Rhizopus oryzae is the most common cause of zygomycosis, a life-threatening infection that usually occurs in patients with diabetic ketoacidosis. Despite standard therapy, the overall rate of mortality from zygomycosis remains >50%, and new strategies for treatment are urgently needed. The activities of caspofungin acetate (CAS) and other echinocandins (antifungal inhibitors of the synthesis of 1,3-␤-D-glucan synthase [GS]) against the agents of zygomycosis have remained relatively unexplored, especially in animal models of infection. We found that R. oryzae has both an FKS gene, which in other fungi encodes a subunit of the GS synthesis complex, and CAS-susceptible, membrane-associated GS activity. Low-dose but not high-dose CAS improved the survival of mice with diabetic ketoacidosis infected with a small inoculum but not a large inoculum of R. oryzae. Fungal burden, assessed by a novel quantitative PCR assay, correlated with increasing inocula and progression of disease, particularly later in the infection, when CFU counts did not. CAS decreased the brain burden of R. oryzae when it was given prophylactically but not when therapy was started after infection. These results indicate that CAS has significant but limited activity against R. oryzae in vivo and demonstrates an inverse dose-response effect. The potential for CAS to play a role in combination therapy against zygomycosis merits further investigation.

Rhizopus oryzae is the most common cause of zygomycosis, a life-threatening infection that usually occurs in immunocompromised patients. A characteristic hallmark of zygomycosis is angioinvasion by the fungus, resulting in thrombosis and subsequent tissue necrosis. Interactions between R. oryzae and vascular endothelial cells are therefore likely of central importance to the organism's pathogenetic strategy. We studied the ability of R. oryzae to adhere to and damage human umbilical vein endothelial cells (HUVECs) in vitro. We report that R. oryzae spores and germ tubes adhere to HUVECs, whereas only spores adhere to subendothelial matrix proteins. Additionally, R. oryzae damages endothelial cells. This endothelial cell damage requires direct contact and subsequent phagocytosis of the fungus. Surprisingly, R. oryzae viability was not required for damage, but phagocytosis was required for dead R. oryzae to cause damage. These results elucidate the nature of R. oryzae-endothelial cell interactions, which are likely central to the angioinvasion and tissue necrosis seen during zygomycotic infections. The fact that dead R. oryzae damage human endothelial cells may, in part, explain the lack of efficacy of fungicidal agents during clinical disease.Rhizopus oryzae is the organism most frequently isolated from patients with zygomycosis (6, 12), a highly destructive and lethal infection in immunocompromised hosts (2, 6, 9, 13). The standard therapy for invasive zygomycosis consists of reversal of the underlying predisposing factors, widespread surgical debridement, and aggressive antifungal medication (2, 6, 13). Unfortunately, despite disfiguring surgical debridement and aggressive therapy with amphotericin B, the overall mortality of zygomycosis remains Ͼ50% (13), and it approaches 100% in patients with disseminated disease (5). Clearly, new strategies to treat zygomycosis are urgently needed.A hallmark of mucormycosis infections is the virtually uniform presence of extensive angioinvasion with resultant vessel thrombosis and tissue necrosis (2,6,9,13). This angioinvasive character is associated with the ability of the organism to spread through viable tissue and to hematogenously disseminate to other target organs. Furthermore, since antifungal agents are carried to the site of infection in the vasculature, adequate blood supply is necessary to deliver these agents to R. oryzae in vivo. Therefore, ischemic necrosis as a result of R. oryzae-mediated angioinvasion is likely an important mechanism by which the fungus survives therapy with fungicidal agents, such as amphotericin B. For these reasons, damage of and penetration through endothelial cells lining blood vessels is likely a critical step in R. oryzae's pathogenetic strategy.We therefore studied the interaction between R. oryzae and endothelial cells. We find that damage to endothelial cells from R. oryzae is dependent upon its adherence to and phagocytosis by endothelial cells. Surprisingly, R. oryzae does not need to be viable to cause endothelial cell damage. ...

The efficacies of liposomal amphotericin B (LAmB) and amphotericin B deoxycholate (AmB) were compared in a diabetic murine model of hematogenously disseminated Rhizopus oryzae infection. At 7.5 mg/kg of body weight twice a day (b.i.d.), LAmB significantly improved overall survival compared to the rates of survival in both untreated control mice (P ‫؍‬ 0.001) and mice treated with 0.5 mg of AmB per kg b.i.d. (P ‫؍‬ 0.047). These data indicate that high-dose LAmB is more effective than AmB in treating murine disseminated zygomycosis.Zygomycosis is a frequently fatal infection that occurs in patients with elevated available levels of iron in serum, such as those treated with deferoxamine, or in patients immunocompromised by diabetic ketoacidosis, organ transplantation, or neutropenia (2, 12). The therapy for invasive zygomycosis includes reversal of the underlying predisposing factors, emergent surgical debridement, and antifungal chemotherapy (5,10,12). Although prospective clinical studies are lacking, amphotericin B deoxycholate (AmB) remains the antifungal therapy of choice for invasive zygomycosis (5, 12), largely because of a historical lack of alternative cidal therapies. Because the fungus is relatively resistant to AmB, high doses are required, frequently resulting in nephrotoxicity and other adverse effects (12). Even when surgical debridement is combined with highdose AmB, the mortality associated with zygomycosis exceeds 50% (12). This mortality rate approaches 100% in patients with disseminated zygomycosis, possibly because surgery to remove the infected foci is not feasible (2). These data emphasize the critical need for more effective antifungal chemotherapy for this lethal infection.The lipid formulations of AmB allow the administration of higher drug doses due to their limited toxicities (1, 4). Scattered case reports have demonstrated successful outcome in patients with zygomycosis treated with lipid-associated AmB (7,8). Since diabetic ketoacidosis represents a major risk factor for the development of zygomycosis infection (5, 12), we used a diabetic mouse model to compare the efficacy of high doses of liposomal AmB (LAmB) against that of AmB in treating hematogenously disseminated zygomycosis caused by Rhizopus oryzae, the most common etiologic pathogen of zygomycosis (11).(This work was presented in part at the 42nd Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, Calif., 27 to 30 September 2002.)R. oryzae 99-880 was obtained from the Fungus Testing Laboratory, University of Texas Health Science Center, San Antonio. This strain was isolated from a brain abscess of a diabetic patient with rhinocerebral zygomycosis. Spores were collected by flooding potato dextrose agar plates (PDA) with 7 ml of endotoxin-free phosphate-buffered saline (PBS) containing 0.01% Tween 80 and gently scrapping the aerial mycelium.Male BALB/c mice (Ն24 g) were rendered diabetic with a single intraperitoneal (i.p.) injection of 210 mg of streptozocin per kg of body weight in 0.2 ml of ice-cold...