Enhancing Trypanosomatids Identification and Genotyping with Oxford Nanopore Sequencing: Development and Validation of an 18S rRNAs Amplicon-Based Method.

Trypanosomatids, including Trypanosoma and Leishmania species, present significant medical and veterinary challenges causing substantial economic losses, health complications, and even fatalities. Diagnosing and genotyping these species and their genotypes is often complex, involving multiple steps. This study aimed to develop an amplicon-based sequencing (ABS) method utilizing Oxford Nanopore long-read sequencing to enhance Trypanosomatid detection and genotyping. The 18S rDNA gene was targeted for its inter-species conservation. The Trypanosomatid-ABS method effectively distinguished between eleven Trypanosoma species (T. evansi, T. theileri, T. vivax, T. rangeli) and six T. cruzi Discrete Typing Units (DTUs), showing strong concordance with conventional methods (kappa index of 0.729, p-value <0.001). It detected co-infections between Trypanosomatid genera and T. cruzi, with a limit of detection of one parasite per mL. The method was successfully applied to human, animal, and triatomine samples. Notably, TcI predominated in chronic Chagas samples, while TcII and TcIV were found in the acute stage. Triatomine vectors exhibited diverse Trypanosomatid infections, with T. dimidiata mainly infected with TcI and occasional TcBat co-infections, and R. prolixus showing TcI and TcII infections, along with T. rangeli co-infections and mixed TcII infections. Animals were infected with T. vivax, T. theileri, and T. evansi. The ABS method's high resolution, sensitivity, and accuracy make it a valuable tool for understanding Trypanosomatid dynamics, enhancing disease control strategies, and enabling targeted interventions.