Quantitative PCR for the Diagnosis of Fungal Infections

David Fredricks, MD

Infections caused by Aspergillus species , Candida species , the Zygomycetes, and other fungi remain a serious problem in patients with cancer and are associated with high mortality.  Cultivation methods fail to detect these fungi in a large percentage of patients with invasive disease, resulting in delayed diagnoses and the failure to select the most optimal antifungal therapy.   We have developed real-time quantitative PCR (qPCR) assays for the detection and quantitation of fungal DNA in clinical samples and are using these molecular assays to facilitate the rapid and accurate diagnosis of infections. Fungal qPCR assays are being applied to blood, bronchoalveolar lavage fluid, and tissue biopsies obtained from hematopoietic cell transplant recipients at risk for invasive fungal infections.   PCR results will be compared to conventional diagnostic methods such as cultivation/histopathology and to other cultivation-independent methods such as galactomannan antigen testing for Aspergillus infections.

Publications:

Sheppard D, Marr KM, Fredricks DN , Chiang L, Doedt T, Filler S. Comparison of three methodologies for the determination of pulmonary fungal burden in experimental murine aspergillosis.   Clinical Microbiology and Infection 2006; 12: 376-380.

Fredricks DN , Smith CA, Meier A. Comparison of six DNA extraction methods for recovery of fungal DNA as assessed by quantitative PCR. Journal of Clinical Microbiology 2005;43:5122-8.

Musher B, Fredricks DN , Leisenring W, Balajee A, Smith C, Marr KA. Aspergillus galactomannan Enzyme Immunoassay and Quantitative PCR for the Diagnosis of Invasive Aspergillosis with Bronchoalveolar Lavage Fluid. Journal of Clinical Microbiology 2004;42;5517-5522.

Imhoff A, Balajee A, Fredricks DN , Englund JA, Marr KA.   Breakthrough fungal infections in stem cell transplant patients receiving voriconazole.   Clinical Infectious Diseases 2004;39:743-6.

Fredricks DN , Relman DA. State-of-the-art clinical article: The application of PCR to the diagnosis of infectious diseases. Clinical Infectious Diseases 1999;29:475-486.

Fredricks DN, Relman DA.   Paraffin removal from tissue sections for PCR analysis.   Biotechniques 1999; 26: 198-200.

Fredricks DN , Relman DA. Improved amplification of microbial DNA from blood cultures by removal of the PCR inhibitor Sodium Polyanetholesulfonate.   Journal of Clinical Microbiology   1998; 36: 2810-2816.