vitro. Human neutrophils partially inhibited the growth of C.albicans. The growth inhibition caused by human neutrophils was augmented by the addition of human Lf at concentrations which did not show any inhibitory effect in the absence of neutrophils. Similar observations were obtained also with the following combinations: human neutrophils + bovine Lf, murine neutrophils + bovine Lf, and murine neutrophils + iron saturated bovine Lf, but not in the case of murine neutrophils + human transferrin. The minimum inhibitory concentration (MIC) of azole antifungal agents, clotrimazole, ketoconazole, fluconazole, and itraconazole was reduced by 1/4 to 1/16 in the presence of a sub-MIC level of each of bovine Lf, bovine Lf pepsin hydrolysate, and the antimicrobial peptide "lactoferricin B" (Lfcin B). Other types of antifungal agents, amphotericin B, nystatin, and flucytosine did not show such combined effects with these Lf-related compounds. The anti-Candida activity of bovine Lf or Lfcin B in combination with clotrimazole was shown to be synergistic by checkerboard analysis. Clinically isolated azole-resistant C. albicans strains were more susceptible to bovine Lf or Lfcin B than azole-susceptible strains. Trailing growth of an azole-resistant strain in the presence of fluconazole was reduced by the addition of sub-MIC levels of bovine Lf or Lfcin B. These results suggest that Lf-related compounds even at relatively low concentrations may function as an antifungal effector in combination with neutrophils thereby modulating azole antifungal efficacies in vivo.

Preparations of human cervical mucus were obtained from fifteen female volunteers with or without vaginal candidiasis. Their content of lactoferrin and inhibitory effects on the growth of Candida albicans were estimated. The concentration of lactoferrin in cervical mucus was measured by enzyme linked immunosorbent assay (ELISA). Lactoferrin concentration of the eleven preparations among them was more than 0.2 mg/ml. The effects of these cervical mucus preparations on Candida growth were examined in vitro. Candida growth was not inhibited by the addition of 1/200 diluted cervical mucus alone, however, anti-Candida activity of murine neutrophils was augmented by such an addition. These results suggest that the combination of neutrophils and cervical mucus may have an important role in defense against Candida infection in the vaginal mucosa.

Effects of itraconazole (ITCZ) on anti-Candida activity of a macrophage (MP)-neutrophil axis were examined in vitro. Murine peritoneal MP, stimulated by an enterococcal preparation FK-23, released tumor necrosis factor a (TNF) into the culture medium in 20 hr-culture period. This level of TNF production by the stimulated MP, which were measured by a bioassay or immunological assay, was heightened by co-stimulation with 0.5 mg/ml of ITCZ. Supernatants obtained from the culture medium of FK-23 and ITCZ-treated MP not only contained about 10 ng/ml of TNF but also had the capacity to enhance anti-Candida activity of murine neutrophils. This enhancement was blocked by pretreatment of the MP-supernatant with anti-TNF antibody. Observation by scanning electron microscope suggested that cell walls of the candida mycelia, the growth of which was inhibited by the neutrophils in the presence of either the MP-supernatant or TNF, sustained similar structural damage. These results suggest that ITCZ augments anti-Candida activity of neutrophils through enhanced production of TNF by MP in vitro.

The combined inhibitory effects of neutrophils and lactoferrins on the growth of Candida albicans were examined. Murine or human neutrophils partially inhibited growth of C. albicans when cultured with C. albicans in vitro. The growth inhibition was augmented by a combination of neutrophils and more than 30 microg/ml of bovine lactoferrin or 1 microg/ml of human lactoferrin, concentrations less than 1/10-1/200 their inhibiting concentrations when used alone. The inhibition of C. albicans was also enhanced by combination of neutrophils and bovine apolactoferrin or iron-bound holo-lactoferrin, but not by transferrin. Combination effects of neutrophils and lactoferrin were also observed in a condition where there was no contact between neutrophils and Candida cells. These results suggest that neutrophils inhibit the growth of C. albicans regardless of whether there is direct contact between them and Candida cells: neutrophil growth inhibition effects were augmented in the presence of a physiological concentration of lactoferrin, perhaps through some action of lactoferrin other than chelation of ferric ion.

Benanomicin A is an antifungal antibiotic produced by Actinomadura spadix. In the present study, we investigated the effect of benanomicin A on the phagocytosis of Candida albicans by murine peritoneal macrophages and on the cell-surface hydrophobicity (CSH) of C. albicans. Although pretreatment of macrophages with benanomicin A had no effect on the phagocytosis, addition of benanomicin A to the culture of macrophages and Candida cells increased the susceptibility of Candida cells to the phagocytosis by the macrophages. Pretreatment of Candida cells with benanomicin A also increased the susceptibility of Candida cells to the phagocytosis. When Candida cells were mixed with benanomicin A, the antibiotic bound irreversibly to Candida cells. These data suggest the possibility that the increased susceptibility of Candida cells to the phagocytosis is mediated by the binding of benanomicin A to Candida cells. Examination of physicochemical property of Candida cell surface showed that the CSH of Candida cells significantly decreased by the treatment with benanomicin A. Thus, binding of benanomicin A to Candida cells may induce biochemical/physicochemical alternation of the surfaces, so that they become more susceptible to phagocytosis by murine macrophages. These properties of benanomicin A, along with its antifungal activity, seem to be beneficial in the treatment of fungal infections.

Effects of a glucose and amino acid mixture prescribed for parenteral alimentation on anti-Candida activity of neutrophils were examined. Neutrophils obtained from peripheral blood of healthy humans inhibited the growth of Candida albicans in vitro. More than 1.0% of glucose inhibited the anti-Candida activity of the neutrophils in a dose-dependent manner. This glucose effect was reduced by the addition of an amino acid mixture clinically prescribed with a carbohydrate solution (PN-twin) in Japan. The amino acid mixture neutralized the suppression of anti-Candida activity of neutrophils by dexamethasone. These results suggest that an amino acid mixture prescribed in an alimentation solution may play a role as a neutralizer of the suppressive action of glucose for anti-Candida activity of neutrophils in a limited area near the top of a catheter in a blood vessel.

The effects of lactoferrin (LF), an antimicrobial protein secreted in body fluids, and its peptides in combination with azole antifungal agents were investigated by the micro-broth-dilution method in a study of Candida albicans. In the case of LF, its pepsin hydrolysate (LFhyd) or the LF-derived antimicrobial peptide Lactoferricin B (LF-B), the concentrations required to inhibit the growth of Candida decreased in the presence of relatively low concentrations of clotrimazole (CTZ). The minimum inhibitory concentration (MIC) of all azole antifungal agents tested was reduced by 1/4-1/16 in the presence of a sub-MIC level of each of these LF-related substances. Polyene and fluoropyrimidine antifungal agents did not show such a combined effect with these LF-related substances. The anti-Candida activity of LF or LF-B in combination with CTZ was shown to be synergistic by checkerboard analysis. These results indicate that LF-related substances function cooperatively with azole antifungal agents against C. albicans.

Anti-Candida activity of murine neutrophils and its regulation by immunomodulators were studied in vitro. Murine neutrophils which were prepared from peritoneal-exudated cells inhibited the growth of Candida albicans at an effector: target (E/T) ratio of 30/1 or above. This anti-Candida activity of neutrophils was augmented by lipopolysaccharide from Escherichia coli, murine tumor necrosis factor (TNF), murine interferon-gamma (IFN-gamma) and murine granulocyte macrophage colony-stimulating factor (GM-CSF) but not by granulocyte colony-stimulating factor (G-CSF) added to the incubation medium. Greater extent of augmentation was obtained when TNF plus GM-CSF or INF-gamma plus GM-CSF were used in combination. These results indicate that anti-Candida activity of murine neutrophils is regulated similarly to that of the human neutrophils reported previously. Therefore murine peritoneal neutrophils can be used as a favorable substitute for human neutrophils in studies on protective machinery against C. albicans infection.