Aspergillus fumigatus and PCR: a panfungal and a species-specific approach

C. Andreutti-Zaugg, K. Jaton, J. Bille, C.H. Lausanne.


Objective: Current diagnosis of invasive fungal infections remains difficult. In order to improve an integrated diagnostic approach (taking into account clinical data, risk factors and mycological methods), a panfungal PCR assay was set up, allowing sensitive and specific detection of yeast and fungal species. Aspergillus fumigatus was chosen as model organism. In parallel, a real time PCR assay specific for A. fumigatus was developed. Methods: Special care was taken in validating the DNA extraction procedure. In blood of patients with invasive aspergillosis, the fungus is probably present as hyphal fragments. To mimic the in vivo fungal cell wall composition, conidia of A. fumigatus were germinated in vitro and grown to 1 mycelial cell. By this mean the fungal cells were countable. Whole blood samples were then spiked with serial dilutions of germinated spores and cell lysis followed by DNA purification was performed. First, erythrocytes were lysed with alcaline citrate-cysteine (Flahaut et al. 1998). Then, to disrupt efficiently the fungal cells the lysing enzyme Novozyme 234 (Calbiochem) was chosen. This multienzyme preparation is active against the two major components of the mycelial cell wall, alpha-1,3 glucan and chitin. Finally, DNA was purified with the QIAamp DNA Mini kit (Qiagen). The DNA was amplified with panfungal primers previously described (Van Burik et al. 1998). The fungal PCR products were detected on an agarose gel containing ethidium bromide and hybridized to an Aspergillus sp.-probe coated on a microtiter plate (Boehringer PCR ELISA kit). In parallel, the same extracted DNA was run on a TaqMan 5700 instrument (Applied Biosystems), targeting the A. fumigatus cytochrome b gene in the mitochondrion. Results: With the panfungal procedure, 10-1 fungal cells were detectable and the Novozyme lysis step increased the yield about 100 times. Moreover, the real time PCR allowed the detection of 1 gene copy: a clear positive result was obtained when DNA corresponding to one fungal cell was amplified. Therefore this method was about 100 times more sensitive than the panfungal approach. Conclusion: The developed extraction procedure using Novozyme 234 and germinated spores, followed by a panfungal amplification or a real time PCR approach allowed highly sensitive detection of A. fumigatus DNA in whole blood samples

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Full conference title: 

11th European Congress of Clinical Microbiology and Infectious Diseases
    • ECCMID 11th (2001)