Adhesion of oral Candida albicans isolates to denture acrylic following limited exposure to antifungal agents

Candidal adherence to denture acrylic surfaces is implicated as the first step in the pathogenesis of Candida-associated denture stomatitis, the most prevalent form of oral candidosis in the West. This condition is treated by topically administered antifungal agents, mainly belonging to the polyenes and azoles. As the intraoral concentrations of antifungals fluctuate considerably due to the dynamics of the oral environment, the effect of short exposure to sublethal concentrations of antifungals on the adhesion of Candida albicans to denture acrylic surfaces was investigated. Seven oral C. albicans isolates were exposed to four-eight times minimum inhibitory concentrations (MIC) of five antifungal drugs, nystatin, amphotericin B, 5-fluorocytosine, ketoconazole and fluconazole, for 1 h. After removing the drug (by repeated washing) the adhesion of these isolates to acrylic strips was assessed by an in vitro adhesion assay. Exposure to antifungal agents significantly reduced the adherence of all seven C. albicans isolates to denture acrylic. The mean percentage reductions of adhesion after limited exposure to nystatin, amphotericin B, 5-fluorocytosine, ketoconazole and fluconazole were 86.48, 90.85, 66.72, 65.88 and 47.42%, respectively. These findings indicate that subtherapeutic doses of antifungals may modulate oral candidal colonization. Further, these results may have an important bearing on dosage regimens currently employed in treating oral candidosis.     

Phenotypic and genotypic characterization of oral yeasts from Finland and the United States

A total of 4-22 isolates of oral yeasts per subjects from 48 yeast-positive Finnish and American subjects (25 females and 23 males) were phenotyped and genotyped to determine the frequency of simultaneous oral carriage of multiple yeast taxa. An oral sample from either periodontal pockets, oral mucosa or saliva was obtained. All subjects yielded Candida albicans and 3 subjects an additional yeast species (Candida krusei, Candida glabrata or Saccharomyces cerevisiae). The API 20C Aux kit distinguished 9 different carbohydrate assimilation profiles among the C. albicans isolates. Thirty-eight of 46 C. albicans biotype I isolates were categorized in a single numerical profile. PCR analysis, using a random primer OPA-03 and a repetitive primer (GACA)4, detected 2 major genotypic groups among the C. albicans isolates; 44 subjects showing isolates with a "typical" PCR-profile and 4 subjects isolates with an "atypical" PCR-profile. The "atypical" PCR-profile was similar to that of Candida dubliniensis. All C. albicans isolates assimilated xylose, except 5, including the 4 with an "atypical" PCR-profile. No difference was found in distribution of oral yeast species, and of C. albicans phenotypes and genotypes between Finnish and American subjects. The present PCR method may offer a rapid and easy means of distinguishing oral Candida species.     

Utility of Fluoroplate Candida for the rapid identification of Candida albicans

BACKGROUND: Candida albicans infections are frequent in immunocompromised patients and a prompt diagnosis could favor an early and proper antifungal treatment. The rapid identification of clinical yeast isolates facilitate this diagnosis. METHODS: The utility of Fluoroplate Candida ready-to-use plates for Candida albicans rapid identification was evaluated with 653 clinical isolates from 23 yeast species, including 307 C. albicans plated onto Fluoroplate Candida agar (Merck, Germany). Rapid identification of C. albicans was based on the hydrolysis of 4-methylumbelliferyl-N-acetyl-beta-D-galactosaminide by the galactosaminidase activity of C. albicans producing white fluorescent colonies under ultraviolet light. Identification on Fluoroplate Candida was confirmed by germ tube, chlamydoconidia formation and API-ATB ID 32C assays. RESULTS: Three hundred and five of 306 isolates showing fluorescent colonies were C. albicans and one was Candida glabrata (false positive). The rest of the isolates showed colonies without fluorescence and with the exception of two false negatives, these isolates were identified as non-C. albicans by other methods. CONCLUSIONS: Fluoroplate Candida allows a rapid and excellent identification of C. albicans showing a sensitivity and specificity of 99.3 and 99.7%, respectively.     

Minimum chemical requirements for adhesin activity of the acid-stable part of Candida albicans cell wall phosphomannoprotein complex

This study was conducted to define adhesive characteristics of the acid-stable moiety of the Candida albicans phosphomannoprotein complex (PMPC) on adherence of this fungus to marginal zone macrophages of the mouse spleen. Complete digestion of the acid-stable moiety (Fr.IIS) of the C. albicans PMPC with an alpha-mannosidase or hydrolysis with 0.6 N sulfuric acid destroyed adhesin activity, as determined by the inability of the soluble digests to inhibit yeast cell adherence to the splenic marginal zone. Fr.IIS adhesin activity was decreased following digestion with an alpha-1,2-specific mannosidase. Oligomannosyls consisting of one to six mannose units, which were isolated from the acid-stable part of the PMPC, did not inhibit yeast cell binding and thus do not function alone as adhesin sites in the PMPC. To gain more insight into the minimum requirements for adhesin activity, PMPCs were isolated from a Saccharomyces cerevisiae wild-type strain and from mutant strains mnn1, mnn2, and mnn4; the PMPCs were designated scwt/Fr.II, scmn1/Fr.II, scmn2/Fr.II, and scmn4/Fr.II, respectively. S. cerevisiae scmn2/Fr.II lacks oligomannosyl side chain branches from the outer core mannan, and scmn2/Fr.II was the only PMPC without adhesin activity. S. cerevisiae scwt/Fr.II, scmn1/Fr.II, and scmn4/Fr.II showed adhesin activities less than that of C. albicans Fr.II. These three S. cerevisiae PMPCs are generally similar to Fr. IIS, except that the S. cerevisiae structure has fewer and shorter side chains. Immunofluorescence microscopy show that the acid-stable part of the PMPC is displayed homogeneously on the C. albicans yeast cell surface, which would be expected for a surface adhesin. Our results indicate that both the mannan core and the oligomannosyl side chains are responsible for the adhesin activity of the acid-stable part of the PMPC.     

Activity of the renin-angiotensin-aldosterone system in euglycemic type I diabetic patients on intensive insulin treatment without diabetic neuropathy

The yeast Candida albicans coaggregates with a variety of streptococcal species, an interaction that may promote oral colonization by yeast cells. C. albicans and Candida tropicalis are the yeasts most frequently isolated from the human oral cavity and our data demonstrate that both these species bind to Streptococcus gordonii NCTC 7869 while two other Candida species (Candida krusei and Candida kefyr) do not. Adherence of C. albicans was greatest when the yeast had been grown at 30 degrees C to mid-exponential growth phase. For 21 strains of C. albicans there was a positive correlation between the ability to adhere to S. gordonii and adherence to experimental salivary pellicle. Whole saliva either stimulated or slightly inhibited adherence of C. albicans to S. gordonii depending on the streptococcal growth conditions. The results suggest that the major salivary adhesins and coaggregation adhesins of C. albicans are co-expressed.     

Binding of Candida albicans yeast cells to mouse popliteal lymph node tissue is mediated by macrophages

We previously reported that Candida albicans yeast cells adhere to the macrophage-rich medullary and subcapsular sinus areas of mouse lymph node tissue. To determine whether the yeast cell-lymph node interaction is mediated by macrophages, the effect of specific elimination of macrophages on yeast cell binding was studied, and yeast cell adherence was correlated with the ingestion of India ink by lymph node cells. Macrophage elimination was done by use of liposome-containing dichloromethylene diphosphonate (L-Cl2MDP). Mice were injected in the hind footpads with the L-Cl2MDP preparation, popliteal lymph nodes were removed 5 days later, and yeast cell adherence was determined by an ex vivo binding assay. As controls, lymph nodes from mice that received footpad injections of either phosphate-buffered saline (PBS) alone or liposome-containing PBS were used. Use of macrophage- and neutrophil-specific monoclonal antibodies in tissue immunostaining showed that the L-Cl2MDP treatment eliminated macrophages but not neutrophils from the medullary and subcapsular sinus areas of the popliteal lymph nodes. A striking reduction of yeast cell adherence occurred with lymph nodes from L-Cl2MDP-treated mice compared with lymph nodes from control animals. The lymph node-yeast cell binding patterns of L-Cl2MDP-treated and control mice were the same regardless of mouse strain, sex, or T-cell competency. Results of India ink experiments, in which India ink was injected into footpads of mice and was rapidly taken up by popliteal lymph node macrophages, showed a strong correlation between yeast adherence and India ink staining of cells. In addition, the interaction of yeast cells with lymph node tissue from normal mice was not significantly affected by the addition of two extracellular matrix proteins, fibronectin and laminin, during the ex vivo adherence assay. These data indicate that medullary and subcapsular sinus lymph node macrophages express an adhesion system similar to that described for mouse splenic marginal zone macrophages.     

Mucocutaneous fungal colonization in HIV-infected children

The prevalence of symptomatic mucocutaneous candidiasis in HIV-infected children is well documented. Information, however, of the carriage rate of potential fungal pathogens is lacking. In this study we determined the fungal colonization rate of multiple mucocutaneous sites from 13 HIV-infected and 12 control children. The rate of yeast and mould colonization and the species of fungal isolates were essentially the same for both groups of patients. However, several HIV-infected children asymptomatic for thrush proved to be colonized by Candida albicans, and disseminated colonization with Trichosporon beigelii occurred in one HIV-infected child. All cultures for dermatophytes were negative. While the carriage rate with fungi other than C. albicans was not increased in the HIV-infected group, the isolates recovered are known pathogens in the immunocompromised host and the colonization of these organisms may be a potential source of infection.     

Identification of hashish samples with inductively coupled high-frequency plasma emission spectrometry and neutron activation analysis and data handling with neuronal networks. 1. Methods for the quantitative determination of characteristic trace elements]

The adherence of six clinical Candida albicans isolates to buccal epithelial cells obtained from AIDS patients, solid organ transplant recipients and healthy individuals was compared. It was shown that Candida albicans bound in significantly greater numbers to epithelial cells obtained from AIDS patients than to those from healthy individuals or transplant patients, and that the adherence capacity varied among the strains tested.     

Strength and will: theoretical and methodological issues from the standpoint of risk in epidemiology and HIV/AIDS prevention

Large granular lymphocytes require adherence to hyphae of Candida albicans to inhibit growth of this fungus. This study was undertaken to identify the lymphocyte surface structures that mediate this adhesion. Monoclonal antibodies specific for epitopes of the alpha subunit (CD11b) and the beta 2 subunit (CD18) of Mac-1 eliminated lymphocyte adhesion to C. albicans hyphae. Significant inhibition of lymphocyte adhesion to C. albicans was also achieved with known protein ligands of Mac-1. These proteins included the extracellular matrix proteins vitronectin, laminin, and fibrinogen as well as two engineered peptides containing RGD (arginine-glycine-aspartic acid) sequences. Carbohydrates including N-acetyl-D-glucosamine which have been demonstrated to inhibit Mac-l-mediated adhesion to whole yeast and yeast zymosan also blocked lymphocyte adhesion to hyphae. These results identify Mac-1 (CD11b/CD18) as the surface structure that mediates lymphocyte adhesion to C. albicans. A model is proposed for lymphocyte Mac-1 activation by microbial ligands.     

Comparison of the rapid yeast plus panel with the API20C yeast system for identification of clinically significant isolates of Candida species

The RapID Yeast Plus system (Innovative Diagnostic Systems, Norcross, Ga.) is a qualitative micromethod employing conventional tests and single-substrate chromogenic tests and having a 4-h incubation period. This system was compared with the API20C (bioMerieux Vitek, Hazelwood, Mo.) system, a 24- to 72-h carbohydrate assimilation method. One hundred thirty-three clinical yeast isolates, including 57 of Candida albicans, 26 of Candida tropicalis, 23 of Candida glabrata, and 27 of other yeasts, were tested by both methods. When discrepancies occurred, isolates were further tested by the Automated Yeast Biochemical Card (bioMerieux Vitek). Germ tube production and microscopic morphology were used as needed to definitively identify yeast isolates. The RapID Yeast Plus system correctly identified 125 yeast isolates, with an overall accuracy of 94% (125 of 133). Excellent correlation was found in the recognition of the three yeasts most commonly isolated from human sources. The test was 99% (105 of 106 isolates) accurate with C. albicans, C. tropicalis, and C. glabrata. The RapID Yeast Plus system compares favorably with the API20C system and provides a simple, accurate alternative to conventional assimilation methods for the rapid identification of the most commonly encountered isolates of Candida species.     

Autoimmune hepatitis and autoimmune manifestations in patients with chronic viral hepatitis

Mucosal adherence and germ tube formation have been considered as important virulence factors of Candida albicans. We investigated 11 clinical isolates (among them six isolates from oesophageal thrush) for quantification of adherence to buccal epithelial cells and germ tube formation in the continuous flow culture in vitro, and correlated the results with the clinical data of the patients. Adherence varied considerably between the different C. albicans strains. Strains recovered from clinically, culturally and serologically confirmed oesophageal thrush adhered stronger to buccal epithelial cells. Isolates from cases with heavy colonisation but clinically without candidosis were less adherent. Only after 30 min germ tube formation was observed in the continuous flow culture. Strains with stronger adherence also showed significantly faster and increased germ tube formation. The patients with oesophageal thrush did not suffer any particular immunosuppression such as HIV infection, although in most cases chronic alcoholism was apparent. We conclude, that in cases with minor immunosuppression the expression of the virulence factors adherence and germ tube formation plays an important role in the pathogenesis of candidosis, whereas it may be of less importance in cases with severe immunosuppression. In the latter they may, however, influence outcome.     

Antitumor effect of electrochemotherapy on colorectal carcinoma in an orthotopic mouse model

Although Candida albicans remains the fungal species most frequently isolated as an opportunistic oral pathogen, other yeast species are often identified in human immunodeficiency virus (HIV)-seropositive patients. Candida dubliniensis phenotypically resembles C. albicans in many respects, yet it can be identified and differentiated as a unique Candida species by its phenotypic and genetic profiles. The purpose of the present study was to prospectively test for the presence of C. dubliniensis among clinical isolates and to determine the clinical and demographic characteristics of patients harboring C. dubliniensis. Over a 90-day period, isolates from 724 patients that were presumptively identified as C. albicans were screened for C. dubliniensis by use of tests for germ tube and chlamydospore production, by detection of an inability to grow at 45 degrees C, by colony color on CHROMagar Candida medium, and by the results of a sugar assimilation test with the API 20C AUX yeast identification system. Among 699 isolates retrieved from those specimens evaluated, 5 from 25 HIV-seropositive patients and 1 isolate from a patient whose HIV status was unknown were shown to be consistent by phenotyping and by electrophoretic karyotyping with the European reference strain of C. dubliniensis. One of the C. dubliniensis isolates had dose-dependent susceptibility to fluconazole (MIC, 16 microg/ml). These results confirm the presence of this interesting species in the United States and support the need for further investigations into the prevalence and pathogenesis of C. dubliniensis.     

Mucin and proteoglycan functions in embryo implantation

OBJECTIVES: To assess the in vitro adherence of Candida albicans to heat-cured hard and soft denture-base materials with varying surface roughness, and to observe the effect of a mixed salivary pellicle on candidal adhesion to these surfaces. METHODS: In vitro adhesion assays on heat-cured acrylic resin (Trevalon), Molloplast B and Novus using the type strain of C. albicans (NCPF 3153A). Surfaces for the assays were prepared using clinically appropriate rotary instruments. Unstimulated, pooled and clarified whole saliva was used to assess its effect on adhesion. RESULTS: Significantly greater adhesion of C. albicans to rough rather than smooth surfaces was found (P < 0.001), as well as increased adhesion to the machined soft lining materials compared with acrylic. Pre-coating denture-base materials with saliva reduced candidal adhesion on all materials. CONCLUSIONS: Rough surfaces on denture-base materials promote the adhesion of C. albicans in vitro. However, saliva reduces adhesion of C. albicans and thus diminishes the effect of surface roughness and free surface energy differences between materials.     

PCR-restriction fragment length polymorphism (RFLP) analyses reveal both extensive clonality and local genetic differences in Candida albicans

Using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method to obtain genotypes for the diploid pathogenic yeast, Candida albicans, we analysed 204 C. albicans isolates from three populations of the Duke University community: two from clinical sources [one from patients infected with human immunodeficiency virus (HIV) and the other from patients without HIV infection], and the third from healthy student volunteers. The results indicated: (i) extensive evidence for clonality within and between populations of C. albicans; and (ii) greater genotypic and gene diversities in the nonclinical population than those derived from clinical specimens, regardless of HIV status. The two clinical populations were genetically more similar to each other than either was to the population consisting of isolates from healthy people. Within each population sample there was a general lack of heterozygotes, and random associations of alleles within and between loci were found in less than 50% of the loci or pairs of loci. These findings were consistent between the two sets of samples analysed: those including all isolates and those including only clone-corrected isolates. Possible mechanisms are presented to explain the observed patterns of genetic variation within and between C. albicans populations.     

Point prevalence of oropharyngeal carriage of fluconazole-resistant Candida in human immunodeficiency virus-infected patients

To estimate the prevalence of both clinically evident and asymptomatic carriage of fluconazole-resistant Candida, we prospectively surveyed 128 adults infected with human immunodeficiency virus (HIV). The patients had an average CD4 cell count of 206/mm3. Ninety-seven isolates of Candida were obtained from the oropharynx of 82 patients (64%). Of these 82 patients, 76% carried C. albicans alone; 18%, both albicans and non-albicans isolates; and 6%, non-albicans species alone. Oropharyngeal candidiasis was evident in only 38 (46%) of the 82 patients for whom a culture was positive and was never seen unless C. albicans was present. When MICs were measured by using the National Committee for Clinical Laboratory Standards M27-T methodology and grouped by using recently proposed breakpoints, we found that eight of the 38 patients with oropharyngeal candidiasis and six of the 44 patients who were asymptomatically colonized carried C. albicans isolates resistant to fluconazole (MIC, > or = 64 micrograms/mL); estimated rates of carriage were 21% (95% confidence interval, 10%-37%) and 14% (95% confidence interval, 5%-27%), respectively. Carriage of resistant isolates of C. albicans by HIV-infected adults is more common than previously suspected, and clinicians should be alert to the possible need for either higher doses of fluconazole or alternative treatment modalities.     

In vitro susceptibility of clinical yeast isolates to fluconazole and terconazole

OBJECTIVE: Fifty clinical yeast isolates, representing equally Candida albicans, Candida krusei, Candida parapsilosis, Candida tropicalis, and Torulopsis glabrata, were tested in vitro for their susceptibility to terconazole and fluconazole. STUDY DESIGN: The minimal inhibitory concentrations of terconazole and fluconazole were determined by use of a proposed standardized broth macrodilution assay. Also, the response of selected yeast isolates to 25 micrograms of either drug was measured by agarose disk diffusion experiments. RESULTS: For all species the minimum inhibitory concentrations for terconazole were significantly lower than those for fluconazole (p < 0.05). In fact, for each individual isolate the minimum inhibitory concentration of terconazole was consistently lower than that of fluconazole. Differences in the geometric mean of terconazole and fluconazole minimum inhibitory concentrations were largest among C. krusei and T. glabrata, followed by C. parapsilosis, C. tropicalis, and C. albicans, in order of decreasing difference. Disk diffusion experiments suggested that terconazole is a more effective fungistatic agent than fluconazole is. CONCLUSION: Terconazole may be more effective than fluconazole against yeast species other than C. albicans.     

Effect of Staphylococcal proteinase on adherence of Candida albicans to mucous membrane cells in vitro

The study was aimed at evaluation of influence of staphylococcal proteinase on adherence of Candida albicans to the cheek mucous membrane cells. Epithelial cells were preincubated with the enzyme which was followed by adherence tests. Significant increase of number of cells of Candida albicans adhering to epithelial cells preincubated with enzyme in concentrations of 10 micrograms/ml and 50 micrograms/ml, was detected. Staphylococcal serine protease seems to play an important role in mixed infections caused by fungi and bacteria.     

Adherence of staphylococci of different hydrophobicity. Study of various intraocular lenses

BACKGROUND: A major goal in research on intraocular lenses (IOL) is the development of new polymers and modifications to reduce foreign-body reactions after implantation. This effect may be achieved by a reduction in the surface hydrophobicity of the polymers. To illustrate the influence of surface modifications on bacterial adhesiveness, the most often isolated organism in "low-grade" postoperative endophthalmitis, Staphylococcus epidermidis, was used. MATERIALS AND METHODS: For this reason three strains of this species, the type strain ATCC 14990 and two clinical isolates (8687, 6579 I) with different hydrophobic surface properties were studied. IOL, used in the experiments were either made of PMMA or silicone with modified surfaces (unpolished, polished, heparinized). The adhesiveness of H3-thymidin-labeled bacteria was calculated/mm2 of lens surface. Each experiment was performed in triplicate and repeated three times. RESULTS: The hydrophobic-type strain showed stronger adherence to unpolished PMMA surface (8000 bacteria per mm2) compared to the polished (5200 bacteria/mm2). In contrast, the hydrophilic strain adhered with 2000 bacteria/mm2 to the unpolished and with 4200 bacteria/mm2 to the polished surface. Polishing PMMA lenses diminished the differences between the three strains. However, surface passivation of silicone lenses increased the adhesion rate of the hydrophilic strain up to 9600 bacteria/mm2. Treatment of PMMA lenses with heparin increased the adhesiveness of the hydrophilic strain and reduced the adhesion rate of the hydrophobic type strain to 250 bacteria/mm2. CONCLUSIONS: It was demonstrated that bacterial adherence to IOL also involves hydrophobic interactions. Obviously, however, that adherence reflects a complex of interactions between the two surfaces.     

Virulence of capsulated and noncapsulated isolates of Pasteurella multocida and their adherence to porcine respiratory tract cells and mucus

The virulence and the adherence to porcine respiratory tract cells and mucus of three toxigenic, capsular type D Pasteurella multocida isolates and their noncapsulated variants were evaluated in the present study. Loss of capsule by P. multocida, verified by transmission electron microscopy after polycationic ferritin labeling, was associated with a massive reduction in virulence of the organisms in mice. Specific-pathogen-free piglets inoculated intranasally with one of the capsulated isolates or its noncapsulated variant developed turbinate lesions characterized by bone resorption and by an inflammation of the mucosa associated with hyperplasia and squamous metaplasia of the epithelium. Infection with the capsulated isolate led to more severe lesions and atrophy of turbinates. The interactions of these P. multocida isolates with porcine respiratory tract cells and mucus were studied in vitro. The presence of capsule resulted in a decrease in binding of respiratory tract mucus were studied in vitro. The presence of capsule resulted in a decrease in binding of respiratory tract mucus to P. multocida isolates as determined by a dot blot assay. The presence of capsule also resulted in a significant decrease in adherence to porcine tracheal rings maintained in culture. The capsule seemed to mask outer membrane components which are involved in adherence. One of these components might be lipopolysaccharide since purified lipopolysaccharide bound respiratory tract mucus and blocked adherence of this microorganism to porcine tracheal rings. Our data indicate that capsular material does not seem to be involved in adherence of P. multocida to respiratory tract cells and mucus, but capsulated isolates are more virulent in mice and also in piglets.     

Use of CHROMagar Candida for genital specimens in the diagnostic laboratory

OBJECTIVE: To evaluate CHROMagar Candida (CA), a new yeast differential medium, for yeast isolation in a clinical laboratory for the routine examination of high vaginal swabs. METHODS: Results of high vaginal swab cultures processed in a standard manner on plates containing equal halves of Sabouraud dextrose agar (SDA) and CA were compared. Non-Candida albicans yeast isolates were further speciated with API 20C AUX or API 32C. To assess the ease of use of CA, laboratory staff lacking in experience of the medium were asked to identify 23 unlabelled yeast cultures on CA by referring to six labelled reference plates. RESULTS: Of the 1784 swab cultures processed, yeasts were isolated from 373 SDA and 368 CA. Of the 78 non-albicans isolates further speciated, CA identified correctly all cultures of C krusei and C tropicalis, and 82% of C glabrata. All the 38 inexperienced laboratory staff achieved 100% accuracy for C albicans and over 90% for C krusei and C tropicalis. CONCLUSIONS: CA is a satisfactory isolation medium for genital specimens, allowing immediate and correct identification of the commonly encountered yeasts and easy recognition of mixed cultures.