Serial ‘deep-sampling’ PCR of fragmented DNA reveals the wide range ofTrypanosoma cruziburden among chronically infected hosts and allows accurate monitoring of parasite load following treatment
Authors: White Brooke E. , Hodo Carolyn L. , Hamer Sarah A. , Saunders Ashley B. , Laucella Susana A. , Hall Daniel B. , Tarleton Rick L. 
Published: June 2024

AbstractInfection with the protozoan parasiteTrypanosoma cruziis 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 ofT. cruziinfection usually averts acute phase death but has the negative consequence that the low-level presence ofT. cruziin 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’) to extend the quantitative range of detectingT. cruziin 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 ofT. cruziin 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 acquiredT. cruziinfection, 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 inT. cruziinfection and Chagas disease.Author SummaryInfection by the protozoanTrypanosoma cruzinormally 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 acquiredT. cruziinfection. 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 inT. cruziinfection, 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.