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Major Research Project RP003

Collaborating Institution: University of Texas Medical Branch, Galveston, TX

Principal Investigator: A. Clinton White, Jr., MD

Title of the Project: Point-of-care Diagnostic Test for Intestinal Protozoa

Co-Investigator: Rebecca Richards-Kortum, PhD, Rice University, Houston, TX

Consultants:

Collaborator:
John McDevitt, PhD – University of Texas-Austin, Austin, TX

Expected Product: A diagnostic device to detect intestinal parasites in clinical samples, to guide treatment at the site of care.

Description:  According to the World Health Organization, diarrheal diseases constitute a global health burden greater than that of HIV/AIDS and are the second leading cause of death in children worldwide, yet the etiology of diarrhea often remains undiagnosed in patients. A variety of enteric pathogens can cause diarrhea, including viruses, bacteria, and protozoa. While viral causes dominate among cases of acute self-limited diarrhea, parasites are more common causes of persistent and chronic diarrhea, which are increasingly recognized as major causes of morbidity. Among the parasites, Cryptosporidium, Giardia intestinalis, and Entamoeba histolytica are most commonly associated with diarrhea. These organisms can all present with a similar clinical picture, typically with watery diarrhea, yet they differ in terms of optimal diagnostic methods and, more importantly, treatment.

The long-range goal of this project is to develop a simple, low-cost test to detect the major diarrhea-causing intestinal protozoa at the point-of-care. In the past year, we have developed and fully characterized a nucleic acid detection scheme employing oligonucleotide-functionalized gold nanoparticles (AuNP) for detection of C. parvum 18S rRNA. We demonstrated a limit-of-detection for this AuNP aggregation assay between 4 x 105 and 4 x 106 copies of RNA per µL, using a short synthetic target and full length in vitro transcribed target. Furthermore, using total nucleic acids purified from C. parvum oocysts in spiked stool, as few as, 2 x 103 oocysts/µL can be detected. We have also developed and characterized sample preparation strategies which can be incorporated as part of this assay.  This includes optimization of nucleic acid extraction methods. We continued our work to examine less-expensive platforms to implement the cryptosporidium nucleic acid detection scheme developed above in low-resource settings. We characterized both paper and nitrocellulose lateral flow platforms as for nucleic acid detection. Ultimately, we will integrate detection of other intestinal parasites including Giardia, Entamoeba histolytica, Cyclospora, and Isospora into a multiplexed assay and migrate towards a microfluidic platform that is compatible for use at the point-of-care. Toward this end, we began to select nucleic acid targets for detection of additional parasite targets. Finally, we established, with supplemental funds, a user facility to assist with rapid prototyping and early stage manufacturing of point-of-care diagnostic devices for WRCE investigators. The facility complements the expertise and fabrication capabilities currently available through the collaboration with Sandia National Laboratories.