The research plan relates to the importance of the observation that relatively late diagnosis of fungal infections in bone marrow transplant (BMT) patients, combined with unsatisfactory treatment options, have led to mortality in greater than 90% of patients with invasive Aspergillus infection and in 40% of patients with Candida bloodstream infection. Moreover, invasive aspergillosis has emerged as the leading cause of infectious death after BMT. Rapid detection of fungal DNA is practical with the application of polymerase chain reaction (PCR) amplification. Three sets of oligonucleotide primer pairs were made, based on homologous areas of the nucleic acid sequence of the 18S ribosomal RNA gene subunit, and therefore function as """"""""universal"""""""" fungal primers. PCR assays will be used to detect DNA from Aspergillus species, Candida species, and other medically relevant fungi in blood specimens before, during, and after evidence of clinical disease. This tool will be used to chart the previously uncharacterized natural history of fungal infections. The time of acquisition of infection, analysis of the source and site of invasion, and determination of the mode of dissemination will be determined for Aspergillus and Candida. PCR assay results will be compared with culture results from non-sterile specimens as a means of examining colonization or invasion of the fungal pathogen. Logistic regression methodology will be utilized to model the sensitivities and specificities of the assays for predicting onset of clinical disease and to examine the assays as a monitor of response to anti-fungal therapy. The PCR methodology defined in these studies will be the critical basis for more sensitive detection of fungal infection and the foundation for improving the serious morbidity and high mortality associated with fungal infection in the BMT setting. Methods for quantitation of PCR assay results will be developed in later years of the project and the response to antifungal therapy will be correlated completed. It is proposed these correlations may lead to methods for determining the intensity and duration of antifungal therapy. The experimental techniques are expected to provide fundamental training in the use of molecular biology to answer other questions of pathogenesis in mycology.
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