Optimization of UltraPCR for detection of and assessment of treatment outcomes in Trypanosoma cruzi infection - Abstract Infection with the protozoan parasite Trypanosoma cruzi is generally controlled but often not eliminated by host immune responses. In humans and many other hosts, this persistent infection ultimately results in muscle tissue damage known as Chagas disease. The success of the immune system in rigorously controlling parasite load in most hosts with T. cruzi infection has the contrary negative consequence that T. cruzi infection is frequently very difficult to routinely directly detect. The major impediment to prevention of life-threatening damage in those with T. cruzi infection is the lack parasite detection methods that can unequivocally establish those that need treatment, and if and when treatment resolves the infection – either in cases of evaluation of new drugs or in hosts being treated with those drugs. Diagnosis of possible infection is generally achieved using a combination of serological tests, which are reliable for detecting prior exposure but provide limited information on whether the infection is active. Methods for assessing treatment efficacy, either in individual subjects or in clinical trial evaluation of new candidate compounds can assess treatment failure but are undependable for assessing treatment success. To address this issue, we have adapted the currently most sensitive parasite detection assay, PCR of abundant repeat sequences in the T. cruzi genome, for increased sensitivity by employing DNA fragmentation to more effectively disperse parasite DNA throughout a DNA sample and performing 100’s of replicate PCR amplifications (“deep-sampling”) to detect that dispersed DNA. These modifications increase the lower range of detection of T. cruzi DNA in blood by several orders of magnitude and allow relative quantification of parasite load in most infected hosts. The goal of this project is to further optimize this amplification approach and to fully integrate it onto the UltraPCR platform, a digital droplet PCR technique with very high DNA loading capacity (at least 40- times that of standard PCR) and which generates 34 million individual partitions for detecting multiplexed, amplification products, giving it an enormous dynamic range. A fully optimized, multiplexed T. cruzi UltraPCR will then be evaluated as a confirmatory diagnostic test and to monitor treatment outcomes in three use settings: 1) seropositive human subjects enrolling or not in a conventional benznidazole-based treatment program in Buenos Aires, Argentina and the U.S. NIH; 2) naturally-infected non-human primates in a clinical trial-style study to help determine eventual optimal human dosing of the benzoxaborale AN15368, and 3) naturally infected dogs being treated with benznidazole using an intermittent dosing protocol and monitored to allow dose modification during the treatment period. These real-world evaluations of the UltraPCR protocol will rigorously assess this platform and provide the basis for its use as an experimental tool for determining the relationship between parasite load, immune responses, and disease severity in T. cruzi infection, as the long-missing method for assessing treatment success in human clinical trials, and ultimately made available in clinical diagnostic labs for use in both veterinary and human settings.
