Abstract

Chronic Trypanosoma cruzi infection, known as Chagas disease, is a major cause of cardiomyopathy in the Americas. Detection of T. cruzi infection is complicated by low parasitemia, which often evades detection with conventional PCR. Deep-sampling qPCR improves sensitivity by running hundreds of replicates, but this approach is too labor- and cost-intensive for diagnostics or routine monitoring.We aimed to develop an improved PCR method for T. cruzi detection from blood samples that would reduce cost and labor, thereby making routine testing for Chagas disease possible.

Methods:
‍We evaluated a multiplexed, single molecule-based PCR platform that performs target amplification inside of ~30 million compartments in a 50 µL reaction and uses automated 3D imaging and analysis to count target-positive compartments. Using blood-extracted DNA from 45 Macaca fascicularis with acquired T. cruzi infections, we assessed detection of both satellite DNA (sDNA) and kinetoplast DNA (kDNA) across matched samples previously tested by deep-sampling qPCR.

Results:
‍Countable PCR matched the sensitivity of 10 to 388 qPCR replicates in a single reaction, achieving limits of detection as low as 4 copies for sDNA and 16 copies for kDNA. The system maintained high linearity across a near million-fold range of input DNA. Matched analysis showed strong correlation to qPCR Cq values, and multiplexed detection of parasite targets enhanced robustness of the assay.