Quantitative PCR assay to measure Aspergillus fumigatus burden in a murinemodel of disseminated aspergillosis: Demonstration of efficacy of caspofungin acetate

Caspofungin acetate (MK-0991) is an antifungal antibiotic that inhibits the synthesis of 1,3-beta -D-glucan, an essential component of the cell wall o f several pathogenic fungi. Caspofungin acetate was recently approved for t he treatment of invasive aspergillosis in patients who are refractory to or intolerant of other therapies. The activity of 1,3-beta -D-glucan synthesi s inhibitors against Aspergillus fumigatus has been evaluated in animal mod els of pulmonary or disseminated disease by using prolongation of survival or reduction in tissue CFU as assay endpoints. Because these methods suffer from limited sensitivity or poor correlation with fungal growth, we have d eveloped a quantitative PCR-based (qPCR) (TaqMan) assay to monitor disease progression and measure drug efficacy. A. fumigatus added to naive, uninfec ted kidneys as either ungerminated conidia or small germlings yielded a lin ear qPCR response over at least 4 orders of magnitude. In a murine model of disseminated aspergillosis, a burden of A. fumigatus was detected in each of five different organs at 4 days postinfection by the qPCR assay, and the mean fungal load in these organs was 1.2 to 3.5 log(10) units greater than mean values determined by CFU measurement. When used to monitor disease pr ogression in infected mice, the qPCR assay detected an increase of nearly 4 log(10) conidial equivalents/g of kidney between days 1 and 4 following in fection, with a peak fungal burden that coincided with the onset of signifi cant mortality. Traditional CFU methodology detected only a marginal increa se in fungal load in the same tissues. In contrast, when mice were infected with Candida albicans, which does not form true mycelia in tissues, quanti tation of kidney burden by both qPCR and CFU assays was strongly correlated as the infection progressed. Finally, treatment of mice with induced disse minated aspergillosis with either caspofungin or amphotericin B reduced the A. fumigatus burden in infected kidneys to the limit of detection for the qPCR assay. Because of its much larger dynamic range, the qPCR assay is sup erior to traditional CFU determination for monitoring the progression of di sseminated aspergillosis and evaluating the activity of antifungal antibiot ics against A. fumigatus.