Dengue and malaria are the most widespread vector-borne human pathogens worldwide, with over half of the world's population living in endemic areas. These infections result in non-specific clinical manifestations that range in severity from a self-limited febrile illness to septic shock and death. A confirmed diagnosis, which relies on accurate laboratory tests, is associated with improved patient outcomes. Available diagnostic tests for these agents, however, often lack sensitivity or the performance characteristics necessary for routine use. Furthermore, multiplex assays that distinguish between dengue and malaria have not been developed, despite significant overlap in the endemic area and clinical presentation of these infections. The objectives of the current application are to utilize sensitive diagnostic tests for dengue in order to identify patient and vral factors associated with severe disease, further delineate dengue epidemiology in endemic regions, and develop a syndrome-based multiplex test for dengue and malaria. In order to accomplish these objectives, samples from cohorts of patients in Nicaragua and Zimbabwe will be studied. Using Nicaraguan children admitted to the hospital for dengue, viral load thresholds and patient risk factors will be identified that portend an increased risk of severe disease. A separate cohort of 2,900 febrile Nicaraguan children diagnosed with a non-dengue illness will be tested for dengue to identify atypical clinical syndromes that warrant specific dengue testing. This cohort of patients will also allow us to establish the sensitivity of serological responses fo the detection of missed dengue infections. Finally, we have developed a dengue and malaria multiplex test that will be employed in the study of these pathogens in the Honde Valley of Zimbabwe, which has experienced a resurgence of malaria over the past decade. This research will build off of work started during my fellowship in infectious diseases at Stanford University. During the past two years, I have designed and validated two real-time dengue RT-PCRs, which have both proven more sensitive than prevailing diagnostics. I am now seeking to employ these assays to study dengue epidemiology and pathophysiology. One of these tests, an internally con- trolled RT-PCR for pan-dengue detection, served as the starting point for the dengue-malaria multiplex assay mentioned above. My co-mentors for this proposal include experts in molecular diagnostics, Dr. Benjamin Pinsky, and dengue pathophysiology and epidemiology, Dr. Eva Harris. I have also assembled a team of collaborators and consultants with a wealth of experience in global health, epidemiology, and diagnostics development. Stanford University represents an ideal environment for the pursuit of clinically oriented research and international studies. While our laboratory contains the equipment necessary for the completion of the research outlined in this proposal, I will still have access to nineteen core facilities on campus o address questions that may arise. Stanford fosters cross-disciplinary collaborations and provides access to leading academic researchers and biotechnology companies focused on the development of new diagnostics. This proposal also includes two international sites located in areas endemic for dengue (Centro Nacional de Diagnostico y Referencia, Managua, Nicaragua) and malaria (Biomedical Research and Training Institute, Harare, Zimbabwe). These sites not only provide access to the clinical material necessary for the study of dengue and malaria, they allow for collaboration with experts in international medicine and capacity building in resource limited settings. The expected outcomes of this proposal include a refined understanding of dengue epidemiology in two endemic regions, the validation of a clinical prediction rule for severe dengue, and the development of a near-care multiplex assay for the most common vector-borne pathogens worldwide. Research performed in pursuit of these aims represents the next step in my professional development, bridging infectious disease fellowship with a career as an independent global health investigator. My co-mentors and collaborators are uniquely qualified to support this research, and three study sites with complimentary resources maximize the opportunities for success.
Over one-half of the world's population resides in areas where dengue and malaria are transmitted, and this affected area continues to expand. Research outlined in this proposal involves the use of highly sensitive tests to study dengue and the development of a new, combined test for the detection of dengue and malaria in a single reaction. These projects aim to improve patient care by identifying individuals at risk for developing severe dengue, generate more accurate estimates of the frequency of dengue infections, and provide a simple but sensitive test for dengue and malaria that can be performed near the point of care.