Final Report: A New Microfluidic System for the Determination of Cryptosporidium Oocysts in Water

EPA Contract Number: 68D01064
Title: A New Microfluidic System for the Determination of Cryptosporidium Oocysts in Water
Investigators: Hitchens, G. Duncan
Small Business: Lynntech Inc.
EPA Contact: Richards, April
Phase: II
Project Period: September 1, 2001 through September 1, 2003
Project Amount: $225,000
RFA: Small Business Innovation Research (SBIR) - Phase II (2001) Recipients Lists
Research Category: Ecological Indicators/Assessment/Restoration , SBIR - Monitoring , Small Business Innovation Research (SBIR)

Description:

Waterborne infectious disease outbreaks have been attributed to a variety of pathogenic bacteria, parasites, and viruses. An alarmingly large number of waterborne infectious diseases resulting from contaminated water sources have occurred during the past decade. Although cases of cryptosporidiosis have been reported much less frequently than those arising from other pathogens, a heightened demand exists for reliable detection of exceedingly low levels of the infecting protozoan, Cryptosporidium parvum, a well-known coccidian parasite that causes self-limiting diarrhea in immunocompetent individuals. Exposure of immunosuppressed persons (e.g., AIDS patients) to the protozoan, however, frequently leads to chronic illness and eventually becomes life threatening. Recent outbreaks of cryptosporidiosis caused by contaminated drinking water and the abundance of C. parvum oocysts in untreated surface water have heightened the need for reliable detection protocols. Within the Continental United States, 26 percent of treated water supplies and more than 80 percent of all surface water sources have tested positive for C. parvum. Although levels of the protozoan generally are very low in water sources (i.e., less than one oocyst per liter), ingestion of as few as 1 to 100 oocysts can cause severe infection. Therefore, to ensure the safety of the general public, it is imperative that sensitive diagnostic methods be developed for C. parvum detection.

Currently available methods for the detection of low numbers of C. parvum oocysts and Giardia cysts, including the currently recommended immunofluorescent antibody (WA) technique, require the processing of large water volumes (thousands of liters), typically via field filtration, which yields variable oocyst and/or cyst recovery because of the effects of the filter type, flow rate, and eluant efficiency. The accuracy of all these methods, however, which use filtration-buoyant density gradient separation and immunofluorescence for detection, is affected by the turbidity of the sample (both inorganic and organic), nonspecific fluorescence by other organisms (algal and yeast cells), the presence of chlorine compounds and/or other disinfectants in water samples, and physical conditions of the samples such as freezing the sample or eluates.

Summary/Accomplishments (Outputs/Outcomes):

Lynntech, Inc.'s method utilizes polymerase chain reaction (PCR) detection principles for the analyses of C. parvum oocysts and successfully addresses the problems stated in this report. The Information Collection Requirements (ICR) Method and Method 1622, both of which currently are in use, are very laborious and cannot be performed in the field because the detection step involves expert immunofluorescence microscopy, which is impractical to perform in the field.

Conclusions:

The ultimate goal of this Phase II research project was to develop a field-portable microbiological water analyzer and a method that will be capable of accurately detecting C. parvum oocysts in water samples in the field. The method combines standard sample filtering techniques (already developed in Method 1622), a new C. parvum oocyst separation/concentration technique, and a new principle for the detection of C. parvum PCR-DNA amplicons.

Supplemental Keywords:

waterborne infectious disease, contaminated water, cryptosporidiosis, Cryptosporidium parvum, C. parvum, coccidian parasite, oocyst, cyst, drinking water, monitoring, analytical, surface water, immunofluorescent antibody, Giardia, filtration, filtration-buoyant density gradient separation, immunofluorescence, ICR Method, Method 1622, polymerase chain reaction, PCR, small business, SBIR., RFA, Scientific Discipline, Water, Ecosystem Protection/Environmental Exposure & Risk, Chemical Engineering, exploratory research environmental biology, Environmental Chemistry, Ecosystem/Assessment/Indicators, Chemical Mixtures - Environmental Exposure & Risk, Ecosystem Protection, Chemistry, Monitoring/Modeling, Ecological Effects - Environmental Exposure & Risk, Environmental Microbiology, Ecological Effects - Human Health, Biochemistry, Environmental Monitoring, Biology, Drinking Water, Environmental Engineering, Ecological Indicators, cryptosporidium parvum oocysts, monitoring, microbial monitoring, pathogens, microbiological organisms, electrochemical , microanalyzer, exposure and effects, electrochemical detection, exposure, microorganisms, analyzer, electrochemical sensor cell, PCR, microbial risk management, microorganism, DNA, sensor, cryptosporidium, microbial


SBIR Phase I:

A New Microfluidic System for the Determination of Cryptosporidium Oocysts in Water  | 2001 Progress Report  | Final Report