Efficacy of ravuconazole in a murine model of vaginitis by Candida albicans.

BACKGROUND: The incidence of vulvovaginal candidiasis, a common infection among healthy women primarily caused by the yeast Candida albicans, has increased significantly in recent years. AIMS: The purpose of this study was to compare the efficacy of ravuconazole (RVC) and fluconazole (FLC) in the treatment of experimental C. albicans vaginitis. METHODS: Forty isolates of C. albicans were screened for their in vitro susceptibility to RVC and FLC. A strain of C. albicans that was resistant to FLC (minimum inhibitory concentration [MIC] of >64 µg/ml) was selected for the in vivo study. Treatment regimens for the murine vaginal infection model were (1) 1, 5, 10, and 20 mg/kg RVC once daily, (2) 20 mg/kg RVC twice daily, (3) 20 mg/kg FLC once daily, and (4) 20 mg/kg FLC twice daily. RESULTS: The geometric means of the MIC values at 48 h for all isolates tested were 0.05 and 0.5 µg/ml for RVC and FLC, respectively. Regimens of either RVC or FLC at 20 mg/kg twice daily were more effective to reduce the load of FLC-resistant C. albicans than single dose administration. CONCLUSIONS: Complete eradication of C. albicans from the vagina was not observed with RVC or FLC treatment in the animal model, although RVC treatment showed a lower fungal concentration 14 days after drug administration.

Murine model of disseminated fusariosis: evaluation of the fungal burden by traditional CFU and quantitative PCR.

Systemic disease is the most severe clinical form of fusariosis, and the treatment involves a challenge due to the refractory response to antifungals. Treatment for murine Fusarium solani infection has been described in models that employ CFU quantitation in organs as a parameter of therapeutic efficacy. However, CFU counts do not precisely reproduce the amount of cells for filamentous fungi such as F. solani. In this study, we developed a murine model of disseminated fusariosis and compared the fungal burden with two methods: CFU and quantitative PCR. ICR and BALB/c mice received an intravenous injection of 1 × 10(7) conidia of F. solani per mouse. On days 2, 5, 7, and 9, mice from each mice strain were killed. The spleen and kidneys of each animal were removed and evaluated by qPCR and CFU determinations. Results from CFU assay indicated that the spleen and kidneys had almost the same fungal burden in both BALB/c and ICR mice during the days of the evaluation. In the qPCR assay, the spleen and kidney of each mouse strain had increased fungal burden in each determination throughout the entire experiment. The fungal load determined by the qPCR assay was significantly greater than that determined from CFU measurements of tissue. qPCR could be considered as a tool for quantitative evaluation of fungal burden in experimental disseminated F. solani infection.

PCR assays for identification of Coccidioides posadasii based on the nucleotide sequence of the antigen 2/proline-rich antigen.

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.