Mixed T Helper1/T Helper2/T Cytotoxic Profile in Subjects with Chronic Chagas Disease with Hypersensitivity Reactions to Benznidazole.

Dermatitis is the most common adverse event during treatment with benznidazole in chronic Chagas disease and is probably mediated by T cells. A set of molecules representative of the different type IV hypersensitivity reactions was evaluated in the circulation and skin biopsies of Trypanosoma cruzi-infected subjects presenting dermatitis during benznidazole administration. Through cytometric bead assays and enzyme-linked immunosorbent assay capture techniques, the serum levels of cytokines, chemokines, proapoptotic molecules, and mediators of the activation and migration of eosinophils and T cells were measured in subjects infected with Trypanosoma cruzi who exhibited skin adverse events (n = 22) and compared with those without adverse events (n = 37) during benznidazole therapy. Serum levels of interleukin- 5 (IL-5), soluble Fas cell surface death receptor ligand (FAS-L), and interferon γ-induced protein (IP-10) significantly increased at 7 to 30 days posttreatment with benznidazole and decreased thereafter in subjects with dermatitis but not in those without dermatitis. Circulating eotaxin levels were lower in subjects with dermatitis than in those without. Two patterns emerged in the skin biopsies: a T helper 1/T cytotoxic profile and a T helper 2/T cytotoxic profile with the presence of CD4+ and CD8+ T cells. Increased low-density lipoprotein (LDL), glutamic-oxaloacetic transaminase (GOT), uremia, and T cell activation emerged as risk factors for the development of dermatitis during benznidazole administration. These results support a delayed-type hypersensitivity reaction to benznidazole, involving CD4+ and CD8+ T cells and eosinophils, and a mixed cytokine profile. This study provides new insights for better management of adverse drug reactions to benznidazole. IMPORTANCE This study identified the risk factors for the development of adverse reactions to benznidazole and identified a set molecule to monitor the appearance of these reactions. This knowledge might improve the safety of benznidazole administration.

Reduced Trypanosoma cruzi-specific humoral response and enhanced T cell immunity after treatment interruption with benznidazole in chronic Chagas disease.

BACKGROUND: Interruption of benznidazole therapy due to the appearance of adverse effects, which is presumed to lead to treatment failure, is a major drawback in the treatment of chronic Chagas disease. METHODS: Trypanosoma cruzi-specific humoral and T cell responses, T cell phenotype and parasite load were measured to compare the outcome in 33 subjects with chronic Chagas disease treated with an incomplete benznidazole regimen and 58 subjects treated with the complete regimen, during a median follow-up period of 48 months. RESULTS: Both treatment regimens induced a reduction in the T. cruzi-specific antibody levels and similar rates of treatment failure when evaluated using quantitative PCR. Regardless of the regimen, polyfunctional CD4+ T cells increased in the subjects, with successful treatment outcome defined as a decrease of T. cruzi-specific antibodies. Regardless of the serological outcome, naive and central memory T cells increased after both regimens. A decrease in CD4+ HLA-DR+ T cells was associated with successful treatment in both regimens. The cytokine profiles of subjects with successful treatment showed fewer inflammatory mediators than those of the untreated T. cruzi-infected subjects. High levels of T cells expressing IL-7 receptor and low levels of CD8+ T cells expressing the programmed cell death protein 1 at baseline were associated with successful treatment following benznidazole interruption. CONCLUSIONS: These findings challenge the notion that treatment failure is the sole potential outcome of an incomplete benznidazole regimen and support the need for further assessment of the treatment protocols for chronic Chagas disease.

The Significance of Discordant Serology in Chagas Disease: Enhanced T-Cell Immunity to Trypanosoma cruzi in Serodiscordant Subjects.

BACKGROUND: Subjects are considered infected with Trypanosoma cruzi when tested positive by at least two out of three serological tests, whereas a positive result in only one of up to three tests is termed "serodiscordant" (SD). Assessment of parasite-specific T-cell responses may help discriminate the uninfected from infected individuals among SD subjects. METHODS: Peripheral blood mononuclear cells from SD and seropositive (SP) subjects, who were born in areas endemic for T. cruzi infection but living in Buenos Aires city, Argentina, at the time of the study, and seronegative unexposed subjects were included for analysis. The function and phenotype of T cells were assessed by interferon-γ (IFN-γ) and interleukin (IL)-2 enzyme-linked immunospot assay and multiparameter flow cytometry. T. cruzi-specific antibodies were quantified by conventional serology and a multiplex assay format. RESULTS: SD subjects exhibited immunity cell responses to T. cruzi but in contrast to SP subjects, T cells in SD subjects more often display the simultaneous production of IFN-γ and IL-2 in response to T. cruzi antigens and have a resting phenotype. SD individuals also have higher IFN-γ spot counts, polyfunctional CD4+ T-cells enriched in IL-2 secreting cells and low levels of antibodies specific for a set of T. cruzi-derived recombinant proteins compared with the SP group. Long-term follow-up of SD individuals confirmed that humoral and T-cell responses fluctuate but are sustained over time in these subjects. T cells in SD subjects for T. cruzi infection did not recognize Leishmania antigens. CONCLUSION: Both T-cell and humoral responses in most subjects assessed by conventional tests as SD for T. cruzi infection indicate prior exposure to infection and the establishment of immunological memory suggestive of a resolved infection.

Treatment Success in Trypanosoma cruzi Infection Is Predicted by Early Changes in Serially Monitored Parasite-Specific T and B Cell Responses.

BACKGROUND: Chagas disease is the highest impact parasitic disease in Latin America. We have proposed that changes in Trypanosoma cruzi-specific immune responses might serve as surrogate indicators of treatment success. Herein, we addressed in a long-term follow-up study whether cure achieved after treatment can be predicted by changes in non-conventional indexes of anti-parasite serological and T cell activities. METHODOLOGY/PRINCIPAL FINDINGS: T. cruzi-specific T cell responses, as measured by interferon-γ ELISPOT and T. cruzi-specific antibodies assessed by ELISA, hemagglutination and immunofluorescence tests as well as by a multiplex assay incorporating 14 recombinant T. cruzi proteins were measured in 33 patients at 48-150 months post-benznidazole treatment. Cure - as assessed by conventional serological tests - was associated with an early decline in T. cruzi-specific IFN-γ-producing T cells and in antibody titers measured by the multiplex serological assay. Changes in the functional status and potential of T. cruzi-specific T cells, indicative of reduced antigen stimulation, provided further evidence of parasitological cure following benznidazole treatment. Patients showing a significant reduction in T. cruzi-specific antibodies had higher pre-therapy levels of T. cruzi-specific IFN-γ- producing T cells compared to those with unaltered humoral responses post-treatment. CONCLUSIONS/SIGNIFICANCE: Monitoring of appropriate immunological responses can provide earlier and robust measures of treatment success in T. cruzi infection.

Clinical characteristics and outcome of 107 adult patients with chronic Chagas disease and parasitological cure criteria.

BACKGROUND: The cure in adult patients with chronic Chagas disease and the relationship between parasitological and clinical evolution is still under debate. The aim of this study was to analyze the clinical, epidemiological and progression features of the disease in a patient population who became serologically negative either spontaneously or post-etiological treatment. METHODS: We included 107 patients over 20 years old with three different confirmed reactive anti-Trypanosoma cruzi serologic tests on admission, and a minimum of two years of follow-up. Patients were assigned to clinical groups according to Kuschnir. Change of clinical group was considered a heart disease progression criterion, and seronegative conversion of two or three as parasitological cure criterion. RESULTS: From 107 patients with parasitological cure, 82 had received treatment (77%) and 25 became spontaneously seronegative (23%). Forty-six (43%) and 61 (57%) patients had two and three negative serological tests, respectively. No differences in clinical groups, ECG, echocardiogram and heart disease progression were found in patients who became negative spontaneously or post-treatment. The clinical progression and ECG changes were observed in 5/107 (5%) and 11/107 (10%) respectively, in a mean of 10 years follow-up. CONCLUSIONS: Adults with chronic Chagas disease can cure, mostly post-etiological treatment, but also spontaneously, showing a favourable clinical outcome.

Allopurinol reduces antigen-specific and polyclonal activation of human T cells.

Allopurinol is the most popular commercially available xanthine oxidase inhibitor and it is widely used for treatment of symptomatic hyperuricaemia, or gout. Although, several anti-inflammatory actions of allopurinol have been demonstrated in vivo and in vitro, there have been few studies on the action of allopurinol on T cells. In the current study, we have assessed the effect of allopurinol on antigen-specific and mitogen-driven activation and cytokine production in human T cells. Allopurinol markedly decreased the frequency of IFN-γ and IL-2-producing T cells, either after polyclonal or antigen-specific stimulation with Herpes Simplex virus 1, Influenza (Flu) virus, tetanus toxoid and Trypanosoma cruzi-derived antigens. Allopurinol attenuated CD69 upregulation after CD3 and CD28 engagement and significantly reduced the levels of spontaneous and mitogen-induced intracellular reactive oxygen species in T cells. The diminished T cell activation and cytokine production in the presence of allopurinol support a direct action of allopurinol on human T cells, offering a potential pharmacological tool for the management of cell-mediated inflammatory diseases.

The impact of socioeconomic conditions on chronic Chagas disease progression.

INTRODUCTION AND OBJECTIVES: The extent to which a patient's socioeconomic conditions determine the persistence or control of chronic Chagas disease has not been previously investigated. The aim of this study was to evaluate the effect of socioeconomic conditions on clinical and serologic measures of disease progression. METHODS: Data on the following socioeconomic variables were obtained by questioning as part of medical history taking at admission: birth in a rural area, time of residence in endemic and urban areas (in years), overcrowding index (i.e. number of inhabitants/number of bedrooms), absence of toilet facilities, years of education, employed or unemployed, and health insurance coverage (i.e. private contributory medical insurance cover). The study endpoints for the Cox regression analysis were: consistently negative results on serologic tests and on tests for markers of cardiomyopathy progression by the end of the study. RESULTS: The study included 801 Argentine patients (mean age 42 years) who were followed up for a mean of 10 years between 1990 and 2005. After adjustment for age and antiparasitic treatment, negative seroconversion was associated with a short time of residence in an endemic area (hazard ratio [HR]=0.97; 95% confidence interval [CI], 0.96-0.99; P=.004), a low overcrowding index (HR=0.82; 95% CI, 0.70-0.97; P=.022) and medical insurance cover (HR=1.46; 95% CI, 1.01-2.09; P=.04). After adjustment for age, sex, ECG abnormalities and antiparasitic treatment, a low rate of cardiomyopathy progression was associated with more years of education (HR=0.88; 95% CI, 0.80-0.97; P=.01) and higher medical insurance cover (HR=0.49; 95% CI, 0.30-0.81; P=.005). CONCLUSIONS: Socioeconomic conditions had a significant effect on chronic Chagas disease progression which was independent of antiparasitic treatment and clinic characteristics.

El impacto de las condiciones socioeconómicas en la evolución de la enfermedad de Chagas crónica / The impact of socioeconomic conditions on chronic Chagas disease progression

Introducción y objetivos. Las condiciones socioeconómicas del huésped no han sido evaluadas como determinantes de la persistencia o el control de la enfermedad de Chagas crónica. El objetivo fue valorar el impacto de las condiciones socioeconómicas sobre la evolución clínica y serológica. Métodos. Las variables socioeconómicas en estudio fueron obtenidas por interrogatorio como parte de la historia clínica de ingreso: nacimiento en área rural, tiempo de residencia en área endémica y urbana (años), índice de hacinamiento (número de habitantes/número de dormitorios), ausencia de instalaciones sanitarias, años de educación, ocupación/desocupación y cobertura social (planes de asistencia médica por aportación privada). La negativización de las pruebas serológicas y los indicadores de progresión de la cardiopatía al concluir el estudio fueron los puntos finales de evaluación para el análisis de regresión de Cox. Resultados. Se incluyó a 801 pacientes, de 42 años de edad y 10 años de seguimiento promedio, en Argentina, entre los años 1990 y 2005. Un aumento de la seroconversión negativa, ajustada para edad y tratamiento etiológico, se asoció con un menor tiempo de residencia en área endémica (hazard ratio [HR] = 0,97 [0,96-0,99]; p = 0,004), menor índice de hacinamiento (HR = 0,82 [0,70-0,97]; p = 0,022) y mayor cobertura social (HR = 1,46 [1,01-2,09]; p = 0,04). Una disminución de la progresión de la cardiopatía, ajustada para edad, sexo, electrocardiograma anormal y tratamiento etiológico, se observó en pacientes con más años de educación (HR = 0,88 [0,80-0,97]; p = 0,01) y con cobertura social (HR = 0,49 [0,30-0,81]; p = 0,005). Conclusiones. Las condiciones socioeconómicas mostraron un significativo impacto sobre la evolución de la enfermedad de Chagas crónica independientemente del tratamiento antiparasitario y las características clínicas (AU)

Introduction and objectives. The extent to which a patient’s socioeconomic conditions determine the persistence or control of chronic Chagas disease has not been previously investigated. The aim of this study was to evaluate the effect of socioeconomic conditions on clinical and serologic measures of disease progression. Methods. Data on the following socioeconomic variables were obtained by questioning as part of medical history taking at admission: birth in a rural area, time of residence in endemic and urban areas (in years), overcrowding index (ie, number of inhabitants/number of bedrooms), absence of toilet facilities, years of education, employed or unemployed, and health insurance coverage (ie, private contributory medical insurance cover). The study endpoints for the Cox regression analysis were consistently negative results on serologic tests and on tests for markers of cardiomyopathy progression by the end of the study. Results. The study included 801 Argentine patients (mean age, 42 years) who were followed up for a mean of 10 years between 1990 and 2005. After adjustment for age and antiparasitic treatment, negative seroconversion was associated with a short time of residence in an endemic area (hazard ratio [HR] = 0.97; 95% confidence interval [CI], 0.96-0.99; P=.004), a low overcrowding index (HR=0.82; 95% CI, 0.70-0.97; P=.022) and medical insurance cover (HR=1.46; 95% CI, 1.01-2.09; P=.04). After adjustment for age, sex, ECG abnormalities, and antiparasitic treatment, a low rate of cardiomyopathy progression was associated with more years of education (HR=0.88; 95% CI, 0.80-0.97;P=.01) and higher medical insurance cover (HR=0.49; 95% CI, 0.30-0.81; P=.005). Conclusions. Socioeconomic conditions had a significant effect on chronic Chagas disease progression which was independent of antiparasitic treatment and clinic characteristics (AU)

Evaluación inmunológica del tratamiento con benznidazol en la enfermedad de Chagas crónica / Immunological Assessment of Benznidazol Therapy in Chronic Chagas Disease

Para determinar el efecto del tratamiento con benznidazol sobre las células T de memoria específica para Trypanosoma cruzi, se seleccionaron 47 pacientes con tres reacciones serológicas positivas para T. cruzi, sin cardiopatía y edades comprendidas entre los 30 y los 50 años. El tratamiento se realizó con benznidazol en dosis de 5 mg/kg/d por 30 días. Se efectuó una evaluación serológica, inmunológica y clínica pretratamiento (tiempo 0) y a los 2, 6 y 12 meses postratamiento. Posteriormente, los controles se hicieron anualmente. La respuesta de linfocitos T frente a un lisado de amastigotas de T. cruzi se evaluó por la técnica de ELISPOT para IFN-ã. La frecuencia de linfocitos T de memoria productores de IFN-ã específicos para T. cruzi disminuyó significativamente en el grupo tratado (n = 33) versus el no tratado (n = 14) 12 meses después del seguimiento. Once de 25 (44%) pacientes que recibieron benznidazol negativizaron la respuesta para IFN-ã. Cuatro de los 11 (36%) pacientes con ELISPOT (+) que negativizaron la respuesta por ELISPOT para IFN-ã también negativizaron la serología convencional a los 2 años postratamiento. Durante el seguimiento no se observaron alteraciones clínicas. Estos hallazgos muestran que el benznidazol es capaz de modular la respuesta celular T de memoria específica para T. cruzi. La medición de la frecuencia de linfocitos T de memoria productores de IFN-ã podría constituir un ensayo más sensible y precoz para determinar el impacto/eficacia del tratamiento específico contra este parásito.

To determine the effect of benznidazol therapy on memory T cells specific for Trypanosoma cruzi, 47 patients between 30 and 50 years old and three positive serological tests for T. cruzi without cardiopathy were selected. Benznidazol was administered in a dose of 5 mg/kg/d during 30 days. Serological, immunological and clinical assessment was performed at basal (time 0) and at 2, 6 and 12 months following treatment, and once a year thereafter. IFN-ã ELISPOT assay was used to evaluate T cell responses against a T. cruzi lysate obtained from amastigotes. The frequency of IFN-ã - producing memory T lymphocytes specific for T. cruzi was significantly lower in the treatment group (n=33) compared to the control group (n=14) 12 months after the therapy. IFN- ã response became negative in 11 patients in the treatment group (44%). Among these 11 patients, conventional serology also became negative in 4 patients (36%) after 2 years of treatment. No clinical manifestations occurred during follow-up. These findings show that benznidazol is capable of modulating T cell responses specific for T. cruzi. Measuring the frequency of memory T lymphocytes producing IFN-ã might become a more sensitive test to determine earlier the impact and/or efficacy of the specific treatment against this parasite.