We report the development and evaluation of a real-time PCR assay using the LightCycler instrument for the detection of C. albicans and A. fumigatus DNA in whole blood. Recently published consensus criteria for the diagnosis of invasive fungal infection (IFI) were used for all patient samples. Unique and published primer pairs were developed and assessed for sensitivity, specificity, and reproducibility to detect C. albicans and A. fumigatus DNA in samples spiked with purified DNA, and whole blood samples from 8 high-risk patients and 45 negative controls. The real-time assay demonstrated an analytical sensitivity of 10 fg of purified C. albicans and A. fumigatus DNA and was found to be specific for each species. The standardized approach was highly reproducible and detected C. albicans and A. fumigatus DNA in two patients with proven IFI and in one patient with a possible IFI. In addition, we report for the first time the use of recently published international consensus criteria for the diagnosis of IFI in the evaluation of a mildy invasive fungal diagnostic assay. Standardized clinical criteria and a more standardized approach to detect fungal DNA in less invasive patient samples, may permit a more reliable comparison of future studies. A rapid real-time detection of fungal DNA in whole blood, combined with standard clinical markers of response, may be more useful for monitoring patients at risk of developing 1171 than other diagnostic methods currently available. (C) 2003 Elsevier Inc. All rights reserved.