Detection of fungal DNA by real-time polymerase chain reaction: evaluation of 2 methodologies in experimental pulmonary aspergillosis

Abstract

The capabilities of 2 quantitative polymerase chain reaction (PCR) assays for detecting pulmonary aspergillosis were analyzed. Both methodologies were real-time (RT) based and were compared with quantitative cultures and galactomannan (GM) antigen detection in a rabbit model of invasive aspergillosis. A total of 106 samples including blood, serum, lung, and brain from 3 controls and 9 infected New Zealand rabbits were analyzed. The RT-PCR methodologies were an Aspergillus fumigatus-specific assay using fluorescent resonance energy transfer technology targeting a highly conserved region of the fungal 18S rRNA gene and a panfungal assay to amplify the internal transcribed spacer regions 1 and 2 from fungal rRNA gene complex, employing SYBRGreen fluorescent dye as a detector. The specificity for both PCR base assays, culture, and GM determination was 100%. The sensitivity of the specific PCR assay was 88.9% in lung samples, 66.6% in serum, 55.5% in blood, and 33.3 in brain specimens. The panfungal assay had a sensitivity of 33.3% in lung and serum samples, being brain and blood specimens invariably negative. Otherwise, 100% of the lungs resulted positive for culture, and all serum samples showed a GM index above 1.0 after 2 days of infection. The specific RT-PCR assay is a reliable technique to detect A. fumigatus DNA in vivo comparable to cultures and GM determination. The panfungal RT-PCR assay exhibited low sensitivity to diagnose invasive aspergillosis in rabbits advising against its clinical introduction.