Presently EPA approved methods (Method 1622 and Method 1623) for detection of Cryptosporidium parvum in drinking water rely upon immunofluorescense of immuno-magnetically captured oocysts. Due to the complexity of these testing methods, offsite reference laboratories perform most of the testing of the U.S. water supply. Furthermore, such tests can be as expensive as $350 - $500 per analysis.<P> Since an easy to perform, inexpensive onsite testing alternative is not presently available, it is understandable why mandated testing for the presence of Cryptosporidium parvum and other waterborne pathogens that pose human health risks is not required in the U.S. Under current circumstances, test results would only be available after the treated water was distributed and consumed. Therefore, sick and infected people would already have appeared in emergency rooms long before test results would be available, as just evidenced in the contamination of a recreational water supply at the Seneca Lake State Park in Geneva, NY with more than 3,000 reported illnesses.<P> We propose, therefore to fully evaluate a molecular biosensor assay, previously developed by us with respect to sensitivity, specificity and comparability to the currently employed EPA Methods 1622 and/or 1623 for use in rapid and inexpensive onsite testing at water treatment plants.<P> This assay relies upon the ability to immunocapture the oocysts according to EPA guidelines, and then to induce a heat shock mRNA that can subsequently be amplified by the isothermic NASBA and then easily be detected in an inexpensive liposome-based biosensor. Such an assay system will provide information not only about the presence, but also the viability status of the oocysts (since nonviable oocysts will not respond to the heat shock).<P> In addition, we are going to adapt the Cryptosporidium assay to a microfluidic biosensor developed previously in our lab for Dengue virus detection in order to obtain a more automated and quantitative device. The availability of such an assay would allow U.S. regulators to consider mandating increased testing that will improve the safety of the U.S. water supply, because so far, of the over 53,000 water treatment utilities in the U.S., less than twenty have been granted Approval Pending status for the analysis of Cryptosporidium under the Safe Drinking Water Act. <OL> <LI>Fully evaluate our biosensor assay, with respect to sensitivity, specificity and comparability to the currently employed 1622/1623 methods, for use in rapid and inexpensive onsite testing at water treatment plants; and produce Crypto-kits for beta-site testing. <LI>Integrate the biosensor into a microfluidic format similar to that developed by us for Dengue virus in order to obtain a more automated and quantitative device that will in the future be developed into a micro-Total Analysis System; produce Crypto-microkits for beta-site testing. <LI> Submit data of in-house and collaborative data to AOAC/ASTM for method validation prior to launch of commercial products. Submission of data and method to EPA for approval.
Non-Technical Summary: Cryptosporidium parvum is a waterborne human pathogen that has caused several outbreaks in recent years in the U.S. and world-wide, with one of the largest outbreaks in the world occurring in Milwaukee in 1993 leading to approximately 100 deaths. Currently EPA approved detection methods are complex and require highly specialized personnel, thus offsite reference laboratories perform most of the testing of the U.S. water supply with costs of $350 - $500 per analysis with results available 7-10 days after treated water is distributed to consumers. Due to this unavoidable delay, mandated testing for the presence of C. parvum and other waterborne pathogens that pose human health risks unfortunately is not required in the U.S. Sick and infected people would already have appeared in emergency rooms long before test results would be available. We propose here to develop a late stage C. parvum biosensor technology into a commercial product. The simple and yet very sensitive and highly specific biosensor will provide results within 4 - 5 hours and can be performed in the water treatment plants directly. Test will cost a fraction of current procedures required and will therefore enable analyses on a routine basis resulting in a much safer water supply. <P> Approach: For several years the co-PIs worked on the development and optimization the CryptoDetect Test System for the detection of viable Cryptosporidium parvum oocysts. The test system relies upon the ability to induce the hsp70 mRNA (i.e., coding for the 70 kDa heat shock protein), which provides the ability to detect only viable oocysts, since nonviable oocysts do not respond to the induction. A specific segment of the mRNA is amplified using NASBA and subsequently detected using the lateral-flow liposome biosensor. Since NASBA amplicons are single stranded, they can immediately be detected via the biosensor, without any additional treatment of the sample. A reporter oligonucleotide probe conjugated to liposomes can hybridize to the single stranded NASBA amplicons. Similarly, a capture oligonucleotide probe immobilized on a lateral flow test strip, captures the entire liposome-target complex so that the presence of the target sequence can be quantitated using a handheld reflectometer. The overall method used in this test system, which can be completed in a total time of 4-5 hours, includes the following steps (after a 10 mL concentrate is obtained from a larger water sample): a) Immunomagnetic capture of the oocysts b) Heat shock c) Disruption of the oocysts and extraction of mRNA d) NASBA amplification of a segment in the hsp70 mRNA sequence e) Liposome biosensor analysis. The following points will be investigated within the course of the two year project: Year 1: 1. Determine if the designed primer pairs and probes are specific for C. parvum and are not prone to false positive reactions against other Cryptosporidium species that do not infect humans or other microbial agents anticipated in drinking water. 2. Determine if the primer pairs and probes will also detect C. hominis 3. Determine if the test system is capable of detecting low numbers of C. parvum and/or C. hominis oocysts against a large background of other microbial agents 4. Determine if the entire test system is capable of detecting low numbers of oocysts in various natural water matrices. 5. Test whether the entire system can distinguish viable oocysts from those killed by UV irradiation and ozonation. 6. Determine if the entire test system will yield performance specifications at least as good as the currently approved Methods 1622/1623. 7. Adapt the Dengue virus microfluidic electrochemical biosensor to C. parvum. Year 2: 1. Prepare CryptoDetect Test System kits for use in water treatment plants for beta site testing. 2. Test the kits by Clancy Environmental Consultants. 3. Test the kits in water treatment plants. 4. Fabricate 10 minipotentiostat and 300 microbiosensors for testing in water treatment plants. 5. Evaluate the use of the microbiosensor in water treatment plants. 6. Prepare quality control protocols to be used as basis for future large volume manufacturing. 7. Summarize data for both systems (in-house for first review approval and collaborative study data of 8 to 10 additional sites for final approval) for ASTM, AOAC test method approval. Submission to EPA for approval with assistance of Clancy Environmental Consultants.