Development of a latex-agglutination assay for the rapid detection of Naegleria fowleri

PROJECT SUMMARY/ABSTRACT Development of a latex-agglutination assay for the rapid detection of Naegleria fowleri As most diagnoses of primary amebic meningoencephalitis are done at the time of autopsy, rapid methods of detection of Naegleria fowleri are urgently needed. In this proposal, we plan to demonstrate proof of concept that the development of a point-of-care assay for the detection of N. fowleri in cerebrospinal fluid (CSF) is feasible. To achieve this goal, we will inoculate rabbits with N. fowleri lysate to produce antisera. Total IgG antibodies will be purified from animal sera and isolated to identify fractions that bind to N. fowleri. These antibodies will be immobilized onto dyed latex microspheres to create an agglutination assay that can detect N. fowleri in CSF in under 15 minutes without the need for additional equipment. We will compare our proposed assay to the current methods of diagnosis, nucleic acid amplification and microscopy, in terms of limit of detection, sensitivity, and specificity using prepared samples and CSF specimens obtained from animals infected with N. fowleri. Cross reactivity of our proposed assay will be assessed by testing against N. fowleri and common bacterial and fungal meningoencephalitis pathogens. Demonstrating proof of concept will allow us to seek future support to refine our methodology to characterize the antibodies and target specific antigens so we can produce monoclonal antibodies that will facilitate large-scale production and kit development. It is the long-term goal of this proposal to incorporate this assay in the clinical setting as part of the screening panel for meningoencephalitis pathogens.