Discrete Typing Units of Trypanosoma cruzi Identified by Real-Time PCR in Peripheral Blood and Dejections of Triatoma infestans Used in Xenodiagnosis Descriptive Study

Abstract

Chagas disease (ChD) is a vector zoonosis native to the American continent caused by the protozoan parasite Trypanosoma cruzi; the biological vectors are multiple species of hematophagous insects of the family Triatominae. A relevant aspect in the host–parasite relationship is the identification of the various genotypes of T. cruzi called discrete typing units (DTU) that circulate in mammals and vectors. In Chile, it has been described that the DTUs TcI, TcII, TcV, and TcVI circulate in infected humans, vectors, and wild animals. Identifying DTUs has acquired clinical importance, since it has been suggested that different genotypes could cause distinct pathologies, circulate in different geographical areas, and present different sensitivities to trypanocidal drugs. In this study, circulating T. cruzi DTUs in peripheral blood and Triatoma infestans dejections used in xenodiagnosis (XD) were amplified by qPCR in 14 Chilean patients with chronic ChD from highly endemic areas. More positive samples were detected by XD compared to peripheral blood samples, and 64.28% of the cases were simple infections and 35.72% mixed, with a statistically significant difference in the frequency of TcV DTU. This study would suggest that T. infestans from Chile is more competent to amplify one DTU over others, probably due to a process of co-evolution.

Parasite Burden of Trypanosoma cruzi in Whole Blood and Buffy Coat Determined by Real-Time PCR in Individuals with Chronic Chagas Disease

“…For the quantification of T. cruzi DNA in clinical samples, a standard curve was prepared from the extraction of DNA from epimastigotes of clones belonging to strains Dm28c (TcI) and Y (TcII), which are DTUs circulating with high frequency in Chile [ 30 , 31 ], from axenic cultures. A 1:1 mixture of the parasite strains was made containing 1 × 10 6 epimastigotes, considering that each parasite cell has approximately 200 fg of DNA; that is, 1,000,000 parasites-eq/mL corresponds to a concentration of 0.2 ng/μL of DNA [ 32 , 33 , 34 , 35 ].…”

“…Subsequently, the mixture was diluted to reach a concentration of 0.2 ng/μL of DNA, obtaining the mother tube from which the different points of the calibration curve were prepared. From the tube with 1 × 10 6 parasites, ten-fold serial dilutions (100,000, 10,000, 1000, 100, 10, 1 and 0.1 par-eq/mL) were prepared, using a pool of negative DNA (from healthy individuals without ChD) [ 17 , 31 ] with the three types of samples (boiled GEB, nonboiled GEB and BC). A standard curve was constructed for each methodology used.…”