Influence of gender and age on course of infection and cytokine responses in mice with disseminated Cryptococcus neoformans infection.

OBJECTIVE: To study the influence of gender and age on the course of infection and the cytokine response in a murine model of disseminated cryptococcosis. METHODS: The course of the infection (survival and fungal load in blood and tissues) as well as pro-inflammatory and anti-inflammatory cytokine responses in plasma and organs were compared according to gender and age in outbred mice previously infected with Cryptococcus neoformans NIH52D. RESULTS: Although survival and fungal load were similar in male and female mice, the expression of all cytokines in plasma and of tumour necrosis factor-alpha and interferon-gamma in spleen was significantly increased in female mice compared to male mice in two independent experiments. Young male mice had a significantly shortened survival, were significantly more infected and had predominant tumour necrosis factor-alpha and interferon-gamma responses in comparison with older male mice. CONCLUSION: Host factors should be taken into account when studying the immune response to experimental C. neoformans infection. Our data support epidemiological and clinical data showing differences in susceptibility to cryptococcosis according to gender and age.

Cas1p is a membrane protein necessary for the O-acetylation of the Cryptococcus neoformans capsular polysaccharide.

The capsule is certainly the most obvious virulence factor for Cryptococcus neoformans. The main capsule constituents are glucuronoxylomannans (GXM). Several studies have focused on the structure and chemistry of the GXM component of the capsule, yet little is known about the genetic basis of the capsule construction. Using a monoclonal antibody specific to a sugar epitope, we isolated a capsule-structure mutant strain and cloned by complementation a gene named CAS1 that codes for a putative membrane protein. Although no sequence homology was found with any known protein in the different databases, protein analysis using the PROPSEARCH software classified Cas1p as a putative glycosyltransferase. Cas1p is a well-conserved evolutionary protein, as we identified one orthologue in the human genome, one in the drosophila genome and four in the plant Arabidopsis thaliana genome. Analysis of the capsule structure after CAS1 deletion showed that it is required for GXM O-acetylation.

Immune mediators in cerebrospinal fluid during cryptococcosis are influenced by meningeal involvement and human immunodeficiency virus serostatus.

Pro- and anti-inflammatory mediators (tumor necrosis factor [TNF]-alpha, interleukin [IL]-6, IL-8, IL-10, and soluble TNF receptor II [sTNFR] II) were measured in cerebrospinal fluid (CSF) before treatment (day 0), and after 2 weeks and 3 months of antifungal therapy in 51 human immunodeficiency virus (HIV)-positive and 7 HIV-negative patients with culture-confirmed cryptococcosis. On day 0, all mediator concentrations, except IL-10 in HIV-positive patients, were higher in patients with meningeal, rather than extrameningeal cryptococcosis or in control subjects (P<.05). For meningitis patients, all mediator levels, except sTNFR II, were higher in HIV-negative than HIV-positive patients (P<.05). Day 0 CSF IL-8 levels were higher in HIV-positive patients receiving antiretroviral therapy than in untreated persons (P<.02). Day 0 sTNFR II levels were higher in HIV-positive survivors at 3 months, and elevated levels were sustained in HIV-positive patients with meningitis. Overall, these data support the idea that inflammatory responses are crucial to the eradication of cryptococcal infections in the central nervous system.

Cytokine profiles of AIDS patients are similar to those of mice with disseminated Cryptococcus neoformans infection.

Cryptococcosis is an hematogenously disseminated meningoencephalitis during which the relationship between the disease severity and the immune response remains unclear. We thus analyzed, by enzyme-linked immunosorbent assay, proinflammatory (tumor necrosis factor alpha [TNF-alpha] and interleukin-6 [IL-6]) and anti-inflammatory (IL-10) cytokine levels in plasma at the time of diagnosis in 51 AIDS patients with culture-proven cryptococcosis. We used a murine model to determine the correlation between cytokine levels and fungal burden in blood and tissues and the kinetics of the immune response and of the formation of cerebral lesions. In AIDS patients, plasma TNF-alpha and IL-10, but not IL-6, levels were significantly higher in the case of fungemia or disseminated infection than in their absence, whereas the presence of meningitis had no influence on these levels. In mice, none of these cytokines were detected within the first day after inoculation. Later on, TNF-alpha and IL-10, but not IL-6, levels in plasma correlated significantly with the fungal burden in the blood and spleen but not the brain. In the brain, cytokine levels were low compared to those in other compartments, and tissue lesions and a degree of infection similar to those observed in humans were seen, further suggesting the relevance of this experimental model. Thus, AIDS patients with cryptococcosis produce an immune response that reflects the dissemination but not the meningeal involvement. This murine model of disseminated cryptococcosis can be used to investigate the pathophysiology of cryptococcosis and new therapeutic approaches.

Fungemia during murine cryptococcosis sheds some light on pathophysiology.

We studied fungemia over time in outbred mice infected with Cryptococcus neoformans and looked at its relationship with the intravenous (i.v.) inoculum size, tissue burden and survival. Fungemia was evaluated by culture of 10 microl of peripheral blood from living mice or by culture of buffy coats from sacrificed animals. For all inoculum sizes studied, fungemia could last several weeks after the i.v. inoculation. Individual susceptibility of outbred mice to cryptococcal infection was evidenced by variations in the course, duration and magnitude of fungemia and tissue localizations. These results suggest that the fungus can recirculate after the initial i.v. inoculation. Fungemia, assessed by culture of buffy coats, correlated with the extent of infection in the spleen, lung or brain (P<<0.001) on day 1 after inoculation but only with yeast burden in lung or spleen on day 8, thus demonstrating that brain reacts differently to C. neoformans infection than other organs. Comparison of blood culture techniques and examination of smears suggest that cryptococci might circulate within leucocytes. Finally, quantitative blood cultures may accurately assess the fungal load during experimental cryptococcosis.

Fluconazole, with or without dexamethasone for experimental cryptococcosis: impact of treatment timing.

The time of initiation of fluconazole treatment with or without dexamethasone, and the impact on mycological outcome and drug pharmacokinetics were assessed in a murine model of disseminated cryptococcosis. Non-infected mice and mice with disseminated cryptococcosis were given saline, dexamethasone, or fluconazole +/- dexamethasone, 1 or 8 days after infection. Cfus were counted in tissues, and fluconazole concentrations were determined in plasma and tissues by HPLC and a bioassay. Despite fluconazole tissue and plasma concentrations which were above the minimal inhibitory concentration, the numbers of cfus in brain and lung tissues were reduced after early (P = 0.002 and 0.04, respectively), but not after late fluconazole treatment. The administration of dexamethasone did not have a deleterious effect on the number of cfus, fluconazole pharmacokinetics or antifungal activity. In conclusion, the size of the fungal burden influences the effective level of fluconazole activity in lung and brain. These results strongly suggest that potential antifungal agents should be studied following both early and late administration in experimental cryptococcosis.

Oral transmission of Candida albicans between partners in HIV-infected couples could contribute to dissemination of fluconazole-resistant isolates.

BACKGROUND: Fluconazole resistance has emerged among Candida albicans isolates and has been associated with the prolonged or repeated use of the drug. This study was designed to discover whether transmission of oral isolates could occur between sexual partners and thereby explain fluconazole resistance in patients never treated with the drug. MATERIALS AND METHODS: The oral flora of 10 HIV-infected couples (five heterosexual and five homosexual) were studied. In vitro susceptibility testing and genotyping (restriction fragment length polymorphism with EcoRI and HinfI) were used to delineate strain relatedness for 230 clones (five clones per sample, one to four samples per patient). RESULTS: The genetic diversity of the clones with one DNA subtype was specific to a given patient or a given couple, except in one case in which unrelated patients shared clones of the same genotype. The persistence of clones between partners was stable over time in six out of 10 couples and only transient in one couple. Fluconazole resistance in isolates from patients who had never been treated with azoles was associated in three patients with the first episode of oropharyngeal candidiasis and treatment failure. CONCLUSION: The observation that couples tended to share genetically indistinguishable clones was highly suggestive of transmission between partners. This phenomenon may, in part, explain the pathogenesis of oropharyngeal candidiasis and the increased frequency of fluconazole resistance both in vitro and in vivo.

Fluconazole concentrations in saliva from AIDS patients with oropharyngeal candidosis refractory to treatment with fluconazole.

Fluconazole (FCZ) has been extensively used as a primary therapy for oropharyngeal candidosis in AIDS patients. Clinical resistance to FCZ is now encountered, often related to decreased susceptibility of the isolate in vitro. We wondered if low levels in saliva play a role in the therapeutic failure, especially in patients complaining of dry mouth. Sixteen AIDS patients treated for oropharyngeal candidosis with FCZ were studied. MICs for the isolates were determined. Serum and saliva samples were collected to measure FCZ levels with a bioassay using paper disks loaded with the clinical specimens. We showed that (i) paper disks were convenient for collecting saliva in patients with dry mouth; (ii) levels in saliva depended on the FCZ dosage regimen but did not correlate with the response to therapy; (iii) correlation between concentrations in saliva and serum was poor and independent of clinical response to treatment, other therapies, or decreased salivation; and (iv) levels in saliva were always lower than MICs in patients who failed to respond to treatment. In conclusion, therapeutic failures are more likely to be related to in vitro resistance of the isolate to FCZ or insufficient dosage regimen than to decreased salivary secretion.

[Uncommon fungal maxillary sinusitis of dental origin due to Scedosporium prolificans]. / Une sinusite maxillaire fongique exceptionnelle d'origine dentaire à Scedosporium prolificans.

This is the first case report of an exceptional maxillary infection due to Scedosporium prolificans. This recently discovered fungus was identified in the sinus. In the literature, it has been observed at different locations. Identification requires careful sample taking for mycology and pathology studies emphasizing the importance in maxillary surgery. This pathogenic fungus is very invasive, particularly in immunodepressed or immunocompromised patients. Therapeutic modalities vary with the patient's immune status.

Trichosporon on humans: a practical account.

Six human pathogenic Trichosporon species are described with respect to criteria for routine identification, epidemiology and clinical origin: T. ovoides, T. inkin, T. asahii, T. asteroides (Fissuricella filamenta), T. cutaneum, and T. mucoides. These species are causative agents of white piedra and cutaneous infections and are involved in systemic, localized or disseminated mycoses, particularly in patients with underlying haematological malignancy. Data on in vitro sensitivity to antifungal drugs are provided.

Phylogenetic relationships of Cryptococcus neoformans and some related basidiomycetous yeasts determined from partial large subunit rRNA sequences.

The genus Cryptococcus was found to be heterogeneous on the basis of partial rRNA sequences. The human-pathogenic species C. neoformans, comprising 4 serotypes and having Filobasidiella neoformans and F. bacillispora as teleomorphs, was found at a relatively large distance from Filobasidium. Serotypes B and C had identical sequences, while in A and D they were different, with D closer to B and C than to A. Filobasidiella depauperata, which lacks a yeast-like anamorph, clustered with F. neoformans. The genus Filobasidium was clearly separated from Filobasidiella and clustered with C. albidus, C. kuetzingii, C. gastricus, C. lupi, C. vishniaciae, C. bhutanensis, C. aerius, C. terreus and C. ater. The latter may represent the anamorph of Filobasidium elegans. The orange to red species of Cryptococcus, as well as C. aquaticus and C. yarrowii, were found completely unrelated with these taxa, C. macerans being affiliated to Cystofilobasidium capitatum. The genus Trichosporon was found relatively homogeneous; it includes C. humicola, C. curvatus and the filamentous species Hyalodendron lignicola. Cryptococcus flavus and C. dimennae probably belong to the Tremellales, though distances between these species are large. The positions of C. laurentii and C. luteolus remains to be determined.

Activity of cilofungin (LY 121019), a new lipopeptide antibiotic, on the cell wall and cytoplasmic membrane of Candida albicans. Structural modifications in scanning and transmission electron microscopy.

Cilofungin, a new biosemisynthetic analog of echinocandin B, inhibits the synthesis of beta-(1,3)-glucan resulting in severe modifications of the cell wall and cytoplasmic membrane of sensitive organisms. The morphological modifications to budding yeast cells, pseudomycelium, mycelium and germ tubes of Candida albicans were studied by scanning and transmission electron microscopy after 3 and 16 h exposure to cilofungin. Changes in yeast cell morphology were apparent after 3 h in 0.1 microgram ml-1 cilofungin but were more marked in 1 and 10 micrograms ml-1 cilofungin. Most of the yeasts failed to separate and formed aggregates. Cracks and discontinuities were present in the cell wall and the cell membrane became undulated and fractured. Inclusions into the periplasmalemma space were observed, along with a release of cellular components. An important inhibition of germ tube formation was noted and the structure of true mycelium and pseudomycelium was severely modified. The budding area of yeast cells was particularly susceptible to damage by cilofungin.

Entry of ketoconazole into Candida albicans.

To define characteristics that determine the entry of ketoconazole (KTZ) into Candida albicans cells, we studied the uptake of [3H]KTZ. The cells rapidly and markedly concentrated the drug: 30% of the final 80-fold intracellular concentration was attained in less than 1 min, and greater than 60% was attained in 10 min. Penetration of [3H]KTZ at an extracellular concentration higher than 0.1875 microM (0.1 microgram/ml) occurred by a simple diffusion mechanism. At lower concentrations, accumulation of the drug was an active, energy-requiring process, dependent at least in part on glycolysis, and pH dependent (optimal pH, 6.6). The active transport system had a high binding affinity (Km = 50 nM) and a high maximum velocity of uptake (Vmax = 1.4 mumol min-1 10(-7) cells). It was not possible to displace intracellular [3H]KTZ with high concentrations of unlabeled KTZ or other antifungal agents. These findings suggest that KTZ is rapidly taken up, highly concentrated, and tightly bound to cellular components of C. albicans.

Enzyme-linked immunosorbent assay (ELISA) in the paracoccidioidomycosis. Comparison with counterimmunoelectrophoresis and erythro-immunoassay.

An enzyme-linked immunosorbent assay (ELISA) for detection and quantification of antibodies anti-Paracoccidioides brasiliensis is described. Polystyrene plates have been used as solid phase to absorb P. brasiliensis metabolic yeast phase antigen. Twenty sera of proven paracoccidioidomycosis, 11 of histoplasmosis due Histoplasma capsulatum, 20 of aspergillosis and 20 human normal sera were tested. Ninety-five percent of the paracoccidioidomycosis sera had O.D. superior to 0.150 (from 0.163 to 2.650) at 1/400 serum dilution. ELISA assay was compared with counterimmunoelectrophoresis and erythro-immunoassay tests; a correlation was observed only with erythro-immunoassay. ELISA test should give new perspectives for the serodiagnosis of paracoccidioidomycosis.

Titration of antibodies to Paracoccidioides brasiliensis by erythro-immunoassay (EIA).

The erythro-immunoassay, a new serological procedure in which a hybrid antibody conjugate is able to bind erythrocytes, was used for the titration of antibodies against P. brasiliensis in sera from patients with paracoccidioidomycosis. A peptide-polysaccharide and a lyophilized yeast culture filtrate of P. brasiliensis were used as antigens. Absorption with dead Candida albicans whole cells was necessary to decrease cross reactions observed with heterologous sera. Erythro-immunoassay provides a sensitive system for titration of antibodies in paracoccidioidomycosis with serum dilutions up to 1:102000.

Magnetic enzyme-linked immunosorbent assay (MELISA) for determination of specific IgG in paracoccidioidomycosis.

A magnetic solid phase enzyme-linked immunosorbent assay (MELISA) for quantification of IgG antibodies to somatic and metabolic antigens of Paracoccidioides brasiliensis was developed. Activation of magnetic polyacrylamide agarose beads with concanavalin A was superior to glutaraldehyde activation, and test sensitivity was higher for somatic than for metabolic antigens. Comparative MELISA, counterimmunoelectrophoresis and erythroimmunoassay tests with sera from 33 proven cases of paracoccidioidomycosis, 14 cases of histoplasmosis and 20 normal human sera showed the MELISA could distinguish antibody levels in paracoccidioidomycosis from those in normal sera; however two sera from histoplasmosis cases cross-reacted in the MELISA. MELISA is a rapid test (5-6 h) and the results suggest it has considerable potential value for assay of anti-P. brasiliensis antibodies.