Real-time PCR Diagnostic Kit for Giardia and Cryptosporidium

DESCRIPTION (provided by applicant): 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 t o 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 sens itive 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 f rom 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 t han 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 m ethod #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 b esides 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/Cryptosporidiu m 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 again st the existing gold standard IFA to enable a 501(k) submission to FDA.