Fungal infections have been reported with increased frequency in severe immunosuppressed patients and ranks as one of the top 10 most common causes of infectious disease-related mortality. There is a pressing need for development of an improved means to diagnose fungal infection. Identification of the fungal species is essential for appropriate clinical decision making concerning both the significance of a particular isolate and the dosage and duration of antifungal therapy. The use of short DNA oligomers with specificity to segments of fungal 18S ribosomal RNA (rRNA) is well documented for in vitro fungal identification by PCR. PCR based technologies have identified the value of DNA/RNA methodologies for in vitro detection of invasive fungal infections in blood, sputum and tissue samples. Thus, an oligomer analogue radiolabeled with an imaging radionuclide such as 99mTc may show preferential localization in sites of fungal infection. We concluded that radiolabeled species specific short oligomers should be investigated as potential agents for specific imaging of fungal infection especially since these agents may be useful for a broad range of patients. The objective of this proposed research is to conduct these investigations to identify the oligomer analogues that can be used with fungi to show specific binding, and that is also compatible with in vivo conditions. Then we will evaluate to what degree radiolabeled oligomers may be useful in vivo for fungal infection detection. This laboratory has experience with infection detection using bacterial mouse models and an extensive background in radiolabeled oligomer analogues for in vivo applications. We wish to continue our studies of infection specific agents and expand it to include fungal infections and complement it with our experience with radiolabeled oligomers to expand upon our other studies. Beginning with species specific oligomer sequences already in the literature, we will evaluate their chemical forms in vitro using morpholino oligomer analogues. Using fungal specific oligomer sequence and their respective fungi we will evaluate their specificity using fluorescence and radiolabeling molecular technologies. As controls we will use a conserved universal fungus binding oligomer and a known non-binding oligomer as negative control. Thereafter, we will test in vivo only those oligomers showing specificity in a mouse model of invasive pulmonary aspergillosis. Oligomer probes will be tested first at the genus level for Aspergillus and for Candida for proof of concept. After the first two years, and we have proven our hypothesis, we will focus on the species specific level to further define sensitivity and specificity of this approach. Finally, we will move towards the development of the oligomers for PET imaging using fluorine-18 and gallium-68 as tracers, plus work with larger animal models, such as the rabbit with endocarditis, and study mice with various fungal infections to establish specificity to facilitate the transition from the laboratory to the clinic. In this way, we expect to confirm our hypothesis that radiolabeled oligomers hold considerable promise as diagnostic agents for fungal infection through imaging. Our goal is to extend this science to diagnostic imaging by investigating whether fungal specific oligomers, specific to segments of the rRNA, can be applied to in vivo applications to identify the location of the fungal infection through imaging. In addition, in the process, the causative fungal agent may also be confirmed. The ultimate goal is to develop a noninvasive molecular imaging molecular marker with specificity to fungal rRNA that once radiolabeled can be used to for the diagnostic imaging of invasive fungal infection using SPECT and PET imaging. We expect this technology to be easily and readily translated into the clinic.
Statement Fungal infections have been reported with increased frequency in severe immunosuppressed patients and ranks as one of the top 10 most common causes of infectious disease-related mortality. Those at high risk for invasive mycotic infections cover a broad range, some of which include solid organ transplant recipients, chemotherapy and allergenic bone marrow transplant recipients, patients with cystic fibrosis or granulomatous disease, HIV-infected patients, as well as patients with extensive surgery or burns, intensive antibiotic therapy, indwelling catheters and diabetes mellitus. A diagnostic noninvasive imaging method capable of detecting the location and possibly identifying the fungal species will significantly contribute to a patient's treatment regime and design of interventional strategies and general patient outcome. In this project we will test the feasibility of a methodology that we are developing for detection of bacterial infection through imaging but now apply it to detection of fungal infections. The aim is to prove that short oligomers complementary to fungal RNA can target fungi in a biological system. With this proven the ultimate goal is to develop a noninvasive molecular imaging molecular marker for the diagnostic imaging of invasive fungal infection that have origins with a range of pathophysiologies. We expect this technology to be easily and readily translated into the clinic. The ultimate goal of this study is to provide evidence that modified oligomers with specificity to fungal 18S or 28S rRNA once radiolabeled can be used to for the diagnosis of fungal infection using SPECT and PET imaging.
|Wang, Yuzhen; Chen, Ling; Liu, Xinrong et al. (2013) Detection of Aspergillus fumigatus pulmonary fungal infections in mice with (99m)Tc-labeled MORF oligomers targeting ribosomal RNA. Nucl Med Biol 40:89-96|