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Antimicrobial Agents and Chemotherapy, June 2004, p . 2288-2291, Vol . 48, No . 6

Posaconazole Therapy of Disseminated Phaeohyphomycosis in a Murine Model

John R . Graybill,1 Laura K . Najvar,1* Elizabeth Johnson,2 Rosie Bocanegra,1 and David Loebenberg3

Department of Medicine, Division of Infectious Diseases, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3900,1 Public Health Service Mycology Reference Laboratory, Bristol, United Kingdom,2 Schering-Plough Research Institute, Kenilworth, New Jersey 07033-13003

Received 18 July 2003/ Returned for modification 18 August 2003/ Accepted 20 February 2004


   ABSTRACT

 
Immunocompetent (nu/+) and athymic (nu/nu) BALB/c mice were infected intravenously with Wangiella dermatitidis and treated with posaconazole . Posaconazole reduced the counts in tissues and prolonged survival . Of particular interest, posaconazole reduced the counts of this neurotropic pathogen in the brain .


   TEXT

 
Phaeohyphomycosis is a collective term describing infection caused by a number of primarily mycelial fungi which produce a melanin pigment . This pigment gives the colonies a dark color (1) . These dark molds are also called dematiaceous fungi . Many infections involve respiratory sites (sinusitis, pneumonia) or skin and subcutaneous lesions following local inoculation (8, 10, 12, 14, 15, 20, 22) . While many localized infections occur in otherwise healthy individuals, there is a predilection for phaeohyphomycosis in patients with solid-organ transplants or corticosteroid therapy, and a few cases have been reported in patients with AIDS (20, 22, 25) . Infection of immune-suppressed patients suggests that cell-mediated immune defenses may be important for the control of phaeohyphomycosis . Disseminated disease can involve multiple tissues . However, some species, including Wangiella dermatitidis, are neurotropic, in that they have a predilection to produce brain abscesses (2, 10, 15, 16, 20, 21, 24) .

Treatment of phaeohyphomycosis commonly involves amphotericin B . The clinical responses to amphotericin B treatment vary, and amphotericin B treatment often fails (10, 20, 21) . Some patients have been given azole antifungals as primary or salvage therapy . Of the antifungal triazoles, itraconazole is highly effective in vitro and is effective clinically in about two-thirds of patients, including some who have failed amphotericin B therapy (21) . Fluconazole is less effective in vitro, is effective in mice, and is occasionally effective clinically (3, 6, 21) . While debridement and resection of focal lesions may be effective for some patients, these are not always possible for patients with widespread dissemination or brain abscesses . For these patients, other alternatives are needed . Posaconazole (Schering-Plough Research Institute) is a broad-spectrum triazole antifungal drug with broad-spectrum activities, including activities against Aspergillus and many other fungal species (4, 5, 9, 11, 13, 23) . In the present studies, we evaluated posaconazole as an alternative therapy for central nervous system infections caused by W . dermatitidis .

A clinical isolate of W . dermatitidis, isolate 8656, was obtained from Paul Szaniszlo, The University of Texas at Austin (7) . W . dermatitidis yeast-like forms were used for the in vitro studies, in which NCCLS methodology M-38A (17) and 80% inhibition (19) were used . MICs were measured at 24 and 48 h of incubation . The MICs of posaconazole were <0.03 and 0.125 µg/ml . Prior to infection, W . dermatitidis was cultured for up to a week on potato dextrose agar at room temperature . Conidia were harvested with isotonic saline, filtered through glass wool to remove fragments of mycelia, counted with a hemacytometer, suspended in isotonic saline, and injected intravenously into unanesthetized mice in a 0.2-ml volume . The infecting inoculum size was confirmed by counting the colonies from serial dilutions .

Six-week-old BALB/c nu/+ and nu/nu mice were obtained from the South Texas Veterans Health Care System, Audie L . Murphy Division, Veterinary Medical Unit Breeding Colony . Responses were evaluated by using measurements of the prolongation of survival and reductions of the counts in brain and spleen tissues . One day after infection the mice were randomized to groups of 7 to 10 mice each . Mice received orally (p.o.) by gavage 0.5% Noble agar (controls) or posaconazole at 0.1, 0.625, 1, 2, 2.5, 5, 10, or 25 mg/kg of body weight suspended in 0.5% Noble agar p.o . once daily . For the survival studies, treatment was continued through day 7 and the mice were observed through day 30 . For the tissue burden studies, the mice were treated from day 1 through day 7 and the survivors were killed 1 day later . The brains and spleens were removed and homogenized in 2 ml of saline . Aliquots of undiluted suspensions and serial 10-fold dilutions of suspensions were plated on Sabouraud dextrose agar, and quantitative counts were calculated . Care was given to using the same intensity and duration of homogenization for each specimen by using a mechanical stirrer . The log rank test was used to assess survival . The Mann-Whitney U test was used for comparisons of counts in tissue . A P value <=0.05 was used for significance .

Figure 1A summarizes the survival data for nu/+ immunocompetent mice . The results of three studies are included . There were 30 control mice, 20 mice each in the groups treated with posaconazole at 5 and 25 mg/kg, and 10 mice each in the additional treatment groups . Posaconazole at >=2.5 mg/kg/day significantly prolonged survival (P > 0.001) . There was a sharp increase from no effect at 0.1 and 1 mg/kg/day to similarly strong levels of protection at 2.5 mg/kg/day and higher doses . Two additional studies with posaconazole were done with athymic mice . The pooled results are presented in Fig . 1B . There were 25 mice in the control group and 10 mice each in the additional posaconazole treatment groups . Posaconazole at >=2.5 mg/kg prolonged survival (P < 0.0001) . The results for the nu/+ and the athymic mice are remarkably similar .


FIG . 1 . Survival study results . (A) Results for nu/+ mice from three studies . The infecting doses ranged from 3.6 x 106 to 3.3 x 107 CFU/mouse . The mice were treated with posaconazole (Posa) at 0.1, 1, 2.5, 5, 10, or 25 mg/kg p.o . for 7 days and were observed for survival through day 30 postinfection . Controls received 0.5% Noble agar p.o . The mice treated with >=2.5 mg/kg/day survived significantly longer than the control mice . (B) Results for athymic mice from three studies . The infecting doses ranged from 9 x 106 to 1.9 x 107 CFU/mouse . Mice were treated with posaconazole at 0.625, 1.25, 2.5, or 5 mg/kg p.o . for 7 days and were observed for survival through day 30 postinfection . Controls received 0.5% Noble agar p.o . The mice treated with >=2.5 mg/kg/day survived significantly longer than the control mice.

 
The burdens in the brain tissues of nu/+ mice are presented in Fig . 2A . The results of two studies in which similar inocula were used were pooled . The results for the mice receiving the two doses of posaconazole were compared with those for the untreated controls . Both regimens reduced the counts in brains significantly compared with those in the brains of the controls (P < 0.0015) . The burdens in spleen tissues are presented in Fig . 2B . Only posaconazole at 25 mg/kg was tested in this study, and it showed a protective effect (P < 0.004) .


FIG . 2 . Tissue W . dermatitidis counts . (A) Brain W . dermatitidis counts . Mice from two studies were infected with 3.6 x 106 to 1.9 x 107 CFU/mouse and were treated from days 1 to 7 . When possible the brains were harvested from mice that succumbed during treatment, and the brains were harvested from all survivors on day 8 . Regimens included posaconazole (Posa) at 2 and 25 mg/kg . Controls received 0.5% Noble agar . Treatment with posaconazole at both doses was superior to the control treatment . (B) Spleen W . dermatitidis counts . Groups of seven nu/+ mice were infected with 1.9 x 107 CFU/mouse and were treated from days 1 to 7 . When possible the spleens were harvested from mice that succumbed during treatment, and the spleens were harvested from all survivors on day 8 . Controls received 0.5% Noble agar . Treatment with posaconazole at 25 mg/kg was superior to the control treatment.

 
The present studies were undertaken to evaluate posaconazole as a possible alternative to amphotericin B for the treatment of Wangiella infections . Posaconazole was consistently effective in the most severe test, prolongation of survival after administration of a lethal inoculum . Posaconazole showed a dose-dependent effect, being highly efficacious at doses of >=2.5 mg/kg/day . Posaconazole was also highly effective in athymic mice . This suggests that immune deficiency may not blunt the beneficial effect of posaconazole treatment . Because the worst site of fungal infection is usually the central nervous system, because W . dermatitidis is neurotropic in humans, and because we could readily detect infection of the brain in untreated control mice, we elected to measure the density of infection in brain tissue . Studies of the burden in brain tissue gave results similar to those obtained in the survival study . Increasing doses of posaconazole showed dose-associated reductions in the counts in brain tissue . The burden in spleen tissue was also reduced by use of a dose of 25 mg/kg .

Relatively little attention has been paid to preclinical studies of phaeohyphomycosis with animal models . There are essentially no data from preclinical studies with voriconazole, although Perfect et al . (18) have found clinical responses in all 11 patients treated with this drug . Several of these patients had central nervous system involvement . Preclinical studies with Ramichloridium mackenzii, another agent with a strong neurotropism, showed that the response to treatment with posaconazole was excellent (2) . The present studies extend this observation to another pathogenic species . However, there are very few data from clinical studies with posaconazole for the treatment of phaeohyphomycosis . In a recent presentation at the 43rd Interscience Conference on Antimicrobial Agents and Chemotherapy (P . Pittisuttithum, V . Gaono-Flores, R . Negroni, I . Sanne, J . R . Graybill, A . Bustamante, R . Hare, and C . Hardalo, Abstr . 43rd Intersci . Conf . Antimicrob . Agents Chemother., abstr . M-978, p . 450, 2003), it was shown that patients with a variety of mycoses responded to posaconazole treatment . Two responders were patients with phaeohyphomycosis . The present studies suggest that posaconazole may be broadly effective against phaeohyphomycetes . Posaconazole prolonged survival and significantly reduced the brain W . dermatitidis counts . The use of posaconazole for the treatment of phaeohyphomycosis, including those patients with central nervous system lesions, is worth exploring in humans .

 


   ACKNOWLEDGMENTS

 
We thank the representatives of Schering-Plough Research Institute for providing posaconazole for these studies .


   FOOTNOTES

 
* Corresponding author . Mailing address: Department of Medicine, Division of Infectious Diseases, Medicine/ID Mail Code 7881, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78229-3900 . Phone: (210) 567-0990 . Fax: (210) 567-0962 . E-mail: najvar{at}uthscsa.edu .


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