Background: In animal models of aspergillosis, assessment of colony forming units (CFU) in organs may underestimate fungal load. Filamentous hyphal masses can produce single colonies when spread and grown on agar plates. A real-time quantitative PCR (TaqMan® ) method has been developed and was used to measure Aspergillus fumigatus burden and evaluate the efficacy of caspofungin, a novel inhibitor of 1,3-beta-D glucan synthesis, in a murine model of disseminated aspergillosis. Methods: A disseminated A. fumigatus infection was induced in C5'-deficient DBA/2 mice by I.V. inoculation of conidia from strain MF5668. Therapy was immediately initiated with either 1.0 mg/kg/dose caspofungin (n=30), 0.5 mg/kg/dose amphotericin B (AmB; n=30), or vehicle (n=60) administered I.P., b.i.d., for 5 days. At selected days up to 35 days post-infection, kidneys removed from euthanized mice (3/group) were homogenized and evaluated for both CFU and A. fumigatus DNA target sequence. Total DNA prepared from homogenates was analyzed using an ABI PRISM® 7700 instrument with PCR primers and probe complementary to the A. fumigatus 18S rRNA gene. Results: When used to monitor disease progression in vehicle-treated infected mice, the TaqMan® assay detected an increase of nearly 4 log10 conidial equivalents/g kidney between days one and four following infection, with a peak burden that coincided with the onset of significant mortality. Traditional CFU methodology detected a marginal increase in fungal load in these same tissues. Treatment with caspofungin or AmB reduced the A. fumigatus burden in kidneys to the limit of TaqMan® detection. Conclusion: Because of its larger dynamic range, the TaqMan® assay is superior to traditional CFU determination for monitoring the progression of disseminated aspergillosis and evaluating the activity of antifungal antibiotics against A. fumigatus.
Full conference title:
- ICAAC 41st