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. Although several partially effective drugs exist to treat the infection, it is estimated that only ~1% of infected subjects receive treatment. The major impediment to the wider use of current drugs, the development of better therapeutics in Chagas disease, and the identification of those who would benefit from treatment and the success of treatment in those individuals, is the absence of reliable methods to definitively determine the presence or absence of infection. 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. The goal of this project is to validate and improve PCR-based tests of cure for T. cruzi infection. The primary application of the results of this study will be to the more definitive evaluation of clinical trial outcomes. Current PCR-detection protocols for diagnosis of T. cruzi infection generally use a very limited (sometimes only one) blood sample and assay a single aliquot from that sample. However, subjects in trials of anti-T. cruzi drugs are generally bled multiple (4-6) times post-treatment and 1-3 aliquots are submitted to PCR, usually with variable results between bleeds and aliquots. The hypothesis underlying the proposed work is that definitive detection of infection via PCR of parasite DNA in blood can be achieved by increasing the sampling of blood, both the number of blood samples taken and the aliquots of that blood that are evaluated by PCR. This hypothesis will be tested in a group of naturally infected rhesus macaques who will be repeatedly sampled over both an extended time frame (1 year) and a more limited period (1 month) with up to 200 aliquots of DNA screened by PCR at each bleed point. Parallel studies using parasite- or parasite-DNA-spiked blood we allow interpretation of the blood parasite levels in the infected animals, and its variation, over months or years. The ultimate outcome of these studies is expected to be a dependable pre-enrollment protocol for participant selection for clinical drug trials and definitive tests at the conclusion of such trials that unequivocally measure treatment efficacy. Such tools will assure that future clinical trials of candidate anti-T. cruzi drugs will provide conclusive results.