An Improved Approach to Trypanosoma cruzi Molecular Genotyping by Next-Generation Sequencing of the Mini-exon Gene

Trypanosoma cruzi, the etiological agent of Chagas disease, is a protozoan parasite usually transmitted by triatomines. As the parasite can infect all mammals and the vectors can be found across a broad range of ecologies, transmission cycles are quite complex, and extensive genetic diversity exists within the parasite population. Seven main evolutionary lineages, named "discrete typing units," have been described, but a large amount of intra-lineage heterogeneity is also observed. To date, typing methods used to elucidate both inter-lineage and intra-lineage diversity have faced limitations, with some approaches unable to determine all levels of diversity and others requiring investigation of numerous markers and often the selective process of isolation of live parasites. Here, we present a method for parasite genotyping using next-generation sequencing of the mini-exon gene marker, to assign lineage and describe intra-lineage diversity directly from biological samples. This approach is sensitive enough to detect the presence of multiclonal infections and low-frequency parasite genotypes within this context, providing an unprecedented description of T. cruzi assemblages in hosts and vectors.