Efficacy of caspofungin and posaconazole in a murine model of disseminated Exophiala infection

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1 Medical Mycology December 2007, 45, Original Articles Efficacy of caspofungin and posaconazole in a murine model of disseminated Exophiala infection ROBERT G. RIVARD, SUZANNE MCCALL, MATTHEW E. GRIFFITH, JOSHUA S. HAWLEY, ROSEANNE A. RESSNER, HIMABINDU BORRA, JAMES E. MOON, MIRIAM L. BECKIUS, CLINTON K. MURRAY & DUANE R. HOSPENTHAL Departments of Medicine and Clinical Investigation, Brooke Army Medical Center, Fort Sam Houston, Texas, USA Introduction Disseminated phaeohyphomycosis is an uncommon infection affecting immunocompetent and immunocompromised individuals in which response to older antifungal agents has been variable. We compared the effect of six days of therapy with caspofungin, posaconazole, and amphotericin B in parallel studies of survival and fungal burden in an immunocompromised mouse model of Exophiala infection. Mice immunocompromised with cyclophosphamide were treated for 6 days starting one day after initiation of infection. Treatment regimens included amphotericin B, caspofungin, and posaconazole. In the survival studies, experimental animals were observed for 14 days. In the fungal burden tests the experimental animals were sacrificed 7 days after infection and brain and kidney burden determined. Treatment with any agent decreased mortality (PB0.05), with 40%, 30%, and 80% observed survival of the animals treated with amphotericin B, caspofungin, and posaconazole, respectively. Amphotericin B and posaconazole treatment resulted in a decrease in fungal burden compared to untreated controls (P B0.05). No reduction in fungal burden was noted in the caspofungin group. All three antifungals evaluated improved survival of immunocompromised mice in this otherwise fatal disseminated phaeohyphomycosis. Amphotericin B and posaconazole reduced fungal burden. Posaconazole and caspofungin appear to have potential for use in treatment of this rare infection. Keywords Systemic fungal infections are an increasing problem in hospitalized patients, especially those who are immunocompromised. Candida and Aspergillus species account for most fungal infections in such patients; however, Received 2 February 2007; Accepted 10 April 2007 This work was presented in part at the 45th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, December Correspondence: Duane R. Hospenthal, Infectious Disease Service (MCHE-MDI), Brooke Army Medical Center, 3851 Roger Brooke Drive, Fort Sam Houston, Texas 78234, USA. Tel: Fax: duane.hospenthal@amedd.army.mil Phaeohyphomycosis, Exophiala, caspofungin, posaconazole, mouse the number of unusual and emerging fungal organisms recovered in these cases is increasing. Phaeohyphomycetes are dark walled (dematiaceous) fungi widely distributed in nature that can cause superficial, subcutaneous, sinus, and central nervous system (CNS) disease in both immunocompromised and immunocompetent individuals. Severe and disseminated infections are usually limited to immunocompromised hosts. Phaeohyphomycetes can also be found on human skin in the absence of clinical disease. Exophiala species are included in this group of fungi and have been reported to cause fungemia, CNS infection, and disseminated disease [16]. Treatment of phaeohyphomycosis, including cases caused by Exophiala species, has had 2007 ISHAM DOI: /

2 686 Rivard et al. poor success and usually includes amphotericin B alone or in combination with other antifungals; infections may require surgery to achieve a cure. Case reports of success with newer antifungals (azoles and echinocandins) have been published but no large trials have been performed [713]. In the present study we evaluated the efficacy of posaconazole, caspofungin, and amphotericin B therapy in immunocompromised mice with disseminated infections caused by Exophiala phaeomuriformis. Materials and methods Three- to four-week-old female ICR (CD-1 ) outbred mice weighing 1824 g were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN). Mice were immunocompromised using intraperitoneal (IP) cyclophosphamide (200 mg/kg) one day prior to and two days after infection. A clinical bloodstream isolate of Exophiala obtained from the University of Texas Health Science Center at San Antonio Fungal Testing Laboratory (San Antonio, TX) was used for the study. The species of this isolate was later identified by DNA sequencing as E. phaeomuriformis. Minimum inhibitory concentrations (MIC) of the study drugs for this organism were low, i.e., amphotericin B, 0.25 mg/ml; caspofungin, 0.06 mg/ml; and posaconazole, 0.06 mg/ml [14]. Aliquots of the organism were stored at 708C until used. These were thawed at room temperature prior to inoculation into yeast extract peptone dextrose broth and incubated at 308C for 4872 h. Samples of the liquid medium cultures were then plated onto solid Sabouraud dextrose agar (SDA) plates (BD, Sparks, MD) and incubated for 4872 h at 358C to produce yeast inoculum. Fungal cells were then harvested from solid media and suspended in normal saline to achieve a final concentration of fungal cells (CFU) per ml. Inoculum concentration was determined using a hemocytometer and verified by quantitative cultures of the suspension before and after infection. Mice were infected with 0.1 ml of the suspension (110 7 fungal cells) via lateral tail vein injection. Preliminary experiments with this inoculum size demonstrated a mortality rate of 6090% with animals dying or becoming moribund between days four and ten after infection. The same inoculum size was used for the survival and fungal burden arms of the experiment. Mice in the treatment groups were administered either conventional amphotericin B, 1 mg/kg/day IP; caspofungin, 10 mg/kg/day IP; or posaconazole, 20 mg/kg/day orally (PO) for six days starting one day after infection. Dosing regimens and routes were selected based on previously reported murine models [1517]. Amphotericin B (Pharm-Tek, Huntington, NY) and caspofungin (Cancidas, Merck & Co., Inc., Whitehouse Station, NJ) were purchased commercially. Posaconazole as oral suspension was obtained from its manufacturer (Schering-Plough Research Institute, Kenilworth, NJ). Response to therapy was evaluated by measuring prolongation of survival and reduction of fungal burden in the brain and kidney in two parallel experiments. One experimental group was observed for survival while the other was sacrificed for fungal burden determination. Each experiment contained three treatment groups (amphotericin B, caspofungin, and posaconazole) and one untreated control group, each composed of 10 mice (40 animals per experiment, 80 total). Subjects in the survival experiment were treated for six days starting one day after infection and then observed until day fourteen. At the end of this observation period all remaining mice were humanely euthanized. Mice in the fungal burden experiment were sacrificed on day seven, 24 h after the last treatment dose to minimize the effect of residual antifungal agent in recovered tissues used for quantitative cultures. The brain and one kidney from each animal were aseptically removed, weighed, and homogenized in 5 ml normal saline. Three 1:10 dilutions were performed, and 0.1 ml of the original homogenate and each dilution was plated on Sabouraud dextrose agar and incubated for five days at 358C. Fungal burdens were calculated from colony counts. Animals in the fungal burden experiment that died or were euthanized before day seven were refrigerated at 2 58C until fungal burden determination. Survival was analyzed using one-way ANOVA, and Student-Newman-Keuls tests. Fungal burden was analyzed using Kruskal-Wallis ANOVA, and Mann-Whitney rank sum tests. The use of animals and protocol were approved by our Institutional Animal Care and Use Committee. Results Treatment with amphotericin B, caspofungin, and posaconazole had a protective effect, prolonging survival in treated subjects (Fig. 1). Forty per cent of subjects survived to the 14th day in the group treated with amphotericin B, 30% in the group treated with caspofungin, and 80% in the group treated with posaconazole. In the untreated control group all subjects died between days four and eight. Using the Student-Newman-Keuls test survival among all treatment groups was significantly improved over that of untreated controls (P B0.05) and survival with posaconazole, but not amphotericin B was significantly different from that of caspofungin (P B0.05).

3 Antifungal therapy of murine Exophiala phaeomuriformis infection 687 Amphotericin B Caspofungin Posaconazole Control Fig. 1 Survival of immunocompromised mice infected with Exophiala phaeomuriformis. Animals were immunocompromised with cyclophosphamide 200 mg/kg one day prior and two days after infection. Solid diamonds, amphotericin B (1 mg/kg/day IP); solid squares, caspofungin (10 mg/kg/day IP); solid triangles, posaconazole (20 mg/kg/day PO); solid circles, controls. Treatment groups received antifungals for six days starting one day after infection In the fungal burden experiment 70% of animals in the group treated with amphotericin B, 50% of those treated with caspofungin, 80% of those treated with posaconazole, and 90% of the untreated controls survived to day seven. At the time of sacrifice, animals in the control group were severely ill (judged by lack of grooming, lack of activity, and weight loss) or moribund compared to the surviving animals in the treatment groups which were only mildly to moderately ill. Although untreated controls in the fungal burden experiment survived one to two days longer than in the survival experiment prior to sacrifice, this was within the range of survival times of four to ten days noted in preliminary experiments. Fig. 2 Fungal burden in the brain (A) and kidney (B) of mice infected with Exophiala phaeomuriformis. Black diamonds each represent one mouse. Percentage survival -1 A 1.00E+07 Colonies per gram 1.00E E E E E+02 B 1.00E+07 Colonies per gram 1.00E E E E E Days after infection (Infection on day 0) Amphotericin B significantly reduced the fungal burden of the brain and kidney (both P B0.01) of treated subjects in comparison to untreated controls (Fig. 2). Treatment with posaconazole also resulted in a significant reduction of fungal burden in these same organs (both P 50.02). Fungal burden was not significantly different between amphotericin B and posaconazole. When comparisons were made excluding the 3 animals that died prior to day 7, amphotericin B still produced a significant reduction in burden compared to control (P 50.01). Removal of the two animals that died early in the posaconazole group resulted in a loss of significance in brain fungal burden change (P 0.07), but not in the kidney (P 50.05). Amphotericin B Caspofungin Posaconazole Control Amphotericin B Caspofungin Posaconazole Control

4 688 Rivard et al. Treatment with caspofungin did not result in a significant reduction in fungal burden in the brain or kidney by either comparison. Discussion There are few studies evaluating the treatment of infection due to Exophiala species in animal models and no clinical trails in humans. The relatively small number of human cases makes clinical trials impractical and unlikely to be performed. The present study was undertaken to evaluate the efficacy of amphotericin B, caspofungin, and posaconazole in the treatment of disseminated infection with Exophiala in an immunocompromised murine model. The three antifungal agents used in our study had a clear protective effect, in each case significantly prolonging the survival of treated animals. The effect was most pronounced with posaconazole. In addition, posaconazole and amphotericin B reduced fungal burdens in the treated animals. Amphotericin B has long been the antifungal of choice for the treatment of serious fungal infections including phaeohyphomycosis. However, outcomes with amphotericin B treatment of phaeohyphomycosis have been poor. Mortality of CNS phaeohyphomycosis was 73% in a review of 101 cases [11]. Four of these cases involved Exophiala species. Three of the four were treated with amphotericin B (one with amphotericin B and itraconazole) with two surviving. The fourth was not treated with antifungals and did not survive. In another review including 72 cases, four of which involved Exophiala species, of disseminated phaeohyphomycosis the overall mortality rate was 79% [12]. All four were treated with amphotericin B (one with amphotericin B and itraconazole), two of which survived. These previously published cases illustrate the poor survival associated with phaeohyphomycosis and the lack of adequate data on optimal therapy. Many of these cases occurred prior to the availability of newer azole (posaconazole and voriconazole) and echinocandin (anidulafungin, caspofungin, and micafungin) antifungal agents. A review of 273 patients with uncommon fungal infections refractory to or intolerant of other licensed antifungal agents reported 66% survival at 90 days with voriconazole therapy [18]. About 75% of these patients had failed to respond to prior antifungal agents. Among this group of patients were five with phaeohyphomycosis (due to Alternaria, Bipolaris, and Exophiala species) who were treated successfully with voriconazole. Another group reported a case of recurrent phaeohyphomycosis due to Exophiala spinifera with five relapses over a ten year period and failure of multiple antifungal agents. Cure was finally achieved after 13 months of therapy with posaconazole [19]. Currently there is little data to support the use of caspofungin (or other echinocandins) in the treatment of infections caused by the dark walled fungi. Although there is in vitro data revealing activity against some of the phaeohyphomycetes, there is not much in vivo data [20]. Caspofungin has been shown to improve survival in a neutropenic rabbit model of pulmonary aspergillosis, but with a paradoxically higher fungal burden [21]. This effect has been postulated to be due to hyphal fragmentation of the organism remaining viable but less invasive after therapy with caspofungin. This effect may account for the increased survival and paradoxical higher fungal burden in animals treated with caspofungin seen in our study. Current human data is very limited, although one published report has documented the use of caspofungin in combination with voriconazole in a patient with cerebral phaeohyphomycosis [22]. Our results appear to support the potential efficacy of caspofungin, but given the lack of activity against the tissue fungal burden we caution against its use in monotherapy without further study. Posaconazole has been shown to have activity in vitro against many agents of phaeohyphomycoses [23]. This new azole has also produced beneficial in vivo results in the experimental treatment of a closely related black mould (Exophiala (Wangiella) dermatitidis), both prolonging survival and reducing fungal burden in the brain and spleen of mice [24]. The current study and recent case reports of successful therapy for phaeohyphomycosis with newer antifungal agents suggest a potential role for posaconazole and caspofungin in the treatment of Exophiala phaeomuriformis and possibly other agents of phaeohyphomycosis. Acknowledgements We would like to thank the Brooke Army Medical Center Department of Clinical Investigation for their support, and specifically John A. Ward for his assistance with statistical analysis of the data. We would also like to thank the University of Texas Health Science Center at San Antonio Fungal Testing Laboratory for providing the fungal isolate. Financial support of this study was provided by Merck & Co., Inc., West Point, PA. Posaconazole was provided by Schering- Plough Research Institute, Kenilworth, NJ.

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