A conventional nested PCR and a real-time LightCycler PCR assay for detection of Coccidioides posadasii DNA were designed and tested in 120 clinical strains. These had been isolated from 114 patients within 10 years in Monterrey, Nuevo Leon, Mexico, known to be endemic for coccidioidomycosis. The gene encoding the specific antigen 2/proline-rich antigen (Ag2/PRA) was used as a target. All strains were correctly identified, whereas DNA from related members of the family Onygenaceae remained negative. Melting curve analysis by LightCycler and sequencing of the 526-bp product of the first PCR demonstrated either 100% identity to the GenBank sequence of the Silveira strain, now known to be C. posadasii (accession number AF013256), or a single silent mutation at position 1228. Length determination of two microsatellite-containing loci (GAC and 621) identified all 120 isolates as C. posadasii. Specific DNA was amplified by conventional nested PCR from three microscopically spherule-positive paraffin-embedded tissue samples, whereas 20 human tissue samples positive for other dimorphic fungi remained negative. Additionally, the safety of each step of a modified commercially available DNA extraction procedure was evaluated by using 10 strains. At least three steps of the protocol were demonstrated to sufficiently kill arthroconidia. This safe procedure is applicable to cultures and to clinical specimens.

The in vitro activity of amphotericin B, fluconazole, flucytosine, itraconazole, voriconazole, and posaconazole was evaluated against 44 clinical isolates of filamentous basidiomycetous fungi. No statistically significant differences were noted between Schizophyllum commune (n ‫؍‬ 5), Coprinus species (n ‫؍‬ 8), Bjerkandera adusta (n ‫؍‬ 14), and sterile, uncharacterized basidiomycetes (n ‫؍‬ 17).

Posaconazole (SCH 56592) was tested against 25 strains of Coccidioides immitis to determine their in vitro susceptibilities. The geometric mean 48-h MIC of posaconazole (POSA) was 0.5 g/ml, the MIC range was 0.25 to 1 g/ml, and the MIC at which 50% of the isolates tested are inhibited (MIC 50 ) and the MIC 90 were 0.5 and 1 g/ml, respectively. The geometric mean 48-h MIC of itraconazole (ITRA) was 0.23 g/ml, the MIC range was 0.125 to 0.5 g/ml, and the MIC 50 and MIC 90 were both 0.25 g/ml. Two strains of C. immitis were selected for in vivo studies on the basis of the POSA 48-h MICs for the isolates. POSA orally administered at 0.01, 0.1, 0.5, 1, 5, and 10 mg/kg of body weight/day was compared with ITRA administered at 10 and 30 mg/kg three times a day. The spleens and livers of mice that died or survived to day 50 were removed to measure the fungal burdens. Mice had >90% survival when they were treated with >0.5 mg of POSA per kg or 30 mg of ITRA per kg. Cultures of whole spleens and livers from mice treated with 10 mg of POSA per kg showed >70% sterilization. No sterilization of whole spleens and livers from mice treated with ITRA was seen. POSA displayed potent in vivo activity against the two strains of C. immitis tested.Amphotericin B remains the drug of choice for the treatment of coccidioidomycosis as well as other systemic mycoses. However, amphotericin B is often toxic and is sometimes ineffective (7). Triazoles have been found to be effective in the treatment of threatening human mycoses. Itraconazole (ITRA) and fluconazole are used for the treatment of patients with mild or moderate manifestations of coccidioidomycosis and following treatment with amphotericin B in patients with severe forms of coccidioidomycosis (8). Therapy can be completed with these antifungal agents, but the response rates are only 50 to 60% (2, 9).These unfavorable conditions have prompted a search for more effective alternatives with improved pharmacokinetic parameters and safety. Posaconazole (POSA; SCH 56592) is a broad-spectrum triazole whose activities against a variety of systemic and opportunistic fungal pathogens are under active evaluation (1,4,6,10,12,15,16,17,19). Lutz et al. (11) reported on the activity of POSA in a murine model of disseminated coccidioidomycosis. POSA exhibited greater in vivo activity than fluconazole and ITRA in both survival and tissue burden studies, with doses of 10 and 25 mg/kg of body weight curing high proportions of infected mice. Pharmacokinetic studies with POSA in mice, rats, rabbits, dogs, and monkeys showed that it has good oral bioavailability (14). The half-lives in mice, rats, and rabbits were 7 to 9 h. In contrast, the halflives in dogs and monkeys were 15 and 23 h, respectively.In this study, we determined the in vitro activities of POSA against isolates of Coccidioides immitis and then compared the lowest and highest MICs of POSA in vivo using a murine model. MATERIALS AND METHODSAntifungal agents. Powders of POSA and ITRA for in vitro use were obtained from Schering-Plough Res...

Caspofungin (Merck Pharmaceuticals) was tested in vitro against 25 clinical isolates of Coccidoides immitis.In vitro susceptibility testing was performed in accordance with the National Committee for Clinical Laboratory Standards document M38-P guidelines. Two C. immitis isolates for which the caspofungin MICs were different were selected for determination of the minimum effective concentration (MEC), and these same strains were used for animal studies. Survival and tissue burdens of the spleens, livers, and lungs were used as antifungal response markers. Mice infected with strain 98-449 (48-h MIC, 8 g/ml; 48-h MEC, 0.125 g/ml) showed 100% survival to day 50 when treated with caspofungin at >1 mg/kg. Mice infected with strain 98-571 (48-h MIC, 64 g/ml; 48-h MEC, 0.125 g/ml) displayed >80% survival when the treatment was caspofungin at >5 mg/kg. Treatment with caspofungin at 0.5, 1, 5, or 10 mg/kg was effective in reducing the tissue fungal burdens of mice infected with either isolate. When tissue fungal burden study results were compared between strains, caspofungin showed no statistically significant difference in efficacy in the organs of the mice treated with both strains. A better in vitro-in vivo correlation was noted when we used the MEC instead of the MIC as the endpoint for antifungal susceptibility testing. Caspofungin may have a role in the treatment of coccidioidomycosis.

Thirty isolates of Pseudallescheria boydii were tested to compare the in vitro activity of posaconazole with those of fluconazole and itraconazole, using NCCLS methods. Posaconazole was evaluated in an immunosuppressed mouse model of disseminated pseudallescheriasis. Posaconazole was more effective than itraconazole and as effective as fluconazole in preventing death and significantly reducing the CFU of P. boydii from tissues.In the last few decades, Pseudallescheria boydii has emerged as an important human pathogen, particularly in the immunocompromised host (1,13,14). The diagnosis of invasive pseudallescheriasis is difficult to make histologically, since the tissue specimens show only septate, branching hyphae that resemble Aspergillus. This is a major problem frequently leading to unsuitable therapy, since amphotericin B (AMB) is the treatment of choice for aspergillosis but P. boydii is resistant to AMB.It has been reported that P. boydii exhibits resistance to AMB in vitro and resistance to flucytosine and that many isolates are only moderately susceptible to miconazole, ketoconazole, and itraconazole (ITRA) (3, 7, 10). There have been some reports of successful treatment of fungal infections with miconazole, ketoconazole, ITRA, and the newer triazole voriconazole (2,4,8). Posaconazole (POS) (SCH 56592) is a new antifungal agent currently undergoing clinical development by Schering-Plough Research Institute (Kenilworth, N.J.). POS is a broad-spectrum triazole and has been shown to have potent in vitro and in vivo activity against Candida spp., Cryptococcus neoformans, Aspergillus spp., and numerous other opportunistic fungi that are likely to be encountered clinically (6,11,12).The purpose of this study was to evaluate the in vitro and in vivo activities of POS against P. boydii. We compared the activity of POS with the activities of the triazoles ITRA and fluconazole (FLU) against a series of P. boydii strains using National Committee for Clinical Laboratory Standards (NCCLS) methods. An animal model of disseminated pseudallescheriasis was used to determine the in vivo efficacies of POS and other drugs.Thirty clinical isolates of P. boydii were used in this study. All isolates were obtained from the Fungus Testing Laboratory, University of Texas Health Science Center at San Antonio.Each isolate of P. boydii was grown for 10 days at 35°C on potato flake agar slants. Isolates were evaluated using the NCCLS broth macrodilution proposed standard reference method M38-P for broth dilution antifungal susceptibility testing of conidium-forming filamentous fungi (9). The conidial suspensions were vortexed and adjusted for a transmittance of 68 to 70% at 530 nm in a spectrophotometer (Spectronic 21; Milton Roy Company); this was verified by plating 10 l of each inoculum onto potato dextrose agar plates, incubating the plates at 35°C, and measuring the resulting growth. POS (Schering-Plough Research Institute) and ITRA (Janssen Pharmaceutica, Beerse, Belgium) were dissolved in polyethylene glycol 400 (Sigma, St. Lo...