New insights into Trypanosoma cruzi genetic diversity, and its influence on parasite biology and clinical outcomes.

Chagas disease, caused by Trypanosoma cruzi, remains a serious public health problem worldwide. The parasite was subdivided into six distinct genetic groups, called "discrete typing units" (DTUs), from TcI to TcVI. Several studies have indicated that the heterogeneity of T. cruzi species directly affects the diversity of clinical manifestations of Chagas disease, control, diagnosis performance, and susceptibility to treatment. Thus, this review aims to describe how T. cruzi genetic diversity influences the biology of the parasite and/or clinical parameters in humans. Regarding the geographic dispersion of T. cruzi, evident differences were observed in the distribution of DTUs in distinct areas. For example, TcII is the main DTU detected in Brazilian patients from the central and southeastern regions, where there are also registers of TcVI as a secondary T. cruzi DTU. An important aspect observed in previous studies is that the genetic variability of T. cruzi can impact parasite infectivity, reproduction, and differentiation in the vectors. It has been proposed that T. cruzi DTU influences the host immune response and affects disease progression. Genetic aspects of the parasite play an important role in determining which host tissues will be infected, thus heavily influencing Chagas disease's pathogenesis. Several teams have investigated the correlation between T. cruzi DTU and the reactivation of Chagas disease. In agreement with these data, it is reasonable to suppose that the immunological condition of the patient, whether or not associated with the reactivation of the T. cruzi infection and the parasite strain, may have an important role in the pathogenesis of Chagas disease. In this context, understanding the genetics of T. cruzi and its biological and clinical implications will provide new knowledge that may contribute to additional strategies in the diagnosis and clinical outcome follow-up of patients with Chagas disease, in addition to the reactivation of immunocompromised patients infected with T. cruzi.

Phenotypic, functional and serological aspects of genotypic-specific immune response of experimental T. cruzi infection.

The complexity and multifactorial characteristics of Chagas disease pathogenesis hampers the establishment of appropriate experimental/epidemiological sets, and therefore, still represents one of the most challenging fields for novel insights and discovery. In this context, we used a set of attributes including phenotypic, functional and serological markers of immune response as candidates to decode the genotype-specific immune response of experimental T. cruzi infection. In this investigation, we have characterized in C57BL/6 J mice, the early (parasitemia peak) and late (post-parasitemia peak) aspects of the immune response elicited by T. cruzi strains representative of TcI, TcII or TcVI. The results demonstrated earlier parasitemia peak for TcII/Y strain followed by TcVI/CL-Brener and TcI/Colombiana strains. A panoramic overview of phenotypic and functional features of the TCD4+, TCD8+ and B-cells from splenocytes demonstrated that mice infected with TcI/Colombiana strain exhibited at early stages of infection low levels of most cytokine+ cells with a slight increase at late stages of infection. Conversely, mice infected with TcII/Y strain presented an early massive increase of cytokine+ cells, which decreases at late stages. The TcVI/CL-Brener strain showed an intermediate profile at early stages of infection with a slight increase later on at post-peak of parasitemia. The panoramic analysis of immunological connectivity demonstrated that early after infection, the TcI/Colombiana strain trigger immunological network characterized by a small number of connectivity, selectively amongst cytokines that further shade towards the late stages of infection. In contrast, the TcII/Y strain elicited in more imbricate networks early after infection, comprising a robust number of interactions between pro-inflammatory mediators, regulatory cytokines and activation markers that also decrease at late infection. On the other hand, the infection with TcVI/CL-Brener strain demonstrated an intermediate profile with connectivity axes more stable at early and late stages of infection. The analysis of IgG2a reactivity to AMA, TRYPO and EPI antigens revealed that at early stages of infection, the genotype-specific reactivity to AMA, TRYPO and EPI to distinguish was higher for TcI/Colombiana as compared to TcII/Y and TcVI/CL while, at late stages of infection, higher reactivity to AMA was observed in mice infected with TcVI/CL and TcII/Y strains. The novel systems biology approaches and the use of a flow cytometry platform demonstrated that distinct T. cruzi genotypes influenced in the phenotypic and functional features of the host immune response and the genotype-specific serological reactivity during early and late stages of experimental T. cruzi infection.

Human Chagas-Flow ATE-IgG1 for advanced universal and Trypanosoma cruzi Discrete Typing Units-specific serodiagnosis of Chagas disease.

The molecular and serological methods available for Discrete Typing Units (DTU)-specific diagnosis of Trypanosoma cruzi in chronic Chagas disease present limitations. The study evaluated the performance of Human Chagas-Flow ATE-IgG1 for universal and DTU-specific diagnosis of Chagas disease. A total of 102 sera from Chagas disease patients (CH) chronically infected with TcI, TcVI or TcII DTUs were tested for IgG1 reactivity to amastigote/(A), trypomastigote/(T) and epimastigote/(E) antigens along the titration curve (1:250-1:32,000). The results demonstrated that "AI 250/40%", "EVI 250/30%", "AII 250/40%", "TII 250/40%" and "EII 250/30%" have outstanding accuracy (100%) to segregate CH from non-infected controls. The attributes "TI 4,000/50%", "EI 2,000/50%", "AVI 8,000/60%" and "TVI 4,000/50%" were selected for DTU-specific serotyping of Chagas disease. The isolated use of "EI 2,000/50%" provided the highest co-positivity for TcI patients (91%). The combined decision tree algorithms using the pre-defined sets of attributes showed outstanding full accuracy (92% and 97%) to discriminate "TcI vs TcVI vs TcII" and "TcI vs TcII" prototypes, respectively. The elevated performance of Human Chagas-Flow ATE-IgG1 qualifies its use for universal and TcI/TcVI/TcII-specific diagnosis of Chagas disease. These findings further support the application of this method in epidemiological surveys, post-therapeutic monitoring and clinical outcome follow-ups for Chagas disease.

Accomplishing the genotype-specific serodiagnosis of single and dual Trypanosoma cruzi infections by flow cytometry Chagas-Flow ATE-IgG2a.

The methods currently available for genotype-specific diagnosis of T. cruzi infection still present relevant limitations, especially to identify mixed infection. In the present investigation, we have evaluated the performance of Chagas-Flow ATE-IgG2a test for early and late differential diagnosis of single and dual genotype-specific T. cruzi infections. Serum samples from Swiss mice at early and late stages of T. cruzi infection were assayed in parallel batches for genotype-specific diagnosis of single (TcI, TcVI or TcII) and dual (TcI+TcVI, TcVI+TcII or TcII+TcI) infections. The intrinsic reactivity to TcI, TcVI and TcII target antigens, including amastigote (AI/AVI/AII), trypomastigote-(TI/TVI/TII) and epimastigote (EI/EVI/EII), at specific reverse of serum dilutions (500 to 64,000), was employed to provide reliable decision-trees for "early" vs "late", "single vs "dual" and "genotype-specific" serology. The results demonstrated that selective set of attributes "EII 500/EI 2,000/AII 500" were able to provide high-quality accuracy (81%) to segregate early and late stages of T. cruzi infection. The sets "TI 2,000/AI 1,000/EII 1,000" and "TI 8,000/AII 32,000" presented expressive scores to discriminate single from dual T. cruzi infections at early (85%) and late stages (84%), respectively. Moreover, the attributes "TI 4,000/TVI 500/TII 1,000", "TI 16,000/EI 2,000/EII 2,000/AI 500/TVI 500" showed good performance for genotype-specific diagnosis at early stage of single (72%) and dual (80%) T. cruzi infections, respectively. In addition, the attributes "TI 4,000/AII 1,000/EVI 1,000", "TI 64,000/AVI 500/AI 2,000/AII 1,000/EII 4,000" showed moderate performance for genotype-specific diagnosis at late stage of single (69%) and dual (76%) T. cruzi infections, respectively. The sets of decision-trees were assembled to construct a sequential algorithm with expressive accuracy (81%) for serological diagnosis of T. cruzi infection. These findings engender new perspectives for the application of Chagas-Flow ATE-IgG2a method for genotype-specific diagnosis in humans, with relevant contributions for epidemiological surveys as well as clinical and post-therapeutic monitoring of Chagas disease.

Performance of TcI/TcVI/TcII Chagas-Flow ATE-IgG2a for universal and genotype-specific serodiagnosis of Trypanosoma cruzi infection.

Distinct Trypanosoma cruzi genotypes have been considered relevant for patient management and therapeutic response of Chagas disease. However, typing strategies for genotype-specific serodiagnosis of Chagas disease are still unavailable and requires standardization for practical application. In this study, an innovative TcI/TcVI/TcII Chagas Flow ATE-IgG2a technique was developed with applicability for universal and genotype-specific diagnosis of T. cruzi infection. For this purpose, the reactivity of serum samples (percentage of positive fluorescent parasites-PPFP) obtained from mice chronically infected with TcI/Colombiana, TcVI/CL or TcII/Y strain as well as non-infected controls were determined using amastigote-AMA, trypomastigote-TRYPO and epimastigote-EPI in parallel batches of TcI, TcVI and TcII target antigens. Data demonstrated that "α-TcII-TRYPO/1:500, cut-off/PPFP = 20%" presented an excellent performance for universal diagnosis of T. cruzi infection (AUC = 1.0, Se and Sp = 100%). The combined set of attributes "α-TcI-TRYPO/1:4,000, cut-off/PPFP = 50%", "α-TcII-AMA/1:1,000, cut-off/PPFP = 40%" and "α-TcVI-EPI/1:1,000, cut-off/PPFP = 45%" showed good performance to segregate infections with TcI/Colombiana, TcVI/CL or TcII/Y strain. Overall, hosts infected with TcI/Colombiana and TcII/Y strains displayed opposite patterns of reactivity with "α-TcI TRYPO" and "α-TcII AMA". Hosts infected with TcVI/CL strain showed a typical interweaved distribution pattern. The method presented a good performance for genotype-specific diagnosis, with global accuracy of 69% when the population/prototype scenario include TcI, TcVI and TcII infections and 94% when comprise only TcI and TcII infections. This study also proposes a receiver operating reactivity panel, providing a feasible tool to classify serum samples from hosts infected with distinct T. cruzi genotypes, supporting the potential of this method for universal and genotype-specific diagnosis of T. cruzi infection.

TcI, TcII and TcVI Trypanosoma cruzi samples from Chagas disease patients with distinct clinical forms and critical analysis of in vitro and in vivo behavior, response to treatment and infection evolution in murine model.

The clonal evolution of Trypanosoma cruzi sustains scientifically the hypothesis of association between parasite's genetic, biological behavior and possibly the clinical aspects of Chagas disease in patients from whom they were isolated. This study intended to characterize a range of biological properties of TcI, TcII and TcVI T. cruzi samples in order to verify the existence of these associations. Several biological features were evaluated, including in vitro epimastigote-growth, "Vero"cells infectivity and growth, along with in vivo studies of parasitemia, polymorphism of trypomastigotes, cardiac inflammation, fibrosis and response to treatment by nifurtimox during the acute and chronic murine infection. The global results showed that the in vitro essays (acellular and cellular cultures) TcII parasites showed higher values for all parameters (growth and infectivity) than TcVI, followed by TcI. In vivo TcII parasites were more virulent and originated from patients with severe disease. Two TcII isolates from patients with severe pathology were virulent in mice, while the isolate from a patient with the indeterminate form of the disease caused mild infection. The only TcVI sample, which displayed low values in all parameters evaluated, was also originated of an indeterminate case of Chagas disease. Response to nifurtimox was not associated to parasite genetic and biology, as well as to clinical aspects of human disease. Although few number of T. cruzi samples have been analyzed, a discreet correlation between parasite genetics, biological behavior in vitro and in vivo (murine model) and the clinical form of human disease from whom the samples were isolated was verified.

Innovations in diagnosis and post-therapeutic monitoring of Chagas disease: Simultaneous flow cytometric detection of IgG1 antibodies anti-live amastigote, anti-live trypomastigote, and anti-fixed epimastigote forms of Trypanosoma cruzi.

This study developed a remarkable methodological innovation (FC-ATE) which enables simultaneous detection of antibodies specific to the three evolutive forms of Trypanosoma cruzi: live amastigote (AMA), live trypomastigote (TRYPO), and fixed epimastigote (EPI) using a differential fluorescence staining as low (AMA), intermediate (TRYPO), and high (EPI). An outstanding performance (100%) was observed in the discrimination of the chagasic (CH) and non-chagasic (NCH) patients. In the applicability of FC-ATE in the diagnosis of Chagas disease, 100% of the CH samples presented positivity in the percentage of positive fluorescent parasites (PPFP) for all the three forms of T. cruzi. Moreover, 94% of the samples of NCH presented negative values of PPFP with AMA and TRYPO, and 88% with EPI. Samples from the NCH group with false-positive results were those belonging to the leishmaniasis patients. Considering the applicability of this technique in post-therapeutic monitoring of Chagas disease, 100% of non-treated (NT) and treated non-cured (TNC) samples were positive with the three T. cruzi evolutive forms, while a percentage of 100% from samples of the treated cured (TC) patients were negative with AMA, 93% with TRYPO and 96% with EPI. The comparison between FC-ATE and two other flow cytometric tests using the same samples of patients NT, TNC and TC showed that the three techniques presented different reactivities, although categorical correlation between the methodologies was observed. Taken together, the results obtained with the novel FC-ATE method have shown an outstanding performance in the diagnosis and post-therapeutic monitoring of Chagas disease.