Trypanosoma Cruzi: Standardization and Validation of Mitochondrial Rt-MTQPCR Diagnosis in Samples From the Amazon

Chagas disease, caused by Trypanosoma cruzi, has great epidemiological importance in the Amazon. Transmitted by more than 130 insect species, T. cruzi has a region called the kinetoplast, delimited by a single mitochondrion rich in circular DNA, with maxi-circles and mini-circles, thus accumulating large quantities of genetic material. In this context, it is essential to establish molecular techniques for the diagnosis of American trypanosomiasis. Therefore, the objective was to develop a molecular diagnostic protocol based on RT-mtqPCR with high sensitivity and specificity for Chagas disease, in order to identify signals that may influence the course of infection in patients with mild and severe forms of the disease. Twenty-five samples were collected ‒ whole blood (17) and stool (8) ‒ from patients with acute Chagas disease, followed by extraction of genetic material. After this step, RT-qPCR was performed using mitochondrial targets designed by the authors, a technique whose differential is the ability to measure the absolute quantities of DNA or RNA, thus allowing discrimination of parasite load levels among patients, followed by analysis of results using the Wilcoxon test and Fisher’s exact test. Regarding clinical findings, the presence of facial edema had a p-value below 0.015, demonstrating significance in the prognosis of patients regarding severity. In addition, among the analyzed samples, the results showed no correlation between parasite load levels in the body and time of disease, as well as with treatment follow-up using appropriate medication. No important differences were found between protozoan concentration in blood or stool samples. However, three (3) samples showed no detectable parasite loads in stool. Furthermore, this project highlights the pioneering use of human stool samples using the described method. Therefore, the application of RT-mtqPCR proved efficient, especially in blood, enabling detection of Trypanosoma cruzi genetic material even at low concentrations. Finally, it would be possible to consider stool analysis to estimate the presence of the protozoan in the gastrointestinal tract, in addition to highlighting the need to develop new treatments for Chagas disease.