Assessment of Aspergillus fumigatus Burden in Pulmonary Tissue of Guinea Pigs by Quantitative PCR, Galactomannan Enzyme Immunoassay, and Quantitative Culture

Abstract

Early diagnosis of invasive pulmonary aspergillosis is problematic in some patient groups due to the lack of rapid, sensitive, specific, and reliable diagnostic tests. Fungal burden and therapeutic efficacy were assessed by survival, quantitative culture (CFU counts), galactomannan enzyme immunoassay (GM-EIA), and quantitative PCR (qPCR) in a new guinea pig model of invasive pulmonary aspergillosis using an aerosol challenge. At 1 day postinfection, qPCR determined that the pulmonary fungal burden was 2 log 10 higher than that determined by CFU counting and increased significantly (P < 0.03) over time. In contrast, the tissue burden assessed by CFU counting did not rise over the course of the study. Therapy with the antifungal drug voriconazole produced statistically significant decreases in pulmonary fungal burden, as detected by CFU counting (P < 0.02), qPCR, and GM-EIA (both P < 0.0002). Daily assessment of the progression of fungal infection in serum was performed by qPCR and GM-EIA. GM-EIA demonstrated a statistically significant reduction in the fungal load on days 6 and 7 in voriconazole-treated animals compared to time-matched controls (P < 0.02). Confirmation of fungal tissue burden by two or more methods should provide a more precise account of the burden, allowing improved assessment of diagnostic and therapeutic strategies in invasive pulmonary aspergillosis.

Interlaboratory and Interstudy Reproducibility of a Novel Lateral-Flow Device and Influence of Antifungal Therapy on Detection of Invasive Pulmonary Aspergillosis

“…]; Mead Johnson, Princeton, NJ) and cortisone acetate (250 mg/kg subcutaneously; Sigma, St. Louis, MO). Additional doses of cyclophosphamide (200 mg/kg) and cortisone acetate (250 mg/kg) were administered on day 3 postinoculation (18). Ceftazidime (100 mg/kg subcutaneously) was administered daily for prevention of bacterial infections.…”

“…Ceftazidime (100 mg/kg subcutaneously) was administered daily for prevention of bacterial infections. Guinea pigs were exposed to A. fumigatus 293 conidia at 1 ϫ 10 8 conidia/ml for 1 h in an aerosol chamber (18,19). In the antifungal treatment, experiment guinea pigs were divided to receive one of five regimens beginning at 1 day postinoculation (n ϭ 8 per group): (i) control, (ii) posaconazole at 10 mg/kg orally (p.o.)…”

“…Tissue fungal burden was measured by enumeration of CFU. Lungs from each animal were weighed, placed into sterile saline containing gentamicin and chloramphenicol, and homogenized, and the number of CFU per gram of lung tissue was determined as previously described (18,21). In addition to serum and BAL fluid, lungs were collected for histopathology.…”

Duality of liver and kidney lesions after systemic infection of immunosuppressed and immunocompetent mice withAspergillus fumigatus

“…Besides quantification of fungal genomic DNA by qPCR, determination of cell wall compounds by chitin assays, CFU determinations, or evaluation of the tissue burden from histopathologic analyses are common. 9 All assays have in common that fungal burden can only be determined from sacrificed animals.…”

Biomarkers for Invasive Aspergillosis: The Challenges Continue

“…BAL fluid has been investigated thoroughly, since it was reported that detection of GM in BAL fluid increased the diagnostic sensitivity from 47% in serum to 85% [66], a comparison of 33 cases found that all 33 BAL specimens, but only nine serum specimens, were positive [67] and that BAL fluid from an established guinea pig model of IA gave a positive result more than 2 days sooner with BAL fluid than with serum [68]. This approach has been investigated for early diagnosis of IA following hematopoietic stem cell transplants [69], after solid organ transplantation [70], in patients with hematological malignancies and pulmonary infiltrates [71], chronic pulmonary disease [72][73][74] as well as in intensive care units [75] and in nonimmunocompromised patients [76].…”