The in vivo activities of posaconazole, itraconazole, and amphotericin B in neutropenic mice with zygomycosis were compared. The in vitro MICs of posaconazole and itraconazole for the strains of Mucor spp. used in this study ranged from 0.125 to 8 g/ml and 0.25 to 8 g/ml, respectively. The in vitro MIC range for amphotericin B is 0.125 to 0.25 g/ml. At twice-daily doses of >15 mg/kg of body weight, posaconazole prolonged the survival of the mice and reduced tissue burden.Zygomycosis is a relatively uncommon but highly aggressive fungal infection that affects diabetics, neutropenic patients, and subjects with burns or iron overload but that only rarely affects healthy people (15,19). Illness may rapidly progress with angioinvasion and tissue infarction. The existing methods for treatment are often ineffective (4, 9, 17). Presently, therapy utilizes aggressive surgical measures and high doses of amphotericin B. There are scattered reports of lipid-associated amphotericin B in salvage, but there is no evidence that these forms are more efficacious than amphotericin B (2, 3, 5). Even with aggressive therapy, mortality is often above 50% (6). Of the alternative antifungals, itraconazole has been effective in vitro, particularly against Absidia spp. One mouse study has also shown some activity against Absidia, but the clinical evidence of efficacy is less clear (2, 6, 10, 16; E. Dannaoui, J. Meletiadis, J. Meis, J. W. Moulton, and P. E. Verweij, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 939, 2000). Posaconazole is a new broad-spectrum triazole with activity against many filamentous fungal pathogens (1,8,12,18). This study describes the in vivo activity of posaconazole against three Mucor spp. isolates in a neutropenic-mouse model.Three clinical Mucor isolates were tested in vitro by the NCCLS microdilution method for triazoles adapted to filamentous fungi (11). The endpoint was a visual MIC at which azoles were seen to reduce growth by 80% compared to that in the drug-free control tube. The MIC of amphotericin B was taken as the least drug producing a visually clear tube. MICs of posaconazole and itraconazole at 48 h were 0.125 and 0.25 g/ml for Mucor ramosissimus strain 98-1763, 0.25 and 0.25 g/ml for Mucor ramosissimus 95-2650, and 8 and 8 g/ml for Mucor circinelloides 00-1194, respectively. The 48-h MIC of amphotericin B was 0.25 g/ml for all three isolates. The isolates were grown on potato flake agar plates at room temperature for 1 week. They were harvested by scraping the plates with sterile isotonic saline and filtering the suspension through glass wool. The inoculum was calculated by determining hemacytometer counts.For the mouse model, we used 18-week-old BALB/c males. One day before infection, mice were rendered neutropenic with single doses of 5-fluorouracil administered intravenously at 150 mg/kg of body weight and with cyclophosphamide administered intraperitoneally at 200 mg/kg. In groups of five uninfected mice, this treatment reduced the neutrophil count from a median pretreatm...

A patient with azole-refractory thrush-esophagitis responded initially to caspofungin, but the treatment eventually failed. In a murine model, caspofungin was effective against two early isolates for which the MICs of caspofungin were low, but it was less effective against a late isolate for which the MIC of caspofungin was greater. We concluded that there is a correlation between in vivo failure and rising in vitro caspofungin MICs.

Early diagnosis of invasive pulmonary aspergillosis is problematic in some patient groups due to the lack of rapid, sensitive, specific, and reliable diagnostic tests. Fungal burden and therapeutic efficacy were assessed by survival, quantitative culture (CFU counts), galactomannan enzyme immunoassay (GM-EIA), and quantitative PCR (qPCR) in a new guinea pig model of invasive pulmonary aspergillosis using an aerosol challenge. At 1 day postinfection, qPCR determined that the pulmonary fungal burden was 2 log 10 higher than that determined by CFU counting and increased significantly (P < 0.03) over time. In contrast, the tissue burden assessed by CFU counting did not rise over the course of the study. Therapy with the antifungal drug voriconazole produced statistically significant decreases in pulmonary fungal burden, as detected by CFU counting (P < 0.02), qPCR, and GM-EIA (both P < 0.0002). Daily assessment of the progression of fungal infection in serum was performed by qPCR and GM-EIA. GM-EIA demonstrated a statistically significant reduction in the fungal load on days 6 and 7 in voriconazole-treated animals compared to time-matched controls (P < 0.02). Confirmation of fungal tissue burden by two or more methods should provide a more precise account of the burden, allowing improved assessment of diagnostic and therapeutic strategies in invasive pulmonary aspergillosis.

Cochleates are lipid-based supramolecular assemblies composed of natural products, negatively charged phospholipid, and a divalent cation. Cochleates can encapsulate amphotericin B (AmB), an important antifungal drug. AmB cochleates (CAMB) have a unique shape and the ability to target AmB to fungi. The minimal inhibitory concentration and the minimum lethal concentration against Candida albicans are similar to that for desoxycholate AmB (DAMB; Fungizone). In vitro, CAMB induced no hemolysis of human red blood cells at concentrations of as high as 500 g of AmB/ml, and DAMB was highly hemolytic at 10 g of AmB/ml. CAMB protect ICR mice infected with C. albicans when the agent is administered intraperitoneally at doses of as low as 0.1 mg/kg/day. In a tissue burden study, CAMB, DAMB, and AmBisome (liposomal AmB; LAMB) were effective in the kidneys, but in the spleen CAMB was more potent than DAMB at 1 mg/kg/day and was equivalent to LAMB at 10 mg/kg/day. In summary, CAMB are highly effective in treating murine candidiasis and compare well with AmBisome and AmB.

In vitro studies have demonstrated that anidulafungin has greater potency than caspofungin against Candida glabrata. However, data from in vivo studies demonstrating that it has superior efficacy are lacking. The objective of this study was to compare the activities of anidulafungin and caspofungin against C. glabrata in a murine model of disseminated candidiasis. Two clinical C. glabrata isolates were used, including one with reduced caspofungin susceptibility. MICs were determined by broth microdilution in the presence and absence of sera. For the animal studies, mice were immunosuppressed with 5-fluorouracil one day prior to intravenous inoculation. Treatment with anidulafungin and caspofungin (0, 0.5, 1, 5, and 10 mg/kg of body weight per day) was begun 24 h later and was continued through day 7 postinoculation. The CFU were enumerated from kidney tissue. According to the standard microdilution methodology, anidulafungin had superior in vitro activity. However, this enhanced potency was attenuated by the addition of mouse and human sera. Caspofungin reduced the kidney fungal burden at lower doses compared to that achieved with anidulafungin in mice infected with the isolate with the lower MIC. Against the strain with the elevated caspofungin MIC, both anidulafungin and caspofungin were effective in reducing the kidney fungal burden at the higher doses studied. Despite the greater in vitro activity of anidulafungin in the absence of sera, both echinocandins were similarly effective in reducing the fungal burden in kidney tissue. The superior in vitro activity of anidulafungin did not confer enhanced in vivo efficacy against C. glabrata.

Previous in vivo studies have reported caspofungin dose escalation to be effective against Candida glabrata with reduced susceptibility. We hypothesized that higher doses of caspofungin would be effective against invasive candidiasis caused by the more virulent species Candida albicans, including isolates resistant to this echinocandin. Immunocompetent mice were inoculated with one of three C. albicans isolates, including one susceptible and two resistant isolates with different FKS1 hot spot 1 point mutations. Mice received daily caspofungin treatment for 7 days and were then followed off therapy for 2 weeks to assess survival. Kidney tissue and blood were collected, and fungal burden and serum (133) survival). In contrast, caspofungin doses as low as 1 mg/kg improved survival (85 to 95%) and reduced tissue burden and (133)-␤-D-glucan concentration against the susceptible isolate (ATCC 90028). These data suggest that caspofungin dose escalation for invasive candidiasis may not be consistently effective against resistant C. albicans isolates, and this may be associated with the virulence of the strain.Echinocandins are safe and effective for the treatment of invasive candidiasis. In clinical trials at approved doses, these agents have been shown to have response rates of between 70 and 75% (15,20,25,26). Overall, the toxicity profile of these agents is quite favorable, with reported adverse event rates similar to that of fluconazole and lower than those of amphotericin B formulations (15,20,29). In fact, a recent study demonstrated that daily doses of caspofungin of 150 mg, or three times the recommended daily dose of 50 mg, were safe and well tolerated (3). Similarly, high doses of micafungin have also been shown to be relatively well tolerated, with few toxicities in different patient populations, including patients with hematologic malignancies undergoing stem cell transplantation and children (12,30,31).As is common with most antimicrobials, isolates of different fungal species have developed resistance to echinocandins.

Evaluating new therapeutic agents for invasive aspergillosis requires animal models that are reproducible among different laboratories. We therefore evaluated a murine model of aerosol infection in two independent laboratories and found a high level of both intra-and interlaboratory reproducibility of survival, fungal burden over time, and the efficacy of liposomal amphotericin B.Murine models of invasive pulmonary aspergillosis have been invaluable for the assessment of novel therapeutics and diagnostics, as well as for the study of disease pathogenesis. A wide variety of these models have been described, including intravenous (3, 6), intranasal (5), intratracheal (13), and inhalation (10-12) infections. Although the availability of such a diverse group of model systems is useful for many specific questions, the methodological differences among models have made direct comparisons of individual studies difficult. Furthermore, there have been very few studies of the interlaboratory variability of results obtained with the same animal model. Demonstration of interlaboratory reproducibility is a critical benchmark in the development of a standardized model of invasive aspergillosis. The availability of such a standardized model would provide a useful benchmark for the evaluation of new diagnostic and therapeutic strategies across geographically separated laboratories.We recently described a simple and reproducible animal model of invasive pulmonary aspergillosis in which mice are infected by inhalation using an aerosol chamber (10). To evaluate the reproducibility of this model, we used it to compare the time course of mortality and fungal burden and the efficacy of liposomal amphotericin B in two different laboratories.Strains and culture conditions. Aspergillus fumigatus Af293 was used for all studies. To obtain conidia for the infections, organisms were grown for 10 days on Sabouraud dextrose agar at 37°C. Conidia were harvested by flooding the plate with phosphate-buffered saline supplemented with 0.1% Tween 80 and concentrated by centrifugation to the desired concentration (10).Animal model. Male BALB/c mice, 20 to 22 g (National Cancer Institute) were used for all experiments. Mice were infected in both laboratories by inhalation in an acrylic aerosol chamber using an identical protocol as described previously (10). Briefly, mice were immunosuppressed with cyclosphosphamide (250 mg/kg on day Ϫ2, relative to infection, and 200 mg/kg on day ϩ3) and cortisone acetate (200 mg/kg on day Ϫ2 and day ϩ3). On the day of infection, mice were exposed in an acrylic chamber for 1 h to an aerosol generated from 12 ml of phosphate-buffered saline supplemented with 0.1% Tween 80 containing 10 9 A. fumigatus conidia per ml. Mice were monitored daily and sacrificed when moribund. To prevent bacterial infection, all immunosuppressed mice received ceftazidime (5 mg/day subcutaneously) from days 1 to 6 after infection. For treatment studies, liposomal amphotericin B (Ambisome; Fujisawa) was administered daily at 10 mg/kg/d by int...

Ramichloridium obovoideum ("Ramichloridium makenziei") is a rare cause of lethal cerebral phaeohyphomycosis. It has been, so far, geographically restricted to the Middle East. BALB/c mice were inoculated with two strains of R. obovoideum intracranially. Therapy with amphotericin B, itraconazole, or the investigational triazole SCH 56592 was conducted for 10 days. Half the mice were monitored for survival and half were killed for determination of the fungal load in brain tissue. Recipients of SCH 56592 had significantly prolonged survival and lower brain fungal burden, and this result was found for mice infected with both of the fungal strains tested. Itraconazole reduced the brain fungal load in mice infected with one strain but not the other, while amphotericin B had no effect on brain fungal concentrations. This study indicates a possible role of SCH 56592 in the treatment of the serious cerebral phaeohyphomycosis due to R. obovoideum.Dematiaceous fungi, the agents of phaeohyphomycosis, cause a variety of clinical syndromes (9). These vary from superficial skin infection to lethal cerebral disease (4,6,8). Several species of the dematiaceous fungi are neurotropic, i.e., have a predilection for central nervous system tissue, causing brain lesions and/or abscesses. These include Cladophialophora bantiana, Wangiella dermatitidis, Chaetomium spp., Exophiala spp., Curvularia spp., Bipolaris spp., and a few other species (1, 2, 4, 5).Ramichloridium obovoideum ("Ramichloridium mackenziei") is a rare cause of cerebral phaeohyphomycosis. It seems to be geographically restricted, as all cases have been reported from the Middle East or natives of the Middle East who lived in other countries (3,7,11). Reported cases have involved both immunocompromised and apparently immunocompetent patients. The infection was lethal for almost all patients with reported cases of infection, despite surgery and, in some cases, antifungal therapy. In the study described here we developed a murine model of infection with R. obovoideum and tested currently available (amphotericin B, itraconazole) and investigational (SCH 56592; Schering-Plough Research Institute Inc., Kenilworth, N.J.) antifungal agents in vitro and in an experimental murine model. MATERIALS AND METHODSPathogen. R. obovoideum ("R. mackenziei") 653 (gift from S. Al-Hedaithy, Riyadh, Saudi Arabia) and 95-1147 (Fungus Testing Laboratory, San Antonio, Tex.) were used for the in vitro testing and the experimental infection of BALB/c nu/ϩ mice. R. obovoideum 653 was also used to establish infection in ICR mice. R. obovoideum strains were grown on potato dextrose agar (PDA) medium plates (BBL, Baltimore Biologics, Cockeysville, Md.) for 14 days at 37°C. Surface growth was washed with sterile 0.85% normal saline and was filtered through layers of sterile glass wool. Homogeneous suspensions of the appropriate conidial concentration were prepared by using hemacytometer counts. The conidial suspension was administered in 0.2 ml for the intravenous inoculations and in 0.06 ml for the int...