Quantitative PCR assay to measure Aspergillus fumigatus burden in a murine model of disseminated aspergillosis: demonstration of efficacy of caspofungin acetate

Abstract

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 of several pathogenic fungi. Caspofungin acetate was recently approved for the treatment of invasive aspergillosis in patients who are refractory to or intolerant of other therapies. The activity of 1,3-beta-D-glucan synthesis inhibitors against Aspergillus fumigatus has been evaluated in animal models 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 developed a quantitative PCR-based (qPCR) (TaqMan) assay to monitor disease progression and measure drug efficacy. A. fumigatus added to naïve, uninfected kidneys as either ungerminated conidia or small germlings yielded a linear 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 progression 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 infection, with a peak fungal burden that coincided with the onset of significant mortality. Traditional CFU methodology detected only a marginal increase in fungal load in the same tissues. In contrast, when mice were infected with Candida albicans, which does not form true mycelia in tissues, quantitation of kidney burden by both qPCR and CFU assays was strongly correlated as the infection progressed. Finally, treatment of mice with induced disseminated 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 superior to traditional CFU determination for monitoring the progression of disseminated aspergillosis and evaluating the activity of antifungal antibiotics against A. fumigatus.

FIG. 1

Quantitation of A. fumigatus conidia or mycelium in mouse kidneys by the CFU and qPCR methodologies. (A) Conidia spiking. (B) Mycelia spiking. Kidney homogenates containing either conidia or mycelia were prepared with 2.5 mL of saline added (CFU; ○) or with 3.6 volumes of saline added per gram of kidney (qPCR; ▵). The qPCR signal represents 2^ΔC_T (A) or CE per gram of kidney (B). CFU are expressed per gram of kidney in both panels.

FIG. 2

Disease progression in A. fumigatus-infected DBA/2J mice monitored by percent survival, CFU, and qPCR analysis. Mice infected with 9.8 × 10⁵ CFU of A. fumigatus were monitored for percent survival (○; n = 60, 23 censored animals), log₁₀ CFU per gram of kidney (□), or log₁₀ CE per gram of kidney (▴). SDA plates were used for CFU enumeration. CFU and CE are the mean ± standard error of three mice, with the exception of CE for days 4 and 6 (n = 5).

FIG. 3

Progression of infection in mice with disseminated candidiasis. DBA/2J mice were infected with C. albicans and assessed for percent survival (○) by the Kaplan-Meier technique (20) or for kidney burden by both a CFU (□) and a qPCR (▴) assay as described in Materials and Methods. Error bars indicate standard errors.

FIG. 4

Effect of caspofungin or AmB on A. fumigatus kidney burden versus that in infected control mice. Animals were infected as described in the legend to Fig. 2. Therapy was administered as described in Materials and Methods. Values (mean ± standard error of three mice unless noted) are log₁₀ CFU per gram of kidney (A) or log₁₀ CE per gram of kidney (B) in the presence of caspofungin therapy (▴), AmB therapy (□), or no therapy (○). The double-headed arrow indicates the duration of the treatment period. On days 4 and 6, five mice from the nontreated group were used for qPCR analysis; on day 35, two mice from the AmB-treated group were used.