Impact of dual infections on chemotherapeutic efficacy in BALB/c mice infected with major genotypes of Trypanosoma cruzi

Abstract

The aim of this work was to investigate the impact of dual infections with stocks of Trypanosoma cruzi major genotypes on benznidazole (BZ) treatment efficacy. For this purpose, T. cruzi stocks representative of the genetic T. cruzi lineages, displaying different susceptibilities to BZ, belonging to the major T. cruzi genotypes broadly dispersed in North and South America and important in Chagas' disease epidemiology were used. Therapeutic efficacy was observed in 27.8% of the animals treated. Following BZ susceptibility classification, significant differences were observed in dual infections on the major genotype level, demonstrating that combinations of genotypes 19+39 and genotypes 19+32 led to a shift in the expected BZ susceptibility profile toward the resistance pattern. Analysis on the T. cruzi stock level demonstrated that 9 out of 24 dual infections shifted the expected BZ susceptibility profile compared with the respective single infections, including shifts toward lower and higher BZ susceptibilities. Microsatellite identification was able to identify a mixture of T. cruzi stocks in 7.7% of the T. cruzi isolates from infected and untreated mice (6.9%) and infected and treated but not cured mice (9.0%), revealing in some mixtures of BZ-susceptible and -resistant stocks that the T. cruzi stock identified after BZ treatment was previously susceptible in single infections. Considering the clonal structure and evolution of T. cruzi, an unexpected result was the identification of parasite subpopulations with distinct microsatellite alleles in relation to the original stocks observed in 12.2% of the isolates. Taken together, the data suggest that mixed infections, already verified in nature, may have an important impact on the efficacy of chemotherapy.

FIG. 1.

Profile of T. cruzi susceptibility to BZ. The left panel shows the cure rates for single and dual infections with T. cruzi lineages (T. cruzi I, T. cruzi, and T. cruzi II). The right panel shows the cure rates for single and dual infections with genotypes 20, 19, 39, and 32. Different letters represent significant differences at P < 0.05 between the observed cure rates of the T. cruzi genetic groups studied. An asterisk represents a shift in the BZ susceptibility profile based on the observed and expected cure rates for dual infections regarding the susceptibility pattern proposed by Filardi and Brener (15). ▪, resistant (cure rates of ≤33%); □, partially susceptible (cure rates of 33% to ≤66%); □, susceptible (cure rates of >66%).

FIG. 2.

BZ susceptibility profile of single and dual infections with T. cruzi stocks of different genotypes. T. cruzi genotypes and stocks in bold are resistant to BZ (genotypes 20 and 39), those in gray are partially susceptibility to BZ (genotypes 19 and 32), and those in normal type are susceptible to BZ (genotypes 19, 39, and 32). Results are expressed as observed cure rates (dual infections) and expected cure rates (based on a theoretical dual infection, expressed as the susceptibility profile of the T. cruzi stock more resistant in the respective single infection). An asterisk represents a shift in the BZ susceptibility profile based on differences between the observed and expected cure rates for dual infections and the observed and expected cure rates regarding the susceptibility profile proposed by Filardi and Brener (15). ▪, resistant (cure rates of ≤33%); □, partially susceptible (cure rates of 33% to ≤66%); □, susceptible (cure rates of >66%). Connecting lines highlight dual infections that show a shift between the observed and expected cure rates.