F. De Bernardis

The secretion of aspartyl proteinase, a virulence enzyme, by isolates of Candida albicans from the oral cavity of HIV-infected subjects

Prevalence, serotype and in vitro secretion of aspartyl proteinase, a virulence enzyme, were studied in Candida isolates from the oral cavity of 337 HIV-infected subjects. Controls were 95 age-sex-matched HIV- (seronegative) subjects, belonging to either HIV-risk categories (47) or to the normal, general population (48). Fungi were isolated from 155 HIV+ subjects. C. albicans was the most prevalent species (85.8% of all isolates). 94.6% of C. albicans isolates were serotype A and all were agglutinated by a monoclonal antibody (AF1) directed against a major mannoprotein immunogen of the candidal cell wall, confirming previous results with C. albicans isolates from non-immunodeficient subjects. With regard to the stage of HIV infection, there were no statistically significant differences in the incidence of oral Candida carriage between asymptomatic (stage II) HIV+ and HIV- subjects, and between stage II and lymphadenopathic (stage III) individuals. Also, the low (3.8%) incidence of oral candidiasis in the subjects of the latter stage was insignificant with respect to stage II subjects. However, the incidence of C. albicans in stage IV (AIDS) subjects (46.8%) was significantly higher than in all other subjects, and in almost all cases, fungal isolation was accompanied by oral thrush and lower CD4+ lymphocyte counts (< 400 × 10°/L).All isolates of C. albicans were proteolytic in vitro, as assessed by scoring the proteinase activity on BSA agar and monitoring the secreted proteinase antigen by a highly sensitive (1 ng) and specific immunoenzymatic assay. However, by both methods, the isolates from subjects at stages III and IV of infection produced more secretory proteinase than the isolates from either HIV+ asymptomatic subjects or HIV- controls. The differences could not be attributed to particular culture media or source of Candida isolation (carriage versus active infection). Thus, the isolates of C. albicans from advanced HIV infection are serologically similar but more proteolytic than the isolates from earlier stages of HIV infection or those from HIV-uninfected subjects. The apparently higher virulence of C. albicans from AIDS subjects may represent a co-factor in determining and/or aggravating oral candidiasis in these patients.

Experimental pathogenicity and acid proteinase secretion of vaginal isolates of Candida parapsilosis

Isolates of Candida parapsilosis from women with or without candidal vaginitis were compared for their ability to produce secretory aspartate (acid) proteinase and their virulence for normal or cyclophosphamide-immunodepressed mice. Although all isolates were strongly proteolytic in vitro, only those from candidosis-affected subjects were appreciably pathogenic for neutropenic mice. In these animals, organ invasion was monitored after challenge with representative isolates of each category. The number of yeast cells in the kidneys of animals infected with an isolate from a subject without candidal vaginitis was approximately one order of magnitude less than that in mice infected with either one of two isolates from patients with candidal vaginitis. Mice infected with either category of C. parapsilosis isolates developed antibodies against a mannoprotein-rich extract of the cell wall, and these antibodies did not cross-react with a chemically similar preparation from Candida albicans. However, only those animals which had been challenged with one of the isolates from a candidosis subject produced a low level of antibodies, detectable by ELISA, against an acid proteinase of C. parapsilosis. These antibodies cross-reacted with a highly purified enzyme preparation of C. albicans. The data demonstrate differences in the potential virulence of different isolates of C. parapsilosis and suggest that the ability to express the acid proteinase in vivo may be related to differences in pathogenicity.

Rat Model of Candida Vaginal Infection

Publisher Summary This chapter presents a rat model of Candida vaginal infection. Several strains of rats have been used in this model including Sprague-Dawley, CD, Wistar, and Alderley Park. No specialized housing or feeding is required, and caring is under the ordinary standards for outbred animal care. Rat vaginal infection by Candida is under strict hormonal control. For the infectious challenge, cells of each Candida strain tested are grown in Winge broth for 48 hours at 28° C on a shaker at 200 rpm. Growth is measured by hemocytometer counts and the yeast suspension is appropriately diluted in physiological saline before animal inoculation. Six days after the first estradiol dose, the animals are inoculated intravaginally with Candida cells in 0.1 ml of saline solution, which is administered to each animal through a syringe equipped with a multipurpose calibrated tip. To favor intravaginal contact and adsorption of fungal cells, the rat is held head-down for 1 minute. For immunological studies and detection of antibodies, samples of vaginal fluids are taken at regular intervals from each animal by gently washing the vaginal cavity with 0.5 ml of sterile saline solution. For the histological examination of vaginal tissue, the vaginas from euthanized Candida-infected rats and control are aseptically removed and fixed in 10% (vol/vol) formalin. Paraffin sections are examined after treatment with PAS and van Giesen stains. A major advantage of the rat model is the possibility of obtaining a relatively large amount of vaginal fluid and other materials for the studies of pathogenesis and immunity in candidiasis.

Detection of Candida mannoproteinemia in patients with neutropenic enterocolitis.

Abstract To assess the role of Candida spp. in the etiology of neutropenic enterocolitis complicating aggressive cytotoxic chemotherapy, a dot immunobinding assay for an immunodominant Candida mannoprotein antigen was employed in 20 patients with hematologic malignancies. Candida antigen was detected in at least one serum sample from 12 (60%) patients. Eleven (92%) patients were cured when an antifungal agent was added to the antibacterial treatment. In eight patients a selective anticandidal therapy with fluconazole was administered on the basis of positive Candida mannoproteinemia, and treatment was successful in all cases but one. Detection of Candida mannoproteinemia seems to be a useful diagnostic tool in patients with neutropenic enterocolitis and represents an additional tool for selecting a less empiric, low toxic antifungal treatment with fluconazole.

Correlation of SfiI macrorestriction endonuclease fingerprint analysis of Candida parapsilosis isolates with source of isolation

SfiI macrorestriction digests from whole chromosome DNA preparations of 46 isolates of Candida parapsilosis from vaginal (20 isolates), blood (23 isolates) and soil (three isolates) sources were examined by CHEF-MAPPER pulsed-field electrophoresis. The isolates were grouped into nine macrorestriction endonuclease fingerprint (MEF) classes according to the number or size of the macrorestriction fragments, or both. The electrophoretic karyotype (EK) was also examined and found to contain 18 karyotypic classes (named A-R). A comparison between SfiI MEF and EK demonstrated that the former correlated much better than the latter with the source of C. parapsilosis isolates. Five SfiI classes (I-V) contained only vaginal isolates (or vaginal and three soil isolates, class I), and the blood isolates were distributed between four classes (VI-IX). This relationship was less evident with the EK classes as several of these were composed of both vaginal and blood isolates (B, G, L and M). The three soil isolates were in class A which also included one vaginal isolate. We conclude that SfiI macrorestriction endonuclease patterns seem to be useful in discriminating among C. parapsilosis isolates, with apparent association with source of isolation.

Clinical and mycological evaluation of fluconazole in the secondary prophylaxis of esophageal candidiasis in AIDS patients. An open, multicenter study

A prospective, multicenter, open study of fluconazole prophylaxis was performed in AIDS patients to evaluate the efficacy and toxicity of the drug in preventing relapses of esophageal candidiasis. To this aim, 99 AIDS patients who presented a first episode of clinically and microbiologically confirmed esophageal candidiasis were enrolled in eleven clinical centers scattered throughout the Italian territory. After resolution of this initial esophagitis, all subjects were given fluconazole, 100 mg/die, and followed up for a 6 month period. Only 7 out of the 99 patients enrolled had a relapse ofCandida esophagitis, during a mean follow-up period of 138.5 days. All relapsing patients had CD4+ cell number <100/µl at baseline. Mild side effects were reported in only eight patients. However, 14 of the 27 subjects from whom serial serum samples were available became (12) or remained (2) antigenemic during fluconazole prophylaxis, independently from relapse, suggesting the persistence of tissue-invasive, proliferatingCandida cells. Overall, the data of this study suggest a beneficial effect of prophylactic maintenance therapy with fluconazole againstCandida esophagitis, particularly in the population with >100 CD4+/µl. However, the data onCandida antigenemia in these patients invite the consideration of a relative inefficiency of the drug to eradicate the microrganism from the esophageal tissue.

Recombinant Streptococcus gordonii for mucosal delivery of a scFv microbicidal antibody.

The gram-positive bacterium Streptococcus gordonii was engineered to express the microbicidal molecule H6, which is an antiidiotypic single chain antibody mimicking a yeast killer toxin. S. gordonii is a human commensal which we developed as a model system for mucosal delivery of heterologous proteins. The in vivo candidacidal activity of both H6-secreting and H6-surface-displaying streptococcal strains were assayed in a well-established rat model of vaginal candidiasis. At day 21 full clearance of Candida albicans infection was observed in 75% of animals treated with the H6-secreting strain, and in 37.5% of animals treated with the strain expressing H6 on the surface, while all animals treated with the control strain were still infected. The observed candidacidal effect was comparable with that observed with the antimycotic drug fluconazole. These data confirm the potential of H6 as a candidacidal agent and show how promising is the approach of using recombinant bacteria for mucosal delivery of biologically active molecules.

Use of 65 kDa mannoprotein gene primers in PCR methods for the identification of five medically important Candida species

We have developed PCR and Multiplex PCR assays for the detection of medically important Candida spp. using different species and genus-specific PCR primers selected within the MP65 gene, a recently cloned gene encoding a mannoprotein adhesin. The genus-specific PCR primers were able to amplify Candida species DNA (100% positivity) whereas DNA from all other isolates tested, belonging to other fungal genera, was not amplified. The species-specific PCR primers allowed differentiation of each of five Candida species by the amplicon length produced. No amplicons were detected using species- or genus-specific primers in several bacterial or human DNA templates. The methods described in this study are reproducible, simple and specific. The total time required for each PCR method was less than 4 h from the extraction to the visualized amplicons after PCR. In conclusion, we developed PCR methods to differentiate the five most medically important Candida species using primers directed to the MP65 gene.

The effect of antimycotics on secretory acid proteinase of Candida albicans.

The effect of antimycotics on secretory aspartate (acid) proteinase, a virulence enzyme of Candida albicans, was investigated. The conditions of the study were such as to induce proteinase production in the stationary phase of growth (25-40 hours), when no antifungal tested, except the polyene derivative methyl partricin, significantly reduced the viability of the culture. Among azole derivatives, fenticonazole (FZ) but not miconazole, fluconazole or ketoconazole, exerted strong inhibition on proteinase, in typical dose-diphasic pattern, (0.01 microgram/ml; 1-10 micrograms/ml). 5-fluorocytosine (5-FC) was also inhibitory at a dose interval 1-10 micrograms/ml. In all cases, the inhibition concerned the synthesis of the enzyme rather that its activity as suggested by the results of comparative ELISA, SDS-PAGE and spectrophotometric methods of proteinase detection. Finally, the inhibition of proteinase production by FZ and 5-FC mainly reflected the effect of these antimycotics on general protein synthesis.

Antemortem diagnosis of an apparent case of feline candidiasis

Candidiasis in cats has always been linked with such predisposing factors as parvovirus infections and antibiotic and chemotherapeutic treatments. Moreover these cases were all diagnosed post-mortem.The clinical observations and the diagnostic procedures used in an antemortem case of probable idiopathic intestinal candidiasis in a cat are reported. The therapeutic measures used and the method of evaluating the efficacy of antimycotic treatment are also described.