A Genome-Wide Association Study Identifies Novel Susceptibility loci in Chronic Chagas Cardiomyopathy.

BACKGROUND: Chagas disease is an infectious disease caused by the parasite Trypanosoma cruzi and is endemic from Latin American countries. The goal of our study was to identify novel genetic loci associated with chronic Chagas cardiomyopathy development in Chagas disease patients from different Latin American populations. METHODS: We performed a cross-sectional, nested case-control study including 3 sample collections from Colombia, Argentina, and Bolivia. Samples were genotyped to conduct a genome-wide association study (GWAS). These results were meta-analyzed with summary statistic data from Brazil, gathering a total of 3413 Chagas disease patients. To identify the functional impact of the associated variant and its proxies, we performed an in silico analysis of this region. RESULTS: The meta-analysis revealed a novel genome-wide statistically significant association with chronic Chagas cardiomyopathy development in rs2458298 (OR = 0.90, 95%CI = 0.87-0.94, P-value = 3.27 × 10-08), nearby the SAC3D1 gene. In addition, further in silico analyses displayed functional relationships between the associated variant and the SNX15, BAFT2, and FERMT3 genes, related to cardiovascular traits. CONCLUSIONS: Our findings support the role of the host genetic factors in the susceptibility to the development of the chronic cardiac form of this neglected disease.

In vitro and in vivo drug combination for the treatment of Trypanosoma cruzi infection: A multivariate approach.

Combination therapies based on the available drugs have been proposed as promising therapeutic alternatives for many diseases. Clomipramine (CLO) has been found to modify the evolution of the experimental infection. The objective of this study was to evaluate the combined effect of benznidazole (BZ) and clomipramine (CLO) against different life-stages of Trypanosoma cruzi in vitro and their efficacy in a murine model. Life-stages of T. cruzi, BZ-partially-resistant (Y) strain, were incubated with BZ and CLO and isobolograms and combination index (CI) were obtained. Swiss mice were infected with trypomastigotes and different treatment schedules were performed, each of which consisted of 30 consecutive daily doses. Treatment efficacy was evaluated by comparing parasitemia, qPCR, survival and histological analysis. These results were analyzed using multivariate analysis to determine the combined effect of the drugs in vivo. CLO + BZ showed synergistic activity in vitro against the clinically relevant life-stages of T. cruzi. The most susceptible forms were the intracellular amastigotes (CI: 0.20), followed by trypomastigotes (CI: 0.60), with no toxicity upon mammalian cells. The combination of both drugs CLO (1.25 mg/kg) and BZ (6.25 mg/kg), in vivo, significantly diminished the parasitic load in blood and the mortality rate. CLO + BZ presented a similar inflammatory response in cardiac and skeletal muscle (amount of inflammatory cells) to BZ (6.25 mg/kg). Finally, the results from the principal component analysis reaffirmed that both drugs administered in combination presented higher activity compared with the individual administration in the acute experimental model.

Quantitative PCR and unconventional serological methods to evaluate clomipramine treatment effectiveness in experimental Trypanosoma cruzi infection.

Clomipramine (CLO), a tricyclic antidepressant drug, has been used for the treatment of mice infected with Trypanosoma cruzi. In this work we evaluated the effectiveness of CLO treatment upon T. cruzi-infected mice in the chronic phase of the experimental infection using Quantitative polymerase chain reaction (qPCR) and recombinant ELISA. Sixty Swiss albino mice were inoculated with 50 trypomastigote forms of T. cruzi (Tulahuen strain). CLO treatment consisted of 5mg/kg/day during 60days by intraperitoneal injection, beginning on day 90 post infection (p.i) when the mice presented electrocardiographic (ECG) alterations compatible with the chronic phase of the disease. The evolution of experimental infection and the treatment efficacy were studied through survival, electrocardiography, serology using a mixture and individual (1, 2, 13, 30, 36 and SAPA) recombinant proteins from epimastigotes and trypomastigotes of T. cruzi; and qPCR on days 180 and 270 p.i. CLO treatment in the chronic phase decreased the parasite load, reduced the levels of antibodies against antigen 13 throughout 270days p.i and reversed the ECG abnormalities in the treated animals, from 100% of the mice with alterations at the beginning of the treatment to only 20% of the mice with alterations by day 270 p.i. This study shows that qPCR and the use of recombinant antigens are more sensitive to evaluate the effectiveness of the treatment and proves that clomipramine may be considered as a new chemotherapy for the chronic phase of the disease.

Chronic indeterminate phase of Chagas' disease: mitochondrial involvement in infection with two strains.

Chagasic cardiopathy has become one of the most frequent causes of heart failure and sudden death, as well as one of the most common causes of cardio-embolic stroke in Latin America. The myocyte response to oxidative stress involves the progression of cellular changes, primarily targeting the mitochondria and modifying therefore the energy supply. In this paper we analysed the effect of the infection of mice with 2 different strains of Trypanosoma cruzi (Tulahuen and SGO Z12) in the chronic indeterminate stage (75 days post-infection), upon the structure and function of cardiac mitochondria. The structural results showed that 83% of the mitochondria from the Tulahuen-infected mice presented an increase in their matrix and 91% of the mitochondria from the SGO Z12-infected group showed a reduction in their diameter (P < 0.05). When the Krebs cycle and mitochondrial respiratory chain functionality was analysed through the measurement of the citrate synthase and complexes I to IV activity, it showed that their activity was altered in all cases in a similar manner in both infected groups. In this paper we have demonstrated that the chronic indeterminate phase is not 'silent' and that cardiac mitochondria are clearly involved in the genesis and progression to the chronic chagasic cardiopathy when different factors alter the host-parasite equilibrium.

SCN5A gene variants as potential markers of the progression of chronic chagasic cardiac alterations.

Host genetic factors have been proposed as determinants of the variable progression of Chagas disease (ChD). Two polymorphisms, H558R and A572D, of the voltage-gated sodium channel α-subunit SCN5A gene were studied in chagasic patients in order to determine their contribution to the susceptibility to the development and/or to the progression of the cardiovascular disease. A total of 104 patients were classified as seronegative or seropositive for Trypanosoma cruzi antibodies. Clinical evaluation, electrocardiograms (ECG) and echocardiograms (Echo) were performed to detect any conduction and/or structural alteration. Patients were classified into: G1: without ECG and/or Echo alterations, G2: with ECG alterations and G3: with ECG and Echo alterations. H558R and A572D polymorphisms were detected by PCR. Cardiac alterations were more frequent in G2 + G3 seropositive patients. For H558R polymorphism, the C allele was significantly increased in seropositive G2 + G3 patients (P = 0.049. OR = 2.08; 95% CI = 1.12-4.33). When comparing the disease cardiac progression (G2 vs G3), the genotypes from the H558R polymorphism were associated to more intense cardiac alterations (P = 0.018). For A572D polymorphism, no associations were found. The results suggest a possible involvement of SCN5A polymorphisms in the susceptibility to chronic ChD and the disease progression, contributing to the elucidation of the molecular mechanism underlying this complex myocardiopathy. In this regard, this is the first work that studies this gene in the context of chagasic cardiomyopathy.

Association of clomipramine and allopurinol for the treatment of the experimental infection with Trypanosoma cruzi.

We have previously shown that clomipramine and allopurinol used separately are effective in preventing chronic chagasic cardiomyopathy. The aim of the present study was to evaluate the effect of the association of clomipramine (Clo--5 mg/kg/day/90 days) and allopurinol (Allo--5, 10, or 15 mg/kg/day/90 days) for the treatment of experimental Chagas disease in the acute stage. Treatment effectiveness was evaluated through parasitemia, survival, electrocardiography, serology, and cardiac histopathology. Groups treated showed no electrocardiographic abnormalities, in contrast to those untreated which presented 25% of mice with conduction alterations. The myocardium of treated mice (Clo, Allo10+Clo, and Allo15+Clo) presented no structural alterations. Cardiac b-receptor affinity was preserved in mice treated with Clo or Clo+Allo at the different doses; receptor density of the Clo and Allo15+Clo groups did not differ from the non-infected group. Anti-cruzipain antibody levels were similar in treated and untreated groups. Survival was significantly increased in the treated groups (p < 0.05), with Clo and all the Clo+Allo groups presenting the highest rates. These results show that the association of clomipramine + allopurinol is effective for Chagas disease treatment and has the same effect as clomipramine alone.

[Goniometry of flexion-extension ankle movement: comparative analysis between bone reference method and neutral 0 method]. / Goniometría del movimiento de flexo-extensión de tobillo: análisis comparativo entre método de referencias óseas y método 0 neutral.

Introduction: Two different methods are used in goniometry of the ankle: the neutral zero method (N0M) and the bone reference method (BRM). In addition, the N0M has a subtype (N0I), with a different technique. Purpose: To determine the average of the amplitude of flexion-extension of the ankle, measured in different body positions, using N0M, N0I and BRM, in young adults of both sexes, with the objective of providing evidence so that the ankle goniometry is more reliable. Material and methods: 190 students from the School of Kinesiology and Physiotherapy were studied, using the three methods of joint measurement in 4 different body positions; the amplitude of flex-extension in an ankle per student was evaluated. Results: In most positions, the measurements were different in the three methods compared (P<0.05). The M0 and M0I methods yielded similar results in some comparisons. The patient's position also significantly influences the result obtained. Dorsal ankle flexion was similar between men and women in most of the methods and positions; the plantar flexion that was greater in women in all cases (P<0.0001). The full flex-extension value, in most cases, was higher in women than in men (P<0.001). Conclusions: Both the method and the patient's position significantly influence the results of the goniometric measurement. Gender influences the joint width of the plantar ankle flexion, regardless of the measurement method used.

Introducción: En la goniometría de tobillo se utilizan dos métodos diferentes, el método cero neutral (M0N) y el método de referencias óseas (MRO). Además, el M0N tiene un subtipo (M0I), con una técnica diferente. Objetivo: Determinar el promedio de la amplitud de flexo-extensión de tobillo, medida en diferentes posiciones corporales, utilizando M0N, M0I y MRO, en adultos jóvenes de ambos sexos, con el objetivo de aportar evidencia para que la goniometría de tobillo sea más fiable. Materiales y Métodos: Se estudiaron 190 alumnos de la Escuela de Kinesiología y Fisioterapia, utilizando los tres métodos de medición articular en 4 posiciones corporales diferentes; se evaluó la amplitud de flexo-extensión en un tobillo por alumno. Resultados: En la mayoría de las posiciones, las mediciones fueron diferentes en los tres métodos comparados (P<0,05). Los métodos M0 y M0I arrojaron resultados similares en algunas comparaciones puntuales. La posición del paciente también influye significativamente en el resultado obtenido. La flexión dorsal de tobillo fue similar entre hombres y mujeres en la mayoría de los métodos y posiciones del paciente, no así la flexión plantar que fue mayor en las mujeres en todos los casos (P<0,0001). El valor completo de flexo-extensión, en la mayoría de los casos fue mayor en las mujeres que en los varones (P<0,001). Conclusiones: Tanto el método como la posición del paciente influyen significativamente en los resultados de la medición goniométrica. El género influye en la amplitud articular de la flexión plantar de tobillo, independientemente del método de medición utilizado.

Lack of Association of IL6 polymorphism with the susceptibility to Chagas disease in Latin American populations.

The aim of the present study was to analyze IL6 rs1800795 genetic variant in the susceptibility to Trypanosoma cruzi infection and in the development of chronic Chagas cardiomyopathy (CCC), in five independent Latin American cohorts. A total of 3,087 individuals from Latin American countries (Argentina, Bolivia, Peru, and two cohorts from Colombia) were studied. In all cohorts, patients were classified as seropositive for T. cruzi antigens (n= 1,963) and seronegative (n= 1,124). Based on clinical evaluation, the seropositive patients, were classified as CCC (n= 900) and asymptomatic (n= 1,063). No statistically significant differences in the frequency of IL6 rs1800795 between seropositive and seronegative, or between CCC and asymptomatic patients, were found. Furthermore, after the meta-analysis no statistically significant differences were observed. Our results do not support a contribution of IL6 rs1800795 genetic variant in the susceptibility to the infection and the development of chronic Chagas cardiomyopathy in the studied populations.

Genetic polymorphisms of IL17A associated with Chagas disease: results from a meta-analysis in Latin American populations.

Genetic factors and the immunologic response have been suggested to determine the susceptibility against the infection and the outcome of Chagas disease. In the present study, we analysed three IL17A genetic variants (rs4711998, rs8193036 and rs2275913) regarding the predisposition to Trypanosoma cruzi infection and the development of chronic Chagas cardiomyopathy (CCC) in different Latin American populations. A total of 2,967 individuals from Colombia, Argentina, Bolivia and Brazil, were included in this study. The individuals were classified as seronegative and seropositive for T. cruzi antigens, and this last group were divided into asymptomatic and CCC. For T. cruzi infection susceptibility, the IL17A rs2275913*A showed a significant association in a fixed-effect meta-analysis after a Bonferroni correction (P = 0.016, OR = 1.21, 95%CI = 1.06-1.41). No evidence of association was detected when comparing CCC vs. asymptomatic patients. However, when CCC were compared with seronegative individuals, it showed a nominal association in the meta-analysis (P = 0.040, OR = 1.20, 95%CI = 1.01-1.45). For the IL17A rs4711998 and rs8193036, no association was observed. In conclusion, our results suggest that IL17A rs2275913 plays an important role in the susceptibility to T. cruzi infection and could also be implicated in the development of chronic cardiomyopathy in the studied Latin American population.

Association of IL18 genetic polymorphisms with Chagas disease in Latin American populations.

Host genetic factors have been suggested to play an important role in the susceptibility to Chagas disease. Given the influence of interleukin 18 (IL-18) in the development of the disease, in the present study, we analyzed three IL18 genetic variants (rs2043055, rs1946518, rs360719) regarding the predisposition to Trypanosoma cruzi infection and the development of chronic Chagas cardiomyopathy (CCC), in different Latin America populations. Genetic data of 3,608 patients from Colombia, Bolivia, Argentina, and Brazil were meta-analyzed to validate previous findings with increased statistical power. Seropositive and seronegative individuals were compared for T. cruzi infection susceptibility. In the Colombian cohort, the allelic frequencies of the three variants showed a significant association, with adjustment for sex and age, and also after applying multiple testing adjustments. Among the Colombian and Argentinean cohorts, rs360719 showed a significant genetic effect in a fixed-effects meta-analysis after a Bonferroni correction (OR: 0.76, CI: 0.66-0.89, P = 0.001). For CCC, the rs2043055 showed an association with protection from cardiomyopathy in the Colombian cohort (OR: 0.79, CI: 0.64-0.99, P = 0.037), with adjustment for sex and age, and after applying multiple testing adjustments. The meta-analysis of the CCC vs. asymptomatic patients from the four cohorts showed no evidence of association. In conclusion, our results validated the association found previously in the Colombian cohort suggesting that IL18 rs360719 plays an important role in the susceptibility to T. cruzi infection and no evidence of association was found between the IL18 genetic variants and CCC in the Latin American population studied.

Mitochondrial involvement in chronic chagasic cardiomyopathy.

The pathogenesis of chronic chagasic cardiopathy is still under discussion; there is considerable evidence that inflammatory infiltrates and their mediators have a direct effect on cardiac cells. Here we studied the structure and function of cardiac mitochondria in chronic chagasic myocardiopathy. Cardiac mitochondrial structure and enzyme activity of citrate synthase and complexes I to IV of the respiratory chain were studied in albino Swiss mice infected with Trypanosoma cruzi (Tulahuen strain or SGO Z12 isolate) on 365 days post-infection (dpi). The presence of parasites in cardiac and skeletal muscle was also investigated. The activity of complexes I to IV was altered in different ways, according to the strain employed (P<0.0001), in relation to the cristae disorganisation and the parasite persistence found in the Tulahuen group, and the chronic inflammatory process described in both groups; citrate synthase activity also increased in both infected groups. Changes in mitochondrial structure were detected in 89% of Tulahuen- and 58% of SGO Z12-infected mice. In this paper we demonstrate that parasite persistence and inflammation are likely to be involved in the structural and functional alterations in cardiac mitochondria from chronically T. cruzi-infected mice, demonstrating that the parasite strain determines different mitochondrial changes in chagasic cardiopathy.

Chemotherapy of chronic indeterminate Chagas disease: a novel approach to treatment.

Treatment of Chagas disease is a controversial issue because the available drugs are highly toxic. Clomipramine is a tricyclic antidepressant drug that inhibits Trypanosoma cruzi's trypanothione reductase, provoking the death of the parasite and preventing the cardiac damage when used for the treatment of acutely infected mice. Here, we studied the effectiveness of clomipramine (5 mg/kg/day for one month) as chemotherapy for T. cruzi-infected mice in the chronic indeterminate stage of the infection. The animals were analyzed in the cardiac chronic phase. Survival of treated animals was 84% while for the untreated ones was 40%; most of the animals presented electrocardiographic abnormalities. Affinity and density of cardiac beta receptors from infected and treated mice were similar to those in the indeterminate phase, showing that clomipramine treatment stopped the increment of functional alterations provoked by the infection, while untreated mice presented affinity and density significantly diminished. Hearts from infected and untreated mice in the chronic stage presented mononuclear cells, necrosis and fiber dissolution while hearts from treated animals showed only isolated inflammatory infiltrates. Present results demonstrate that clomipramine used in the chronic indeterminate phase of the T. cruzi infection modified the natural evolution of the chagasic cardiopathy.