Assessment of Aspergillus fumigatus in guinea pig bronchoalveolar lavages and pulmonary tissue by culture and realtime polymerase chain reaction studies

Abstract

In this study we pursued a diagnostic target in Aspergillus fumigatus (AF) by using qualitative Realtime PCR combined with proprietary DNA primers and a hydrolysis probe specific for this fungal target. Qualitative Realtime PCR is a diagnostic tool that utilizes Realtime PCR technology and detects the presence or absence target specific DNA within a predetermined detection range. Respiratory tissue and fluids from experimentally infected guinea pigs were tested by extracting DNA from the samples which were amplified and detected using AF specific DNA primers and probe. This study included qualitative evaluations of all specimens for the presence of the DNA of AF. The findings in the tissues after AF infection were compared to the numbers of spores in aerosolized samples used to inoculate the animals. Results demonstrated that the specific probe and primer set could detect the presence or absence of AF DNA in the sample. The qualitative detection limit of the assay ranged from 6 × 10(4) copies to 6 copies. Since blood cultures are rarely positive for Aspergillosis, our data indicate that qualitative Realtime PCR, in combination with the appropriate DNA primers and probe can serve as an effective diagnostic tool in the early detection of fungal infections.

Keywords: Aspergillus fumigatus; Realtime PCR; bronchoalveolar lavages.

Figure 1

Comparison of infected groups to numbers of Aspergillus fumigatus colony forming units (CFU) by culture present in the lung.

Figure 2

Aspergillus fumigatus PCR dilution series demonstrating the qualitative detection limit.

Figure 3

Aspergillus fumigatus PCR dilution Standard Curve demonstrating efficiency and linearity.