Quantification and Speciation of Fungal DNA in Clinical Specimens Using the LightCycler Instrument

Abstract

Invasive fungal infections have become a major cause of morbidity and mortality in immunocompromised patients such as bone marrow and solid organ transplant recipients, patients receiving intense chemotherapy, AIDS patients, patients with cystic fibrosis, neonates, and severe burn patients [1]. Invasive aspergillosis has become a leading cause of death, mainly among hematology patients. The incidence is estimated to be 25% and the mortality is up to 90% in patients with acute leukemia [2]. Early diagnosis is essential for appropriate and successful antifungal therapy. Conventional tests for the detection of Aspergillus spp., such as blood culture and serology, have limited sensitivity and specificity. Diagnostic assays based on in vitro amplification and speciation of fungal DNA show promising sensitivity and specificity, indicating potential for the early diagnosis of invasive fungal infections [3–5]. However, published assays require 6–8 h for fungal DNA extraction, followed by a minimum of 9 h for DNA amplification and amplicon detection. Post-PCR analysis is mainly based on qualitative or semi-quantitative methods such as gel electrophoresis or hybridization by Southern blot or PCR-ELISA techniques.