SBIR-STTR Award

This proposal aims to develop a collaboration between IDX Labs and the University of Virginia that will take real-time PCR tests for Cryptosporidium and Giardia from the research arena to the clinical lab. These intestinal protozoa cause diarrhea worldwide, with an estimated 2 million infections per year in the United States. The investigators in this proposal have developed and patented real time PCR tests for (1) Giardia that sub-genotypes into the major Assemblages A and B and (2) Cryptosporidium that speciate the parasite into the major pathogens Cryptosporidium hominis, parvum, and meleagridis. The tests are robust, with comparable sensitivity vs. gold-standard antigen detection by ELISA, and have been validated on over 400 patients from Bangladesh and Tanzania. However the tests have been designed for research purposes and require modifications to make them widely usable in clinical care. In aim 1 of this proposal we will optimize our DNA extraction method from stool into a high-throughput magnetic bead format involving Giardia and Cryptosporidium-specific DNA capture at downstream portions of the amplified 18S rRNA gene; this method provides both increased specificity (by requiring specific sequence for binding) and sensitivity (via elution of DNA away from PCR inhibitory substances in stool). In Aim 2 we will convert our individual genotype- specific PCR assays into a two-color single-vessel reaction that detects and distinguishes Cryptosporidium and Giardia without genotype information, which in the short-term remains clinically irrelevant for the low endemicity U.S. scenario. The reaction will continue to utilize Scorpion probes, a powerful technology for detection of DNA in stool because of the dual specificity provided at both primer and probe. We will adapt this new single-vessel test onto a commercial real-time PCR platform (e.g., Cepheid's SmartCycler). The innovation of this proposal lies not in PCR, but in the widely translatable DNA purification technology and in the concept of bringing the emerging field of molecular diagnostics to underfunded small-market infectious diseases. A Phase II application is anticipated to validate the final diagnostic test product against immunoassay for an FDA 501 (k) medical device application

This two-year proposal seeks small business support for IDX Labs LLC to continue collaborating with Dr. Houpt at the University of Virginia to bring sensitive real-time PCR (qPCR) tests for Giardia and Cryptosporidium to U.S. labs. According to the CDC, these parasites infect up to 2 million Americans annually, and we have developed robust sensitive qPCR tests for both infections that have been used successfully on hundreds of patients in Bangladesh and Tanzania. During Phase I these assays were converted into a Cryptosporidium/Giardia multiplex assay that detected all known human subspecies and was workable on two major qPCR platforms (Roche LightCycler and BioRad iCycler). During Phase I we also determined the most sensitive DNA extraction procedure among the commercially available kits, and then improved the sensitivity of PCR detection further through magnetic target-specific DNA capture. This Phase II application will now finalize the target DNA extraction procedure from stool for highest throughput and collect validation data from the field at our site in Tanzania in order to prepare an FDA 501(k) application, which we predict will allow a strong market entry. We hypothesize that our technique will be more sensitive than the typical gold standard Merifluor Cryptosporidium/Giardia IFA, and we will utilize a third tiebreaker PCR test to resolve any discrepant results. Secondarily, the test will also be validated on water samples versus the EPA's Giardia/Cryptosporidium method #1623, given the market and need for improved tests for these protozoa on drinking and recreational water samples. The innovation of this application lies in the DNA capture extraction technology, a platform translatable to other clinical specimens besides stool, and the concept of bringing state-of-the-art diagnostics beyond antigen detection to under-funded parasitic infections of waterborne health importance. This Phase II application will advance our development of a Giardia/Cryptosporidium real time PCR kit that incorporates highly sensitive target-specific DNA extraction with a highly optimized multiplex qPCR assay. During Phase II the final sample-to-result kit will be completed and performance will be validated on clinical samples against the existing gold standard IFA to enable a 501(k) submission to FDA.

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