Detection and measurement of fungal burden in a guinea pig model of invasive pulmonary aspergillosis by novel quantitative nested real-time PCR compared with galactomannan and (1,3)-β-D-glucan detection

Abstract

We developed and assessed the diagnostic value of a novel quantitative nested real-time (QNRT) PCR assay targeting the internal transcribed spacer region of ribosomal DNA (rDNA) in a guinea pig model of invasive pulmonary aspergillosis. Groups of 5 immunosuppressed animals that were infected using an aerosol chamber with Aspergillus fumigatus conidia were humanely terminated 1 h postinoculation and at days 3, 5, 7, and 11 postchallenge, and lung tissue, bronchoalveolar lavage (BAL) fluid, whole blood, and serum samples were collected. The QNRT PCR results obtained with the serum and BAL fluid were compared to those achieved with galactomannan and (1→3)-β-d-glucan assays. High fungal burden levels were detected by QNRT PCR in both lung tissue and BAL fluid in all infected animals at each time point, and the sensitivity of each assay in BAL fluid was 100% by day 3 and remained so through the remainder of the study. The sensitivity of detection of fungi in whole blood and serum samples was significantly lower, and some samples remained negative by all three assays despite the advanced stage of the infection. From these data, we can conclude that this novel QNRT PCR method was highly sensitive for the detection of A. fumigatus from different types of samples in this model. In addition, BAL fluid samples appeared to be the most suitable for the early diagnosis of invasive pulmonary aspergillosis. When testing serum, the use of a combination of available assays may increase the possibility of early detection of this opportunistic mycosis.

Fig 1

ClustalW multiple-sequence alignment of the region detected by tested QNRT PCR. The primers for the external round (solid lines) and the primers and TaqMan-MGB probe (dashed-lines) are boxed. The GenBank accession numbers for the sequences shown are as follows: A. fumigatus, AY214446; A. flavus, AY214444; A. niger, AY213632; P. chrysogenum, AY213669; P. commune, AY213672; C. cladosporioides, AY213640. The asterisks indicate 100% sequence similarity among all presented species.

Fig 2

Comparison of fungal loads detected in BAL fluid samples with single-round and nested PCRs. Filled circles denote nested PCR data. Empty circles denote single-round PCR data.

Fig 3

Time courses of fungal burdens as detected by all three methods. Results are presented as means ± standard deviations. (A) QNRT PCR detection in BAL fluid (gray), lung tissue (white), and whole blood (dashed boxes). (B) QNTR-PCR detection in serum. (C) GM detection in BAL fluid (gray) and serum (white). (D) BG detection in BAL fluid (gray) and serum (white).