Development of a Quantitative PCR Assay to Measure Aspergillus fumigatus Burden in a Murine Model of Disseminated Aspergillosis: Demonstration of the Efficacy of Caspofungin

J. C. BOWMAN, G. K. ABRUZZO, J. W. ANDERSON, A. M. FLATTERY, C. J. GILL, V. B. PIKOUNIS, D. M. SCHMATZ, P. A. LIBERATOR, C. M. DOUGLAS

Author address: 

Merck and Co., Inc., Rahway, NJ.

Abstract: 

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.
2001

abstract No: 

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Full conference title: 

ICAAC 41st
    • ICAAC 41st