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Journal of Clinical Microbiology, February 2000, p. 586-590, Vol. 38, No. 2
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Quantification of Fungal DNA by Using Fluorescence Resonance Energy Transfer and the Light Cycler System

Juergen Loeffler,1,* Norbert Henke,1 Holger Hebart,1 Diethard Schmidt,1 Lars Hagmeyer,1 Ulrike Schumacher,2 and Hermann Einsele1

Medizinische Klinik, Abteilung II,1 and Hygieneinstitut,2 Abteilung Medizinische Mikrobiologie, Eberhard-Karls-Universität, 72076 Tübingen, Germany

Received 6 August 1999/Returned for modification 14 October 1999/Accepted 10 November 1999

The Light Cycler technique combines rapid in vitro amplification of DNA in glass capillaries with real-time species determination and quantification of DNA load. We have established a quantitative PCR protocol for two clinically important pathogens, Candida albicans and Aspergillus fumigatus. The sensitivity of the assay was comparable to those of previously described PCR protocols (5 CFU/ml). Specific detection of C. albicans and A. fumigatus could be achieved. The assay showed a high reproducibility of 96 to 99%. The assay was linear in a range between 101 and 104 Aspergillus conidia. As capillaries do not have to be reopened for post-PCR analysis, the risk of carryover contaminations could be minimized. The Light Cycler allowed quantification of the fungal loads in a limited number of clinical specimens from patients with hematological malignancies and histologically proven invasive fungal infections. Five of nine positive samples had fungal loads between 5 and 10 CFU/ml of blood, two of nine positive samples had fungal loads between 10 and 100 CFU/ml of blood, and two of nine samples had fungal loads of more than 100 CFU/ml of blood. All samples were also found to be PCR positive by PCR-enzyme-linked immunosorbent assay analysis.

* Corresponding author. Mailing address: Medizinische Klinik, Abt. II, Labor Prof. Dr. med. H. Einsele, Otfried-Mueller-Str. 10, 72076 Tuebingen, Germany. Phone: 49 7071 2987355. Fax: 49 7071 293179. E-mail: juergen.loeffler{at}med.uni-tuebingen.de.

Journal of Clinical Microbiology, February 2000, p. 586-590, Vol. 38, No. 2
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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