Diagnostic of Aspergillosis from Bronchoalveolar Lavage Samples by Combining a Mechanical DNA Extraction and a TaqMan-Based Realtime PCR

M. RUHNKE1, C. SCHEER 1, J. Hí„NFLER 1, C. A. SCHMIDT 1, G. MASCHMEYER 1, S. SCHWARTZ 2

Author address: 

1Charite, Berlin, Germany, 2Benjamin Franklin Hospital, Berlin, Germany.

Abstract: 

Background: Different DNA-extraction methods and PCR assays are available for detecting Aspergillus fumigatus DNA in bronchoalveolar lavage (BAL) samples of patients with invasive aspergillosis. These methods are often time consuming and emphasize the need to develop a rapid DNA Isolation assay. Furthermore, they generally do not give quantitative results that could be used to control antifungal therapy. Methods: We have developed a new rapid assay which yields results within six hours. This was achieved by combining high-speed cell disruption using a commercially available mechanical extraction procedure (FastPrep) with a realtime PCR assay based on TaqMan technology. In addition to being fast, this combination also produces quantitative results due to the nature of the TaqMan assay. Quantification was achieved by comparing to DNA derived from plasmids. For the realtime PCR, newly designed primers and a probe specific for Aspergillus fumigatus were used. Results: The new method was used to measure 108 BAL-samples from three different sets of patients with either proven, suspected or excluded aspergillosis. In patients with proven aspergillosis 16 samples from 10 patients were tested positive for Aspergillus fumigatus DNA with a median of 21168 copies per µl. One of the patients was tested at two different times and showed a 20-fold reduced result (66 vs. 1325 copies per µl) after antifungal therapy. In patients with suspected aspergillosis 15 samples from 16 patients were tested positive with a median of 18 copies per µl while 24 samples were tested negative. Out of 68 samples from patients with excluded aspergillosis 4 were tested positive with a median of 7 copies per µl. Conclusions: Quantification of Aspergillus-DNA in BAL may correlat with infection and may be used for monitoring. Aspergillus species other than Aspergillus fumigatus are not detected by the assay.
2002

abstract No: 

M-904

Full conference title: 

42nd Interscience Conference on Antimicrobial Agents and Chemotherapy
    • ICAAC 42nd