A Hybrid Pathogen Detection System

Cryptosporidium is a resilient waterborne protozoan pathogen that causes gastrointestinal disease and is one of five major causes for 5 million deaths each year worldwide. Recently, a Cryptosporidium outbreak sickened more than 1,700 people, mostly children and teenagers, in New York. Cryptosporidium oocysts are of particular interest in the water industry because the infectious dose is low (1 to 132 oocysts) and does not respond to common drug treatments. Self-contained microelectrochemical assays are desirable because precise detection can be performed down to a single oocyst on colored and turbid samples. In addition, electrochemical detection that depends only on loss and gain of electrons immediately adjacent to surface-bound assay components provides rapid signals even for very low analyte concentrations based on short transport distances for reporter molecules. In Phase I of this research project, Vegrandis, LLC, successfully demonstrated its chip-based, self-contained, microelectrochemical hybrid assay (SMEHA). The assay involved an immunoassay followed by a DNA hybridization assay to confirm viability of the oocysts. Both assays, which took approximately 90 minutes to complete, were demonstrated in an 8 x 3 array of 50 µm diameter cavities. Phase I of this project proved the ability to detect live C. parvum oocysts down to 5 oocysts/10 L of pelleted surface water samples in less than 90 minutes. This Phase II project proposes to develop disposable SMEHA cartridges for the quantification and viability assessment of waterborne pathogens to meet the escalating need for fast warning of pathogen outbreaks and possible dispersal of bioterrorism agents. Although the disposable SMEHA cartridges can be specified for nearly any pathogen of interest, this Phase II project will focus on the detection of Cryptosporidium oocysts and Giardia cysts. Both protozoans seriously threaten the nation’s water supply because they resist ordinary water treatment processes and do not respond to common antibiotics. Because of the speed and specificity of the proposed device, investigators will be able to act quickly to suspected Cryptosporidium or Giardia contamination. Both the source and the path of contamination flow can be efficiently traced. The development of the disposable SMEHA cartridge and reagent kits will advance the state-of-the-art over existing U.S. Environmental Protection Agency Methods 1622 and 1623 by providing a previously unavailable combination of speed, sensitivity, affordability, ease-of-use, and testing for viability of waterborne pathogens.