The benznidazole (BZ) and itraconazole (ITC) susceptibilities of a standard set of Trypanosoma cruzi natural stocks were evaluated during the acute phase and the chronic phase of experimental chagasic infection in BALB/c mice. Twenty laboratory-cloned stocks representative of the total phylogenetic diversity of T. cruzi, including genotypes 20 and 19 (T. cruzi I) and genotypes 39 and 32 (T. cruzi II), were analyzed. Our results demonstrate important differences among stocks that could be pointed out as markers of biological behavior. Members of the T. cruzi I group were highly resistant to both BZ and ITC, whereas members of the T. cruzi II group were partially resistant to both drugs, despite their susceptibilities to ITC during the chronic phase of infection. The resistance to BZ observed for T. cruzi I was mainly triggered by genotype 20 isolates, whereas resistance to ITC was due to both genotype 20 and 19 isolates. Two polar patterns of response to BZ observed for genotype 39 isolates had a major impact on the partial resistance pattern observed for members of the T. cruzi II group. Genotype 32 isolates showed a typical profile of susceptibility. The correlation between the response to treatment and phylogenetic classification of T. cruzi stocks was clearer for ITC than for BZ. In conclusion, the data presented show a correlation between phylogenetic divergence among T. cruzi stocks and their susceptibilities to chemotherapeutic agents in vivo. Our results warn of the necessity to take into account the lesser genetic subdivisions of T. cruzi stocks since the upper subdivisions (T. cruzi I and II) show a great deal of heterogeneity for in vivo drug susceptibility.

Twenty Trypanosoma cruzi stocks attributed to the 19, 20, 39, and 32 clonal genotypes were comparatively studied in BALB/c mice during the acute and chronic phases of the infection to test the working hypothesis that T. cruzi clonal structure has a major impact on its biological properties. Fourteen parameters were assayed: (1) infectivity; (2) prepatent period; (3) patent period; (4) maximum of parasitemia; (5) day of maximum of parasitemia; (6) parasitemia; (7) mortality, (8) percentage of positive hemoculture, (9) tissue parasitism; (10) inflammatory process during the acute phase of the infection; (11) mortality, (12) percentage of positive hemoculture; (13) tissue parasitism; and (14) inflammatory process during the chronic phase of the infection. Statistical comparison showed that the results are overall consistent with the working hypothesis that biological differences are proportional to the evolutionary divergence among the genotypes. Thus, closely related genotypes (19 vs 20 and 32 vs 39) show in general fewer differences than distantly related groups (19 or 20 vs 32 or 39) except for the comparison between 19 and 32. The working hypothesis is even more strongly supported by the result of the nonparametric Mantel test, which showed a highly significant correlation (P = 2.3 x 10(-3)) between biological differences and genetic distances among all pairs of stocks. These data taken together emphasize that it is crucial to take into account the phylogenetic diversity of T. cruzi natural clones in all applied studies dealing with diagnosis, drug and vaccine design, epidemiological surveys, and clinical diversity of Chagas' disease. Index Descriptors and Abbreviations: Trypanosoma cruzi; phylogenetic distance; biological properties; clonal theory; multilocus enzyme electrophoresis (MLEE); randomly amplified polymorphic DNA (RAPD); acute phase (AP); chronic phase (CP); days after inoculation (d.a.i.); liver infusion tryptose (LIT); gastrointestinal tract (GIT); genitourinary tract (GUT); percentage of infectivity (%INF); percentage of mortality during the acute phase (%MORT AP); percentage of mortality during the chronic phase (%MORT CP); prepatent period (PPP); patent period (PP); maximum of parasitemia (MP); day of maximum of parasitemia (DMP); parasitemia (PAR); percentage of positive hemoculture during the acute phase (% + HC AP); percentage of positive hemoculture during the chronic acute phase (% + HC CP); tissue parasitism (TP); inflammatory process (IP); tissue parasitism during the acute phase (TP AP); tissue parasitism during chronic phase (TP CP); inflammatory process during acute phase (IP AP); inflammatory process chronic phase (IP CP); Mann-Whitney test (MW); Kruskal-Wallis (KW); Kolmogorow-Smirnov test (KS).

BackgroundChagas disease is an emergent tropical disease in the Brazilian Amazon Region, with an increasing number of cases in recent decades. In this region, the sylvatic cycle of Trypanosoma cruzi transmission, which constitutes a reservoir of parasites that might be associated with specific molecular, epidemiological and clinical traits, has been little explored. The objective of this work is to genetically characterize stocks of T. cruzi from human cases, triatomines and reservoir mammals in the State of Amazonas, in the Western Brazilian Amazon.Methodology/Principal FindingsWe analyzed 96 T. cruzi samples from four municipalities in distant locations of the State of Amazonas. Molecular characterization of isolated parasites from cultures in LIT medium or directly from vectors or whole human blood was performed by PCR of the non-transcribed spacer of the mini-exon and of the 24 S alfa ribosomal RNA gene, RFLP and sequencing of the mitochondrial cytochrome c oxidase subunit II (COII) gene, and by sequencing of the glucose-phosphate isomerase gene. The T. cruzi parasites from two outbreaks of acute disease were all typed as TcIV. One of the outbreaks was triggered by several haplotypes of the same DTU. TcIV also occurred in isolated cases and in Rhodnius robustus. Incongruence between mitochondrial and nuclear phylogenies is likely to be indicative of historical genetic exchange events resulting in mitochondrial introgression between TcIII and TcIV DTUs from Western Brazilian Amazon. TcI predominated among triatomines and was the unique DTU infecting marsupials.Conclusion/SignificanceDTU TcIV, rarely associated with human Chagas disease in other areas of the Amazon basin, is the major strain responsible for the human infections in the Western Brazilian Amazon, occurring in outbreaks as single or mixed infections by different haplotypes.