An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease.

Randomly amplified polymorphic DNA (RAPD) has emerged as an effective genetic marker for analysis of Trypanosoma cruzi population variability. This method has been used to study the genetic variability of Mexican T. cruzi stocks and to relate these results to previous classifications. High clonal diversity was observed among the Mexican populations: 24 RAPD types were scored among 56 stocks analyzed. Only two stocks (3.6%) belonged to the T. cruzi II lineage, while all others belonged to T. cruzi I. The robustness of these clusters was statistically highly significant. Mexican T. cruzi I stocks formed a homogeneous group with reduced genetic distances among its members. Parasites from this group were isolated from both domestic and sylvatic cycles over a broad geographic area in Mexico. The two Mexican stocks classified as T. cruzi II (isolated from sylvatic cycles) were of the same RAPD type, although they were not closely related to the three reference T. cruzi II stocks circulating in domestic cycles in Argentina, Brazil, Bolivia, and Chile. These stocks were also unrelated to the formerly named Zymodeme III.Trypanosoma cruzi, the agent of Chagas' disease, which is widely distributed in Latin America, shows considerable genetic polymorphism (14). Extensive studies carried out by using multilocus enzyme electrophoresis have demonstrated that natural populations of T. cruzi have a clonal structure (21), which has led to subdivision of the taxon into two major lineages, T. cruzi I and T. cruzi II (1,13,18,20). Each lineage is genetically heterogeneous, and despite failure to identify subdivisions within T. cruzi I, five subgroups of T. cruzi II have been proposed as discrete typing units (DTU) on the basis of randomly amplified polymorphic DNA (RAPD) analysis (1, 5). These six DTU have been proposed as a reference framework for genetic variability and for biological characterization studies of T. cruzi stocks.A few studies have attempted to characterize Mexican T. cruzi stocks by genetic methods. Seventeen stocks were analyzed using HindIII restriction fragment length polymorphisms associated with rRNA gene spacers and kinetoplast DNA maxicircle polymorphisms (9,12,22). These studies identified two groups of Mexican stocks, which correlate with geographic origin, although assignment to a T. cruzi lineage was not possible. The same stocks were also analyzed by multilocus enzyme electrophoresis, demonstrating a high degree of heterogeneity that was not related to geographic origin (12). Analysis of minicircle kDNA variability among Mexican stocks showed no correlation between the sequence homologies of the hypervariable region of minicircles and clustering (2), contrasting with results obtained for South American stocks (3, 4).In the present study, 56 Mexican stocks, isolated from diverse hosts and with diverse geographic origins, were analyzed by RAPD. Relevant phylogenetic analyses were used to assess population variability and structure, and to evaluate the taxonomic position of Mexican stocks and their corresp...

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An enzyme-linked immunosorbent assay to diagnose Chagas' disease by a serological test was performed with Trypanosoma cruzi recombinant antigens (JL8, MAP, and TcPo). High sensitivity (99.4%) and specificity (99.3%) were obtained when JL8 was combined with MAP (JM) and tested with 150 serum samples from chagasic and 142 nonchagasic individuals. Moreover, JM also diagnosed 84.2% of patients in the acute phase of T. cruzi infection.

It is has been shown that the majority of T. cruzi strains isolated from Mexico belong to the T. cruzi I (TCI). The immune response produced in response to Mexican T. cruzi I strains has not been well characterized. In this study, two Mexican T. cruzi I strains were used to infect Balb/c mice. The Queretaro (TBAR/MX/0000/Queretaro)(Qro) strain resulted in 100% mortality. In contrast, no mortality was observed in mice infected with the Ninoa (MHOM/MX/1994/Ninoa) strain. Both strains produced extended lymphocyte infiltrates in cardiac tissue. Ninoa infection induced a diverse humoral response with a higher variety of immunoglobulin isotypes than were found in Qro-infected mice. Also, a stronger inflammatory TH1 response, represented by IL-12p40, IFNγ, RANTES, MIG, MIP-1β, and MCP-1 production was observed in Qro-infected mice when compared with Ninoa-infected mice. We propose that an exacerbated TH1 immune response is a likely cause of pathological damage observed in cardiac tissue and the primary cause of death in Qro-infected mice.