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-␤-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-␤-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.Life-threatening fungal infections have become more prevalent as the population of immunocompromised patients has increased (6, 7). The value of existing therapies is tempered by such factors as a lack of sufficient spectrum, toxic side effects, or emerging resistance (13, 31, 37). Currently there are several antifungal agents in clinical development, including novel triazoles and members of a new class of compounds that inhibit the synthesis of 1,3-␤-D-glucan, an essential polysaccharide of the fungal cell wall. Caspofungin acetate (MK-0991) is the first compound of this class to be approved for therapeutic use. The development of caspofungin for treatment of aspergillosis has been supported by results from animal models of disseminated or pulmonary disease. The efficacy of caspofungin in t...

Collaborative Cross mice and their power to map host susceptibility toAspergillus fumigatusinfection

“…Postmortem fungal load analysis indicated that the spleen, kidney, and liver, but not the lung or brain, were the target organs for systemic Af293 infection (data not shown). Susceptible mice showed fatigue, confusion, and limited motor activity leading ultimately to paralysis, which could be due to Af invasion of the brain, as is the case in humans (Latgé 1999, Soubani andChandrasekar 2002) and in animal models (Bowman et al 2001). Resistant mice were normally active during the course of infection, with fewer neurological or other deleterious symptoms (data not shown).…”

Chlorine gas exposure increases susceptibility to invasive lung fungal infection

“…Lung A. fumigatus burden was analyzed with real-time PCR measurement of the A. fumigatus 18S rRNA [GenBank accession no. AB008401 (11)] and quantified using a standard curve of A. fumigatus conidia as previously described (36). As a validation of the real-time PCR method, heat-killed A. fumigatus did not yield a signal by real-time PCR and were unable to grow on potato dextrose agar plates (36).…”

Strain-Dependent Variation in 18S Ribosomal DNA Copy Numbers in Aspergillus fumigatus

“…The primers and probe for the A. fumigatus FKS1 gene were designed according to Costa et al (10). The primers and probe for the 18s rDNA sequence were also based on a previous study (6). The FKS1 gene was chosen because it is a known single-copy gene in A. fumigatus involved in ␤(1-3) glucan synthesis (3) and was used as an internal control.…”

“…from a diverse variety of sources, including clinical specimens. Investigators studying IA with animal models routinely use qPCR to measure fungal load (17,27), including response to drug treatment (6,42). Bioaerosol quantitation of Aspergillus spp., particularly in the hospital environment, is also amenable to qPCR (32).…”