Biomedical subjects

F De Bernardis

Publications and source records attributed to F De Bernardis.

At least 37 records · Page 2Linked to original sources

PubMed1996

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.

Blood↗

PubMed1996

Elevated aspartic proteinase secretion and experimental pathogenicity of Candida albicans isolates from oral cavities of subjects infected with human immunodeficiency virus.

Isolates of Candida albicans from the oral cavities of subjects at different stages of human immunodeficiency virus (HIV) infection or uninfected controls were examined for (i) production of aspartic proteinase(s), a putative virulence-associated factor(s); (ii) the presence in the fungal genome of two major genes (SAP1 and SAP2) of the aspartic proteinase family; and (iii) experimental pathogenicity in a murine model of systemic infection. It was found that the fungal isolates from symptomatic patients secreted, on average, up to eightfold more proteinase than the isolates from uninfected or HIV-infected but asymptomatic subjects. This differential property was stably expressed by the strains even after years of maintenance in stock cultures. Moreover, representative high-proteinase isolates were significantly more pathogenic for mice than low-proteinase isolates of C. albicans. The characters high proteinase and increased virulence were not associated with a single molecular type or category identifiable through DNA fingerprinting or pulsed-field electrophoretic karyotype, and both SAP1 and SAP2 genes were present in both categories of isolates, on the same respective chromosomes. In conclusion, our data suggest that during HIV infection more-virulent strains or biotypes of C. albicans which are identifiable by direct analysis of virulence determinants are selected. It also appears that the biotype switch to increased aspartic proteinase and virulence properties occurs before the HIV-infected subject enters the symptomatic stage and overt AIDS.

Animals↗

PubMed1996

[Comparison of the effects of fenticonazole and econazole on the aspartic proteinase secreted by Candida albicans].

In this note, the effect of fenticonazole on secretory aspartic proteinase of the human opportunistic fungus Candida albicans is shown, in a comparison with econazole. Both antigenic and enzymatic assays demonstrate that fenticonazole, in contrast to econazole, greatly reduces the production of the virulence enzyme by stationary-phase C. albicans. This inhibitory effect was specific and was not mediated by the inhibition of fungal growth. These results confirm fenticonazole's unique property, already shown in previous studies.

Antifungal Agents↗

PubMed1995

Biotype diversity of Candida parapsilosis and its relationship to the clinical source and experimental pathogenicity.

Environmental, vaginal, and blood isolates of Candida parapsilosis were biotyped by karyotype analysis in pulsed-field gel electrophoresis. Morphotype and resistotype were also determined as was aspartyl proteinase secretion and pathogenicity in a systemic mouse infection model. Overall, the karyotype patterns consisted of 6-9 chromosome bands (> 3.0-0.6 Mb) with limited clustering, since most isolates had unique chromosome profiles. Major clusters C. parapsilosis, differing by source of isolation and in experimental pathogenicity, could be discriminated by morphoresistotyping. The morphotypes of isolates from subjects with candidemia ranged from one that caused elevated mortality in the normal mouse to those that were totally avirulent in the neutropenic animal. Among clinical isolates, secretion of aspartyl proteinase was higher in vaginitis than in candidemia isolates and did not correlate with the experimental pathogenicity. These results emphasize the biotype diversity of C. parapsilosis and have potentially important epidemiologic and pathologic implications.

Animals↗

PubMed1995

Expression of Candida albicans SAP1 and SAP2 in experimental vaginitis.

Several strains of Candida albicans were compared for their ability to cause vaginal infection in a rat model, and their vaginopathic potentials were correlated with the expression of two aspartyl proteinases genes (SAP1 and SAP2) and adherence in vivo to the vaginal epithelium. Dot blot reactions and Northern blot analysis with RNA extracted from the vaginal fluid of rats infected with the highly vaginopathic strains H12 and 10261 demonstrated the expression of both SAP1 and SAP2 during the first week of infection. In contrast, neither gene was expressed during infection by a nonvaginopathic strain (N), even though the organism could be recovered during the first 24 h postinfection. A moderately vaginopathic strain (P) also expressed both genes, but the level of SAP1 mRNA appeared to decrease prior to that of SAP2. Neither gene was expressed, even by the highly vaginopathic strains, after the first week of infection, concomitant with a decrease in the number of organisms recovered from the vaginas. Analysis of in vivo adherence showed that the nonvaginopathic strain (N) adhered to vaginal epithelial cells less readily than the highly vaginopathic strain (H12) and moderately vaginopathic strain (P). Thus, in addition to its inability to express SAP1 and SAP2 in vivo, the nonvaginopathic strain does not colonize host cells to the same extent as the other strains tested. Our results demonstrate the early in vivo expression of two aspartyl proteinase gene during candidal vaginitis and suggest its association with the establishment of a vaginal infection.

Animals↗

PubMed1995

Rats clearing a vaginal infection by Candida albicans acquire specific, antibody-mediated resistance to vaginal reinfection.

Oophorectomized, estrogen-treated rats were susceptible to experimental vaginal infection by Candida albicans. After spontaneous clearing of the primary infection, the animals were highly resistant to a second vaginal challenge with the fungus. The vaginal fluid of Candida-resistant rats contained antibodies directed against mannan constituents and secretory aspartyl proteinase(s) of C. albicans and was capable of transferring a degree of anti-Candida protection to naive, nonimmunized rats. This passive protection was mediated by the immunoglobulin fraction of the vaginal fluid and was substantially abolished by preabsorption of the vaginal fluid with C. albicans, but not with Saccharomyces cerevisiae, cells. Vaginal anti-mannan antibodies were also produced by active immunization with heat-killed cells of C. albicans or with a mannan extract when administered via the vaginal route. The protection conferred was comparable to that resulting from clearing of the primary infection. In summary, the data suggest that acquired anticandidal protection in this vaginitis model is mediated at least in part by antibodies, among which those directed against the mannan antigen(s) might play a dominant role.

Animals↗

PubMed1994

Idiotypic intravaginal vaccination to protect against candidal vaginitis by secretory, yeast killer toxin-like anti-idiotypic antibodies.

The principles of idiotypic (Id) vaccination were used to immunize against vaginitis caused by Candida albicans, a widespread and sometimes intractable disease in women. To this aim, a murine mAb (KT4, IgG1) neutralizing in vitro the anti-Candida activity of a yeast killer toxin (YKT) was used as an Id vaccine to elicit Abs with toxin-like activity in a rat vaginitis model. Nonimmunized and isotype-matched, irrelevant mAb-immunized rats served as controls. An effective protection was obtained in Id-vaccinated animals, as demonstrated by a highly significant decrease in vaginal Candida CFU compared with controls. The protection was associated with rising vaginal titers of anti-idiotypic Abs (IdAb), prevalently of the IgA isotype, that were able to passively transfer the protective state to nonimmunized animals. The vaginal IdAb possessed YKT-like activity because they were able to kill in vitro the challenging fungal cells, and this killing was neutralized by the mAb KT4. Overall, these data demonstrate that secretory IdAb elicited by intravaginal Id vaccination with mAb KT4 protected the rats from the infectious challenge with Candida albicans by molecular mimicking YKT activity as its internal image.

Animals↗

PubMed1994

Prospective study of Candida colonization, use of empiric amphotericin B and development of invasive mycosis in neutropenic patients.

The association between colonization with Candida spp., subsequent occurrence of invasive candidiasis and empiric use of amphotericin B was investigated prospectively in 139 neutropenic patients with hematologic malignancies. Treatment with amphotericin B was required in 67% of patients colonized in multiple non-contiguous body sites (multicolonized) versus 31% of patients colonized in single or contiguous sites (monocolonized) and in 21% of non-colonized patients (p = 0.0037 and p = 0.00026, respectively). Invasive candidiasis was documented in 22.2% of multicolonized versus 4.8% of monocolonized patients and in none of the non-colonized patients (p = 0.035 and p = 0.0036, respectively). Analysis of the spectrum of colonizing Candida spp. showed that multicolonized subjects were colonized with increased frequently by Candida albicans compared to monocolonized subjects, and that the association between multicolonization, invasive candidiasis and amphotericin B usage was statistically significant in patients colonized by Candida albicans but not in patients colonized by other Candida species. The association between Candida multicolonization and the occurrence of Candida infection seems to be confirmed by a double-blind placebo-controlled study performed in a small subgroup of the multicolonized patients treated with fluconazole.

Adolescent↗

PubMed1994

Modulation of cell surface-associated mannoprotein antigen expression in experimental candidal vaginitis.

The monoclonal antibody (MAb) AF1 recognizes an oligosaccharide epitope present on highly immunogenic and immunomodulatory mannoproteins (MP) of Candida albicans. The expression of this epitope (AF1-MP) during experimental candidal vaginitis was studied in two strains of C. albicans (3153 and CA-2) which were equally vaginopathic but differed in the mode of hypha formation in the vagina. In both strains, immunofluorescence of vaginal samples, taken 1 h after challenge, revealed an intense, MAb AF1-specific labelling of the yeast cells. This labelling was very scarce in fungal cells taken at 24 h and on subsequent days during the development of filamentous forms. Electron-microscopic gold immunolabelling observations showed that molecules carrying AF1-MP spanned the entire cell wall in the initial yeast cells but were absent on the cell surface and in the outermost, capsular layer of the cell wall of the germ tubes and filamentous forms. In both strains, at any time and for any form of intravaginal growth, AF1-MP was clearly expressed in the cytoplasm and cytoplasmic vesicles, and was fully incorporated into the inner layers of the cell wall. As seen by immunofluorescence, the vaginal fluid from C. albicans-infected rats did not hinder the expression of AF1-MP on the yeast cells surface in vitro. In electron-microscopic gold immunolabelling, a hypha-specific MAb (3D9) labelled the surface of the hyphal but not of the yeast cells of C. albicans harvested from rat vagina. Overall, these data strongly suggest that cell surface expression of MP antigen is modulated during intravaginal growth and morphogenesis of C. albicans.

Animals↗

PubMed1993

Idiotypic vaccination: immunoprotection mediated by anti-idiotypic antibodies with antibiotic activity.

Anti-Id antibodies were raised in mice against a monoclonal antibody (MoAb KT4) that neutralized the in vitro activity of a Pichia anomala yeast killer toxin. Monoclonal antibody was administered to BALB/C syngeneic mice with different schedules of immunization before intravenous challenge with increasing amounts of yeast killer toxin-sensitive Candida albicans cells. The course of candidosis was studied in comparison with mice non-immunized and immunized with an isotype-matched unrelated MoAb subdivided into control groups. Protection was reflected by statistically significant increases in survival rate of mice immunized with MoAb KT4 which showed variable serum levels of yeast killer toxin-like anti-Id antibodies. MoAb KT4 affinity chromatography purified mouse anti-Id antibodies were capable of killing in vitro the yeast cells of the Candida albicans strain used for the experimental infection. This is the first report of antimicrobial protection that exploits the role of anti-idiotypic antibodies presumably acting in vivo as antibiotics (idiotypic vaccination).

Animals↗

PubMed1993

Filamentous growth and elevated vaginopathic potential of a nongerminative variant of Candida albicans expressing low virulence in systemic infection.

The vaginopathic potential and the intravaginal morphology of a nongerminative variant of Candida albicans, strain CA-2, were studied in a rat vaginitis model. Although it expressed low virulence in systemic infections, strain CA-2 was capable of causing a vaginal infection of the same duration and extent as that obtained in rats challenged with the germ-tube-forming strain C. albicans 3153 from the stock collection or with a fresh clinical isolate of C. albicans from a case of human vaginitis. During the experimental infection, the CA-2 cells did not maintain their yeast morphology but gave rise to single enlarged-elongated elements (1 to 2 days) which grew predominantly as coarse, short, pseudomycelium-like filaments (2 to 3 days) and then as long threads (7 days). These latter filaments were ultimately indistinguishable from the hyphal filaments formed by the germ-tube-forming strains, which, however, initially developed in the vagina by typical germ tube formation. This peculiar morphological development of strain CA-2 was not observed in organs of systemically infected mice, where, in contrast to strain 3153 which formed typical hyphae, strain CA-2 maintained a typical pattern of yeast growth. Vaginal isolates of strain CA-2 taken at different days of infection were found to be identical to the challenging CA-2 cells, in terms of biochemical characteristics, inability to form germ tubes in any medium at 37 degrees C in vitro, echinocandin resistance, DNA biotype, and low virulence in systemic infections in mice. Thus, experimental vaginitis by strain CA-2 is associated with a peculiar filamentous growth in the vagina, through an apparently novel morphological development bypassing classical germ tube formation but ultimately leading to ordinary hyphae. The elevated vaginopathic potential of strain CA-2, in contrast to its low virulence in systemic infection, also suggests that different Candida virulence factors (and host responses) come into play in local and disseminated candidal infections.

Acute Disease↗

PubMed1993

Use of a monoclonal antibody in a dot immunobinding assay for detection of a circulating mannoprotein of Candida spp. in neutropenic patients with invasive candidiasis.

A dot immunobinding assay for the detection of a circulating mannoprotein (MP) antigen of Candida species in the sera of neutropenic patients in a hematological setting is described. The technique is based on the use of a monoclonal antibody which recognizes an oligomannoside epitope shared by distinct MP of pathogenic Candida species. The sensitivity of the assay for antigen detection in serum was between 2 and 5 ng/ml, and MPs from Candida albicans, Candida tropicalis, Torulopsis glabrata, and Candida parapsilosis, but not Candida krusei, could be detected. A retrospective analysis of sera from patients with proven invasive candidiasis versus sera from controls (Candida-colonized and noncolonized subjects) revealed that the novel assay has sufficient sensitivity, specificity, and predictive values to be of potential diagnostic significance.

Antibodies, Monoclonal↗

PubMed1992

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 (less than 400 x 10(6)/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.(ABSTRACT TRUNCATED AT 250 WORDS)

Acquired Immunodeficiency Syndrome↗

PubMed1992

Fluconazole treatment of catheter-related right-sided endocarditis caused by Candida albicans and associated with endophthalmitis and folliculitis.

An unusual case of catheter-related right-sided endocarditis, endophthalmitis, and extensive folliculitis, apparently caused by a single DNA biotype of Candida albicans, was successfully treated with a 6-month course of fluconazole plus two intravitreous doses of amphotericin B. The patient was a 21-year-old man who underwent colectomy for diffuse polyposis and developed the clinical syndrome just described following total parenteral nutrition for the treatment of purulent anal fistulas. Fluconazole was initially administered at a daily dose of 200 mg, with 600 mg daily given after 4 weeks. Clinical improvement resulted, with no relapse during 14 months of follow-up. Sequential measurements by an enzyme-linked immunosorbent inhibition assay demonstrated that levels of circulating mannoprotein antigen of C. albicans fell from 75 ng/mL to less than 1 ng/mL after the institution of fluconazole therapy. These observations seem to confirm previous reports on the efficacy of fluconazole as sole therapy for candidal endocarditis and suggest a role for serological studies in clinical monitoring of severe candidal infections.

Adult↗

PubMed1991

DNA fingerprinting and electrophoretic karyotype of environmental and clinical isolates of Candida parapsilosis.

The endonuclease restriction pattern (DNA fingerprinting) and the electrophoretic karyotype of 16 Candida parapsilosis isolates from environmental and clinical sources were investigated. DNA from both whole cells and separated mitochondria was digested with enzymes, including EcoRI, BamHI, KpnI, BglII, HpaII, PvuII, and HindIII. Regardless of their source and pathogenic properties, all isolates showed a uniform, reproducible, and overlapping whole-cell DNA fingerprinting with each endonuclease digest. Mitochondrial DNA fragments were, in all cases, major contributors to the total cellular DNA restriction pattern. In contrast, the electrophoretic karyotype generated by rotating field gel electrophoresis (RFGE) or contour clamped homogeneous field electrophoresis (CHEF) showed a remarkable polymorphism among the isolates. This polymorphism concerned the smaller molecular size section of the karyotype (range, 1.8 to 0.7 Mb), where at least two to five chromosomal bands could be consistently detected by both RFGE and CHEF. Larger (greater than or equal to 3.0 to 1.9 Mb) chromosome-sized DNA bands (four in CHEF and three in RFGE) were quite distinct and common to all isolates. Thus, seven karyotype classes could be defined, on the basis of both the number and size of putative chromosomes. The three categories of isolates (soil, vaginal, and hematological) were not randomly distributed among the seven classes. In particular, the four hematological isolates had a karyotype pattern which was clearly distinct from that shown by the three environmental isolates, and of the nine vaginal isolates only one shared a class with isolates from another source (soil). Although tentative, the classification was totally consistent with the independent and reproducible results obtained by the two pulse-field electrophoretic techniques employed. It is suggested that the electrophoretic analysis of the karyotype might be particularly useful for epidemiological and pathogenicity studies on biotypes of C. parapsilosis.

Candida↗

PubMed1991

Vaginopathic and proteolytic Candida species in outpatients attending a gynaecology clinic.

Non-pregnant, non-diabetic outpatients were examined for the presence of pathogenic vaginal yeasts to determine if a correlation existed between a specific yeast and clinical disease. Yeasts were isolated as single vaginal species from 186 of 228 subjects with clinically diagnosed candidal vaginitis, as well as from 122 out of 380 asymptomatic, age-matched controls. Apart from Candida albicans and C glabrata, other prevalent species were C krusei, C parapsilosis and Saccharomyces cerevisiae which accounted for 9.2%, 6.0% and 5.4%, and 9.0%, 2.4% and 19.7%, of yeasts from patients and carriers, respectively. Only C albicans and C parapsilosis were significantly more common in those with vaginitis. Only the isolates of these two species secreted aspartyl proteinase in vitro, and the amount of the enzymes secreted by the isolates from patients was significantly higher than that secreted by the isolates from carriers. These two species consistently produced vaginal infection in pseudoestrus rats, whereas none of the non-proteolytic species tested (C glabrata, C krusei, and S cerevisiae) colonised the vagina in these rats. Proteinase secretion correlated with experimental vaginal infection; it could also be a reliable factor for distinguishing clinically active infection from asymptomatic fungal carriage.

Adult↗

PubMed1990

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.

Antifungal Agents↗

PubMed1990

Evidence for a role for secreted aspartate proteinase of Candida albicans in vulvovaginal candidiasis.

The presence of the secretory aspartate (acid) proteinase in the vaginal fluid of candidal vaginitis patients and controls was studied by ELISA and immunoblot (Western blot). In addition, a proteinase-deficient mutant strain of Candida albicans (IR24) was compared with the wild-type parent strain (10261) for ability to infect the vagina of pseudoestrus rats under estradiol treatment. Among the 67 women examined, proteinase was detected only in 22 harboring C. albicans (range, 42-233 ng/ml of vaginal fluid), at concentrations significantly higher in the 14 vaginitis patients than in the 8 asymptomatic fungal carriers. Western blots confirmed the presence of only one protein band of approximately 43 kDa, corresponding to that of the purified proteinase, in the ELISA-positive vaginal fluids. Experimental vaginal infection was significantly more extensive and persistent in rats infected with the proteinase-producer strain than in those challenged with the proteinase-deficient mutant, and the enzyme was detected in the vaginas of the former but not of the latter animals. Both strains 10261 and IR24 developed hyphal forms to a roughly similar extent during infection, and both showed a comparable adherence in vitro to vaginal and buccal epithelial cells. The clinical and experimental evidence support a role for secretory proteinase as a virulence factor in the pathogenesis of candidal vaginitis.

Adult↗