Serial 'deep-sampling' PCR of fragmented DNA reveals the wide range of<i>Trypanosoma cruzi</i>burden among chronically infected hosts and allows accurate monitoring of parasite load following treatment

Abstract Infection with the protozoan parasite Trypanosoma cruzi is generally well-controlled by host immune responses, but appears to be rarely eliminated. The resulting persistent, low-level infection results in cumulative tissue damage with the greatest impact generally in the heart in the form of chagasic cardiomyopathy. The relative success in immune control of T. cruzi infection usually averts acute phase death but has the negative consequence that the low-level presence of T. cruzi in hosts is challenging to detect unequivocally. Thus, it is difficult to identify those who are actively infected and, as well, problematic to gauge the impact of treatment, particularly in the evaluation of the relative efficacy of new drugs. In this study we employ DNA fragmentation and high numbers of replicate PCR reaction ('deep-sampling') and to extend the quantitative range of detecting T. cruzi in blood by at least 3 orders of magnitude relative to current protocols. When combined with sampling blood at multiple time points, deep sampling of fragmented DNA allowed for detection of T. cruzi in all infected hosts in multiple host species. In addition, we provide evidence for a number of characteristics not previously rigorously quantified in the population of hosts with naturally acquired T. cruzi infection, including, a > 6-log variation between chronically infected individuals in the stable parasite levels, a continuing decline in parasite load during the second and third years of infection in some hosts, and the potential for parasite load to change dramatically when health conditions change. Although requiring strict adherence to contamination-prevention protocols and significant resources, deep-sampling PCR provides an important new tool for assessing new therapies and for addressing long-standing questions in T. cruzi infection and Chagas disease. Author Summary Infection by the protozoan Trypanosoma cruzi normally results in a life-long, but low-level parasitization of muscle tissues, often leading to chagasic heart disease. A major challenge in the Chagas disease field has been the difficulty in detecting and quantifying parasite load in infected hosts. In this study we show that collection of serial blood samples and performance of sometimes high numbers of replicate PCR reactions on fragmented blood DNA, allows detection and quantification of relative parasite load in non-human primates, dogs, and humans with naturally acquired T. cruzi infection. This 'deep-sampling' approach reveals a mostly stable, 100,000-fold or greater difference in parasite load among chronically infected hosts and can detect alterations in parasite levels due to changes in health status or following therapeutic treatment in individual hosts, thus providing a powerful tool for assessing treatment outcomes in T. cruzi infection, including for evaluation of new therapeutics. Additionally, the ability to accurately and sensitively monitor parasite load in hosts provides the means to address highly contentious issues in the Chagas field, including the relative role of parasites and hosts in establishing the persistent parasite burden and the relationship between parasite burden and the presence and severity of clinical disease.