Development and Validation of a Quantitative Real-Time PCR Assay Using Fluorescence Resonance Energy Transfer Technology for Detection of Aspergillus fumigatusin Experimental Invasive Pulmonary Aspergillosis

ABSTRACTInvasive pulmonary aspergillosis (IPA) is a frequently fatal infection in immunocompromised patients that is difficult to diagnose. Present methods for detection of Aspergillusspp. in bronchoalveolar lavage (BAL) fluid and in tissue vary in sensitivity and specificity. We therefore developed an A. fumigatus-specific quantitative real-time PCR-based assay utilizing fluorescent resonance energy transfer (FRET) technology. We compared the assay to quantitative culture of BAL fluid and lung tissue in a rabbit model of experimental IPA. Using an enzymatic and high-speed mechanical cell wall disruption protocol, DNA was extracted from samples of BAL fluid and lung tissues from noninfected and A. fumigatus-infected rabbits. A unique primer set amplified internal transcribed spacer regions (ITS) 1 and 2 of the rRNA operon. Amplicon was detected using FRET probes targeting a unique region of ITS1. Quantitation of A. fumigatusDNA was achieved by use of external standards. The presence of PCR inhibitors was determined by use of a unique control plasmid. The analytical sensitivity of the assay was =10 copies of target DNA. No cross-reactivity occurred with other medically important filamentous fungi. The assay results correlated with pulmonary fungal burden as determined by quantitative culture (r= 0.72, Spearman rank correlation; P= 0.0001). The mean number of genome equivalents detected in untreated animals was 3.86 log10(range, 0.86 to 6.39 log10) in tissue. There was a 3.53-log10mean reduction of A. fumigatusgenome equivalents in animals treated with amphotericin B (AMB) (95% confidence interval, 3.38 to 3.69 log10; P= 0.0001), which correlated with the reduction of residual fungal burden in lung tissue measured in terms of log10CFU/gram. The enhanced quantitative sensitivity of the real-time PCR assay was evidenced by detection of A. fumigatusgenome in infarcted culture-negative lobes, by a greater number of mean genome equivalents compared to the number of CFU per gram in tissue and BAL fluid, and by superior detection of therapeutic response to AMB in BAL fluid compared to culture. This real-time PCR assay using FRET technology is highly sensitive and specific in detecting A. fumigatusDNA from BAL fluid and lung tissue in experimental IPA.