Invasive fungal infections continue to affect about 15-20% of hematopoietic stem cell transplant (HSCT) recipients, with devastating consequences. Aspergillus and Candida are currently the most common fungi causing disease. Conventional methods for detecting these fungal infections are slow, insensitive, or both. The poor yield from conventional diagnostic tests fosters an approach wherein many patients are treated with prophylactic or empiric antifungal medication, increasing costs, drug resistance, drug interactions, and toxicities. Quantitative PCR (qPCR) is a rapid and sensitive method for detecting fungal pathogens based on amplification of fungal DNA. qPCR can be used to identify fungal pathogens and to measure the amount of fungal DNA present in tissues. Candida and Aspergillus qPCR assays have been developed which can detect femtogram amounts of fungal DNA. These assays have been successfully applied to clinical samples such as blood, liver biopsies, lung biopsies, and bronchalveolar lavage fluid. ? ? -Is qPCR more sensitive than culture for the detection of Candida and Aspergillus infections? A bank of blood and tissue samples from HSCT patients has been collected and will be tested with Candida and Aspergillus qPCR assays. Hypotheses: qPCR is more sensitive than cultivation and levels of fungal DNA in tissues help predict clinical course and response to antifungal medications. ? ? -Is screening blood from HSCT patients useful for early diagnosis of fungal infections? A prospective study of fungal qPCR will be performed in posttransplant patients using blood samples drawn every 3 days. Fungal qPCR will also be performed on BAL fluid and other tissue samples as they become available. Hypotheses: Candida and Aspergillus qPCR of blood will provide early evidence of fungal infection, paving the way for future intervention studies. Fungal qPCR performed on BAL fluid will increase the yield from bronchoscopy, reducing the need for open lung biopsies. ? ? -Are qPCR assays for non-Aspergillus moulds useful for detecting emerging fungal pathogens in HSCT patients receiving prophylaxis with broad-spectrum azole antifungals? The fungal pathogens causing disease in HSCT recipients are likely to change with the introduction of drugs such as voriconazole for prophylaxis. The existing qPCR platform will be adapted for the detection of non-Aspergillus moulds and applied to tissue samples from high-risk patients. Hypothesis: Fungal 18S rDNA qPCR employing a broad range fungal probe, or a system of multiple specific probes, will be useful for detecting emerging fungal pathogens in the transplant setting. ? ?
| Khot, Prasanna D; Ko, Daisy L; Fredricks, David N (2009) Sequencing and analysis of fungal rRNA operons for development of broad-range fungal PCR assays. Appl Environ Microbiol 75:1559-65 |
| Khot, Prasanna D; Fredricks, David N (2009) PCR-based diagnosis of human fungal infections. Expert Rev Anti Infect Ther 7:1201-21 |
| Khot, Prasanna D; Ko, Daisy L; Hackman, Robert C et al. (2008) Development and optimization of quantitative PCR for the diagnosis of invasive aspergillosis with bronchoalveolar lavage fluid. BMC Infect Dis 8:73 |
| Fredricks, David N; Smith, Caitlin; Meier, Amalia (2005) Comparison of six DNA extraction methods for recovery of fungal DNA as assessed by quantitative PCR. J Clin Microbiol 43:5122-8 |
