Concentration of antifungal agents within host cell membranes: a new paradigm governing the efficacy of prophylaxis.

Posaconazole prophylaxis has proven highly effective in preventing invasive fungal infections, despite relatively low serum concentrations. However, high tissue levels of this agent have been reported in treated patients. We therefore hypothesized that the intracellular levels of antifungal agents are an important factor in determining the success of fungal prophylaxis. To examine the effect of host cell-associated antifungals on the growth of medically important molds, we exposed cells to antifungal agents and removed the extracellular drug prior to infection. Epithelial cells loaded with posaconazole and its parent molecule itraconazole, but not other antifungals, were able to inhibit fungal growth for at least 48 h and were protected from damage caused by infection. Cell-associated posaconazole levels were 40- to 50-fold higher than extracellular levels, and the drug was predominantly detected in cellular membranes. Fungistatic levels of posaconazole persisted within epithelial cells for up to 48 h. Therefore, the concentration of posaconazole in mammalian host cell membranes mediates its efficacy in prophylactic regimens and likely explains the observed discrepancy between serum antifungal levels and efficacy.

Monitoring and impact of fluconazole serum and cerebrospinal fluid concentration in HIV-associated cryptococcal meningitis-infected patients.

OBJECTIVES: The aim of the present study was to assess fluconazole pharmacokinetic measures in serum and cerebrospinal fluid (CSF); and the correlation of these measures with clinical outcomes of invasive fungal infections. METHODS: A randomized trial was conducted in HIV-infected patients receiving three different regimens of fluconazole plus amphotericin B (AmB) for the treatment of cryptococcal meningitis. Regimens included fluconazole 400 mg/day+AmB (AmB+Fluc400) or fluconazole 800 mg/day+AmB (AmB+Fluc800) (14 days followed by fluconazole alone at the randomized dose for 56 days); or AmB alone for 14 days followed by fluconazole 400 mg/day for 56 days. Serum (at 24 h after dosing) and CSF samples were taken at baseline and days 14 and 70 (serum only) for fluconazole measurement, using gas-liquid chromatography. RESULTS: Sixty-four treated patients had fluconazole measurements: 11 in the AmB group, 12 in the AmB+Fluc400 group and 41 in the AmB+Fluc800 group. Day 14 serum concentration geometric means were 24.7 mg/L for AmB+Fluc400 and 37.0 mg/L for AmB+Fluc800. Correspondingly, CSF concentration geometric means were 25.1 mg/L and 32.7 mg/L. Day 14 Serum and CSF concentrations were highly correlated with AmB+Fluc800 (P<0.001, r=0.873) and AmB+Fluc400 (P=0.005, r=0.943). Increased serum area under the curve (AUC) appears to be associated with decreased mortality at day 70 (P=0.061, odds ratio=2.19) as well as with increased study composite endpoint success at days 42 and 70 (P=0.081, odds ratio=2.25 and 0.058, 2.89, respectively). CONCLUSION: High fluconazole dosage (800 mg/day) for the treatment of HIV-associated cryptococcal meningitis was associated with high serum and CSF fluconazole concentration. Overall, high serum and CSF concentration appear to be associated with increased survival and primary composite endpoint success.

Comparison of three methodologies for the determination of pulmonary fungal burden in experimental murine aspergillosis.

Quantitative culture, quantitative PCR and the galactomannan enzyme immunoassay (EIA) were compared for their ability to determine the pulmonary fungal burden in a murine model of invasive aspergillosis. Quantitative culture of specimens containing hyphae under-represented the absolute fungal burden in established infection when compared with the two other methods. The best correlation was observed between the two non-culture methods. Higher variability was observed with the galactomannan EIA when compared with quantitative PCR. Collectively, these data suggest that quantitative PCR is the preferred method for determination of the pulmonary fungal burden in experimental aspergillosis.

Acetylsalicylic acid reduces vegetation bacterial density, hematogenous bacterial dissemination, and frequency of embolic events in experimental Staphylococcus aureus endocarditis through antiplatelet and antibacterial effects.

BACKGROUND: Platelets are integral to cardiac vegetations that evolve in infectious endocarditis. It has been postulated that the antiplatelet aggregation effect of aspirin (ASA) might diminish vegetation evolution and embolic rates. METHODS AND RESULTS: Rabbits with Staphylococcus aureus endocarditis were given either no ASA (controls) or ASA at 4, 8, or 12 mg. kg-1. d-1 IV for 3 days beginning 1 day after infection. Vegetation weights and serial echocardiographic vegetation size, vegetation and kidney bacterial densities, and extent of renal embolization were evaluated. In addition, the effect of ASA on early S aureus adherence to sterile vegetations was assessed. In vitro, bacterial adherence to platelets, fibrin matrices, or fibrin-platelet matrices was quantified with either platelets exposed to ASA or S aureus preexposed to salicylic acid (SAL). ASA at 8 mg. kg-1. d-1 (but not at 4 or 12 mg. kg-1. d-1) was associated with substantial decreases in vegetation weight (P<0.05), echocardiographic vegetation growth (P<0.001), vegetation (P<0.05) and renal bacterial densities and renal embolic lesions (P<0.05) versus controls. Diminished aggregation resulted when platelets were preexposed to ASA or when S aureus was preexposed to SAL (P<0.05). S aureus adherence to sterile vegetations (P<0.05) or to platelets in suspension (P<0.05), fibrin matrices (P<0.05), or fibrin-platelet matrices (P<0.05) was significantly reduced when bacteria were preexposed to SAL. CONCLUSIONS: ASA reduces several principal indicators of severity and metastatic events in experimental S aureus endocarditis. These benefits involve ASA effects on both the platelet and the microbe.

Endothelial cells, tissue factor and infectious diseases.

Tissue factor is a transmembrane procoagulant glycoprotein and a member of the cytokine receptor superfamily. It activates the extrinsic coagulation pathway, and induces the formation of a fibrin clot. Tissue factor is important for both normal homeostasis and the development of many thrombotic diseases. A wide variety of cells are able to synthesize and express tissue factor, including monocytes, granulocytes, platelets and endothelial cells. Tissue factor expression can be induced by cell surface components of pathogenic microorganisms, proinflammatory cytokines and membrane microparticles released from activated host cells. Tissue factor plays an important role in initiating thrombosis associated with inflammation during infection, sepsis, and organ transplant rejection. Recent findings suggest that tissue factor can also function as a receptor and thus may be important in cell signaling. The present minireview will focus on the role of tissue factor in the pathogenesis of septic shock, infectious endocarditis and invasive aspergillosis, as determined by both in vivo and in vitro models.

Predictors of poor clinical outcome of cryptococcal meningitis in HIV-infected patients.

The aim of this study was to identify baseline prognostic factors for poor clinical outcome of HIV-associated cryptococcal meningitis. We conducted a trial in Thailand and the USA comparing low- and high-dose concomitant use of amphotericin B and fluconazole for HIV-associated cryptococcal meningitis to amphotericin B followed by fluconazole. Subjects who were either alive and cerebrospinal fluid (CSF) culture-positive or dead were considered to have a poor outcome. At day 14, baseline characteristics associated with poor outcome included: low weight, high CSF cryptococcal antigen (CrAg) titre and low CSF white blood cell (WBC) count. At day 70, the associated baseline characteristics included: CSF CrAg titre >1:1024 and low Karnofsky performance status. Overall, consistent with published findings, low weight, high CSF CrAg titre and low CSF WBC counts at baseline were predictors for poor clinical outcome. In addition, we found that low Karnofsky performance status was predictive of poor outcome. Prompt management with appropriate antifungal therapy for this particular group of patients may improve the outcomes.

Interactions of Aspergillus fumigatus with vascular endothelial cells.

Invasive aspergillosis is characterized by two different types of angioinvasion. During pulmonary aspergillosis, hyphae are initially outside of the pulmonary vasculature and they invade the endothelial cell lining of the blood vessels by passing from the abluminal to the luminal surface. Some of these hyphal fragments can break off and circulate in the bloodstream. In severely immunocompromised hosts, these blood-borne hyphal fragments adhere to the luminal surface of the endothelial cells and they penetrate the endothelial cell lining of the vasculature by passing from the luminal to the abluminal surface. We have set up in vitro models of luminal and abluminal endothelial cell invasion by Aspergillus fumigatus. Luminal invasion by hyphae results in both endothelial cell damage and stimulation of tissue factor expression. Abluminal invasion causes less endothelial cell damage than luminal invasion, but greater induction of endothelial cells genes encoding cytokines, leukocyte adhesion molecules and tissue factor. These differences in the endothelial cell response to luminal versus abluminal infection may indicate significant differences in the pathogenesis of hematogenously disseminated versus locally invasive versus aspergillosis.

Current strategies for treating invasive candidiasis: emphasis on infections in nonneutropenic patients.

The increasing incidence of nosocomial candidal infections is a pivotal problem for patients who do not have neutropenia. In contrast with previous principles, candidemia--even in nonneutropenic patients--should be treated with systemic antifungal agents except in rare circumstances. Amphotericin B remains the agent of choice for treatment of hematogenously disseminated candidiasis and for candidemia, although the optimal dosage and duration of this therapy are poorly defined. Therapy with fluconazole is an alternative for patients who are intolerant to amphotericin B; the efficacy of fluconazole compared with that of amphotericin B in hematogenous candidal infections is unknown but is currently being evaluated. Removal of prosthetic devices that are infected with Candida is necessary in nearly all instances to cure the infection. Similarly, removal of an indwelling catheter whose use is associated with candidemia, as opposed to leaving the catheter in place during antifungal therapy, may increase survival rates of patients and lower their rates of complication. Antifungal prophylaxis may be useful for patients who are undergoing transplantation of an organ.

Severe candidal infections in neutropenic patients.

The incidence of candidal infections in patients with cancer is increasing, and drug-resistant fungi are being isolated more frequently. Diagnosis of hematogenously disseminated candidiasis remains difficult. Characteristic clinical presentations, such as endophthalmitis and chronic hematogenously disseminated candidiasis, are inconstant and may not develop until after neutrophil recovery. Blood cultures are insensitive for detecting candidemia. Growth of Candida species in even one blood culture is strongly suggestive of hematogenously disseminated candidiasis. Serological tests to diagnose this disease remain experimental. Whenever feasible, central venous catheters should be removed from patients with candidemia. Amphotericin B is the treatment of choice for acute and chronic hematogenously disseminated candidiasis. The roles of azoles and liposomal amphotericin B in treating these diseases are currently undefined. Prophylactic use of antifungal agents decreases the incidence of documented fungal infections in neutropenic patients but does not improve overall survival and may increase the likelihood of infections by resistant fungi.

Candida albicans adherence to endothelial cells.

Mechanisms of adherence to vascular endothelial cells by microorganisms on a molecular level can be elucidated by using monoclonal antibodies, purified cell wall constituents, and receptor analogues. Since these agents are expensive and available in limited quantities, a microsystem for probing adherence mechanisms to these cells has become essential. We studied techniques to accurately quantify the adherence of L-[35S]methionine-labeled Candida albicans to human umbilical vein endothelial cells in a 96-well microtiter plate system while avoiding specific problems related to Candida coadherence and avid binding to plastic. The endothelial cells were grown on a collagen matrix in individually detachable microwells enabling the determination of the number of adherent organisms from radioactive counts of the entire well. This procedure has the critically important advantage of obviating the need to remove adherent Candida from the wells. Expressing adherence to endothelial cell monolayers as the percentage of total organisms added to each well significantly decreases the variability of the assay.

Eukaryotic translation initiation factor-6 enhances histamine and IL-2 production in mast cells.

Eukaryotic translation initiation factor (eIF)-6 is known to be important in ribosome biogenesis. Previously, we have discovered that eIF-6 mRNA is induced in lung in a murine model of asthma. We also found that there was enhanced eIF-6 expression in mast cells stimulated with PMA plus calcium ionophore. Therefore, we hypothesized that the induction of eIF-6 enhances the production of bioactive mediators by mast cells upon allergic stimulation. In the current study, we found that eIF-6 mRNA was rapidly induced in murine mast cells stimulated by Fc epsilon RI cross-linking, which is a major physiologic stimulant for mast cells. eIF-6 was also induced in human mast cells upon stimulation. The increase in eIF-6 gene expression in murine mast cells was blocked by therapeutic agents such as dexamethasone and cyclosporin A. To determine the location and function of eIF-6, murine mast cells were transfected with a construct that overexpressed enhanced green fluorescent protein-tagged eIF-6. These experiments demonstrated that eIF-6 was localized predominantly in the nucleolus of the mast cells. Also, overexpression of enhanced green fluorescent protein/eIF-6 enhanced the production of histamine and IL-2, but not IL-4 by stimulated murine mast cells. These results suggest that eIF-6 regulates the production of selected bioactive mediators in allergic diseases. This is the first demonstration of a biologic function of eIF-6 in mammalian cells.

Reversible fluconazole resistance in Candida albicans: a potential in vitro model.

To study the development and potential mechanisms of antifungal resistance in relation to antifungal exposure, reversible fluconazole resistance was examined in vitro. Candida albicans ATCC 36082 blastospores were passed in liquid yeast nitrogen base medium containing either 4, 8, 16, or 128 micrograms of fluconazole per ml, and susceptibility testing was performed after each passage. High-level fluconazole resistance (50% inhibitory concentration, > 256 micrograms/ml) developed in the isolates after serial passage in medium containing 8, 16, or 128 micrograms of fluconazole per ml, but not in isolates passed in 4 micrograms of fluconazole per ml. Reduced susceptibility was noted within four to seven passages, which was equivalent to 14 to 19 days of exposure to the drug. However, all isolates returned to the susceptible phenotype after 8 to 15 passages in medium lacking the drug; thus, fluconazole resistance was reversible in vitro. In vivo, organisms retained the resistant phenotype after a single passage in the rabbit model of infective endocarditis. Restriction digest profiles and karyotypic analysis of the parent strain and selected fluconazole-resistant and -susceptible isolates from each group were identical. Investigations into the molecular mechanisms of this reversible resistance failed to reveal increased accumulation of mRNA for 14 alpha-demethylase, the target enzyme for fluconazole, or for the candidal multidrug transporters CDR1 and BENr. This process of continuous in vitro exposure to antifungal drug may be useful as a model for studying the effects of different antifungal agents and dosing regimens on the development of resistance and for defining the mechanism(s) of reversible resistance.

Role of hyphal formation in interactions of Candida albicans with endothelial cells.

The ability to change from yeast to hyphal morphology is a major virulence determinant of Candida albicans. Mutants with defined defects in filamentation regulatory pathways have reduced virulence in mice. However, is it poorly understood why hyphal formation is critical for C. albicans to cause hematogenously disseminated infections. We used recently constructed mutants to examine the role of hyphal formation in the interactions of C. albicans with endothelial cells in vitro. These interactions included the ability of the mutants to invade and injure endothelial cells. Because the formation of hyphae may influence the host inflammatory response to C. albicans, we also investigated the capacity of these mutants to stimulate endothelial cells to express E-selectin and intercellular adhesion molecule 1. We infected endothelial cells with C. albicans strains containing homozygous null mutations in the following filamentation regulatory genes: CLA4, CPH1, EFG1, and TUP1. Whereas the wild-type strain formed true hyphae on endothelial cells, we found that neither the Deltaefg1 nor the Deltacph1 Deltaefg1 double mutant germinated. The Deltatup1 mutant formed only pseudohyphae. We also found that the Deltaefg1, Deltacph1 Deltaefg1, and Deltatup1 mutants had significantly reduced capacities to invade and injure endothelial cells. Therefore, Efg1p and Tup1p contribute to virulence by regulating hyphal formation and the factors that enable C. albicans to invade and injure endothelial cells. With the exception of the Deltacph1 Deltaefg1 mutant, all other mutants stimulated endothelial cells to express at least one of the leukocyte adhesion molecules. Therefore, the combined activities of Cph1p and Efg1p are required for C. albicans to stimulate a proinflammatory response in endothelial cells.

Mechanisms of the proinflammatory response of endothelial cells to Candida albicans infection.

Endothelial cells can influence significantly the host inflammatory response against blood-borne microbial pathogens. Previously, we found that endothelial cells respond to in vitro infection with Candida albicans by secreting interleukin 8 (IL-8) and expressing E-selectin, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1). We have now examined the mechanisms mediating this endothelial cell response. We determined that C. albicans stimulated endothelial cells to synthesize tumor necrosis factor alpha (TNF-alpha), which in turn induced these infected cells to secrete IL-8 and express E-selectin by an autocrine mechanism. Expression of VCAM-1 was mediated not only by TNF-alpha but also by IL-1alpha and IL-1beta, all of which were synthesized by endothelial cells in response to C. albicans. These three cytokines remained primarily cell associated rather than being secreted. Candidal induction of ICAM-1 expression was independent of TNF-alpha, IL-1alpha, and IL-1beta. These observations demonstrate that different proinflammatory endothelial cell responses to C. albicans are induced by distinct mechanisms. A clear understanding of these mechanisms is important for therapeutically modulating the endothelial cell response to C. albicans and perhaps other opportunistic pathogens that disseminate hematogenously.

Interferon-gamma protects endothelial cells from damage by Candida albicans.

Endothelial cells activated with interferon-gamma (IFN-gamma) have been shown to inhibit the replication of Toxoplasma gondii. To determine if this cytokine protects endothelial cells from damage by Candida albicans, human umbilical vein endothelial cells were pretreated with IFN-gamma and infected with C. albicans; endothelial cell damage was measured by the release of 51Cr. Pretreatment with IFN-gamma decreased the extent of endothelial cell injury caused by C. albicans by up to 100% +/- 8.2%. This diminution of endothelial cell damage was confirmed by scanning electron microscopy. The degree of protection was dependent on the concentration of IFN-gamma, with maximum protection occurring at 13 units/mL. Higher concentrations of IFN-gamma were toxic to the endothelial cells. Pretreating the endothelial cells with this cytokine had no effect on candidal germination and growth, suggesting that IFN-gamma stimulates endothelial cells to become resistant to or inhibit the action of candidal virulence factors.

Modulation of interactions of Candida albicans and endothelial cells by fluconazole and amphotericin B.

Using an in vitro model of intravascular infection, we examined the effects of exposure to subinhibitory concentrations of fluconazole and amphotericin B on the ability of Candida albicans to adhere to and damage human umbilical vein endothelial cells. Incubation of the organisms for 18 h in 0.5x the MICs of fluconazole and amphotericin B inhibited endothelial cell adherence by 22 and 91%, respectively (P less than 0.001 for each drug). Candida-induced endothelial cell injury was also decreased by exposing the organisms to the antifungal drugs while in contact with the endothelial cells. Fluconazole inhibited damage by approximately 50% at concentrations ranging from 0.25x to 5x the MIC (P less than 0.01 for each concentration). Exposure to amphotericin B at 0.5x the MIC completely blocked the ability of the organisms to injure endothelial cells. The capacities of the antifungal agents to inhibit endothelial cell injury paralleled their abilities to suppress candidal germination. Organisms exposed to up to 5x the MIC of fluconazole had diminished, but still detectable, germ tube production and elongation, whereas incubation in 0.5x the MIC of amphotericin B completely abrogated germination. In addition to their direct effects on the growth of C. albicans, fluconazole and amphotericin B may decrease the ability of the fungus to disseminate hematogenously by inhibiting the organisms' capacity to adhere to and injure endothelial cells.

New model of oropharyngeal candidiasis in mice.

We established a straightforward murine model of oropharyngeal candidiasis. Mice were immunosuppressed with cortisone acetate, anesthetized, and then inoculated by placing cotton wool balls saturated with Candida albicans sublingually for 2 h. A prolonged, reproducible infection was induced. This model may be useful for antifungal screening or pathogenesis studies.

Cell extracts of Candida albicans block adherence of the organisms to endothelial cells.

Cell extracts of Candida albicans were fractionated by concanavalin A affinity chromatography. Eluted mannosylated proteins (fraction II) and nonbinding, nonmannosylated proteins (fraction I) were collected and assayed directly for inhibition of adherence of C. albicans to endothelium. Fraction II blocked blastospore adherence to endothelial cells. Fraction I blocked both blastospore and germ tube adherence to endothelial cells. Monoclonal antibody OKM-1 (anti-CR3) and an anti-C. albicans monoclonal antibody, CA-A (anti-CR2), reacted in Western blots with proteins from fraction I, suggesting the presence of the CR2- and CR3-like proteins that have been previously identified on C. albicans germ tubes.

Comparison of fluconazole and amphotericin B for treatment of disseminated candidiasis and endophthalmitis in rabbits.

We compared the efficacy of intravenous fluconazole (80 mg/kg of body weight per day) with that of amphotericin B (1 mg/kg/day) for the long-term treatment of endophthalmitis in rabbits with disseminated candidiasis. After 17 days of therapy, fluconazole decreased the fungal colony counts of the choroid-retinas significantly more than did the saline control (P less than 0.05); however, after 24 days of fluconazole therapy, this treatment effect was lost and fluconazole was no more effective than saline. In contrast, treatment for 24 days with amphotericin B reduced the vitreous and choroid-retina fungal colony counts significantly more than either fluconazole or saline (P less than 0.05 for both treatment groups). After 17 days of therapy, indirect ophthalmoscopy revealed less severe eye involvement in both antifungal treatment groups than in saline controls; however, this difference reached statistical significance only for the amphotericin B-treated rabbits (P less than 0.05). Also, there was a trend towards worsening eye lesions, as seen by indirect ophthalmoscopy, in the fluconazole-treated rabbits after 24 days of therapy, which roughly paralleled the quantitative culture results. Despite the presence of negative choroid-retina cultures, some rabbits in all treatment groups had persistently visible eye lesions, indicating that ophthalmoscopic resolution of Candida endophthalmitis may lag behind lesion sterilization. Amphotericin B was superior to fluconazole in the treatment of Candida endophthalmitis in this model.