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 three 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, including humans, macaques, and dogs. 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 therapies and for addressing long-standing questions in T. cruzi infection and Chagas disease.
Keywords: Chagas disease; PCR; Trypanosoma cruzi; infectious disease; microbiology; test of cure; trypanosoma cruzi.
Chagas disease is a dangerous tropical illness caused by single-cell parasites known as Trypanosoma cruzi. In most cases, if not treated immediately, the infection becomes chronic: the immune system of the host greatly reduces the number of parasites present in the body yet fails to fully eradicate them. Current diagnostic approaches often fail to detect these low numbers of parasites. More broadly, without a reliable way to measure Trypanosoma cruzi levels, researchers and clinicians struggle to test new treatments as well as determine whether patients carrying more parasites tend to develop more severe disease. In response, White et al. aimed to develop a new way of measuring parasitic loads. They based their approach on standard PCR (polymerase chain reaction), an experimental method that amplifies specific DNA sequences in proportion to their initial levels in a sample. This allows researchers to not only pinpoint the presence of the parasites, but also to assess their relative number However, the approach has limited sensitivity: if the amount of target DNA initially collected is too low, it may not be detected. To bypass this limitation, White et al. adopted a ‘deep-sampling PCR’ approach and made several crucial changes. First, they collected several samples from the same patient, therefore increasing the overall sampling volume. Second, they fragmented the sample DNA before the amplification step in order to disperse the target DNA, thus increasing the chances of its detection in each PCR reaction. Third, they performed as many as 400 PCRs per sample. These modifications greatly improved sensitivity compared to usual approaches. White et al. used their newly improved method to examine Trypanosomas cruzi levels in infected macaques over long periods. The results show that parasitic burden can be stable over a year, but vastly differs between individuals (with some having a million times more parasites than others). Similar findings were also observed in humans and dogs. The method developed by White et al. is likely to be too labour intensive to be routinely used in diagnostic laboratories; however, it represents an important and immediate advance for researchers testing new compounds to treat Chagas disease.
© 2025, White et al.