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.

New Scheme of Intermittent Benznidazole Administration in Patients Chronically Infected with Trypanosoma cruzi: Clinical, Parasitological, and Serological Assessment after Three Years of Follow-Up.

In a pilot study, we showed that the intermittent administration of benznidazole in chronic Chagas disease patients resulted in a low rate of treatment suspension and therapeutic failure, as assessed by quantitative PCR (qPCR) at the end of treatment. Here, a 3-year posttreatment follow-up study of the same cohort of patients is presented. The treatment scheme consisted of 12 doses of benznidazole at 5 mg/kg of body weight/day in two daily doses every 5 days. Parasite load, Trypanosoma cruzi-specific antibodies, and serum chemokine levels were measured prior to treatment and after a median follow-up of 36 months posttreatment by DNA minicircle kinetoplastid and nuclear DNA satellite sequence qPCR methods, conventional serological techniques, a Luminex-based assay with recombinant T. cruzi proteins, and a cytometric bead array. At the end of follow-up, 14 of 17 (82%) patients had negative qPCR findings, whereas three of 17 (18%) had detectable nonquantifiable findings by at least one of the qPCR techniques. A decline in parasite-specific antibodies at 12 months posttreatment was confirmed by conventional serological tests and the Luminex assays. Monocyte chemoattractant protein 1 levels increased after treatment, whereas monokine induced by gamma interferon levels decreased. New posttreatment electrocardiographic abnormalities were observed in only one patient who had cardiomyopathy prior to treatment. Together, these data strengthen our previous findings by showing that the intermittent administration of benznidazole results in a low rate of treatment suspension, with treatment efficacy comparable to that of a daily dose of 5 mg/kg for 60 days.

Perturbed T cell IL-7 receptor signaling in chronic Chagas disease.

We have previously demonstrated that immune responses in subjects with chronic Trypanosoma cruzi infection display features common to other persistent infections with signs of T cell exhaustion. Alterations in cytokine receptor signal transduction have emerged as one of the cell-intrinsic mechanisms of T cell exhaustion. In this study, we performed an analysis of the expression of IL-7R components (CD127 and CD132) on CD4(+) and CD8(+) T cells and evaluated IL-7-dependent signaling events in patients at different clinical stages of chronic chagasic heart disease. Subjects with no signs of cardiac disease showed a decrease in CD127(+)CD132(+) cells and a reciprocal gain of CD127(-)CD132(+) in CD8(+) and CD4(+) T cells compared with either patients exhibiting heart enlargement or uninfected controls. T. cruzi infection, in vitro, was able to stimulate the downregulation of CD127 and the upregulation of CD132 on T cells. IL-7-induced phosphorylation of STAT5 as well as Bcl-2 and CD25 expression were lower in T. cruzi-infected subjects compared with uninfected controls. The serum levels of IL-7 were also increased in chronic chagasic patients. The present study highlights perturbed IL-7/IL-7R T cell signaling through STAT5 as a potential mechanism of T cell exhaustion in chronic T. cruzi infection.

New Scheme of Intermittent Benznidazole Administration in Patients Chronically Infected with Trypanosoma cruzi: a Pilot Short-Term Follow-Up Study with Adult Patients.

There is a clinical need to test new schemes of benznidazole administration that are expected to be at least as effective as the current therapeutic scheme but safer. This study assessed a new scheme of benznidazole administration in chronic Chagas disease patients. A pilot study with intermittent doses of benznidazole at 5 mg/kg/day in two daily doses every 5 days for a total of 60 days was designed. The main criterion of response was the comparison of quantitative PCR (qPCR) findings prior to and 1 week after the end of treatment. The safety profile was assessed by the rate of suspensions and severity of adverse effects. Twenty patients were analyzed for safety, while qPCR was tested for 17 of them. The average age was 43 ± 7.9 years; 55% were female. Sixty-five percent of treated subjects showed detectable qPCR results prior to treatment of 1.45 (0.63 to 2.81) and 2.1 (1.18 to 2.78) parasitic equivalents per milliliter of blood (par.eq/ml) for kinetoplastic DNA (kDNA) qPCR and nuclear repetitive sequence satellite DNA (SatDNA) qPCR, respectively. One patient showed detectable PCR at the end of treatment (1/17), corresponding to 6% treatment failure, compared with 11/17 (65%) patients pretreatment (P = 0.01). Adverse effects were present in 10/20 (50%) patients, but in only one case was treatment suspended. Eight patients showed mild adverse effects, whereas moderate reactions with increased liver enzymes were observed in two patients. The main accomplishment of this pilot study is the promising low rate of treatment suspension. Intermittent administration of benznidazole emerges a new potential therapeutic scheme, the efficacy of which should be confirmed by long-term assessment posttreatment.

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.

Immune exhaustion in chronic Chagas disease: Pro-inflammatory and immunomodulatory action of IL-27 in vitro.

In chronic Chagas disease, Trypanosoma cruzi-specific T-cell function decreases over time, and alterations in the homeostatic IL-7/IL-7R axis are evident, consistent with a process of immune exhaustion. IL-27 is an important immunoregulatory cytokine that shares T-cell signaling with IL-7 and other cytokines of the IL-12 family and might be involved in the transcriptional regulation of T-cell function. Here, we evaluated the expression and function of IL-27R in antigen-experienced T cells from subjects with chronic Chagas disease and assessed whether in vitro treatment with IL-27 and IL-7 might improve T. cruzi-specific polyfunctional T-cell responses. In vitro exposure of PBMCs to T. cruzi induced a downregulation of IL-27R in CD4+ T cells and an upregulation in CD8+ T cells in subjects without heart disease, while IL-27R expression remained unaltered in subjects with more severe clinical stages. The modulation of IL-27R was associated with functional signaling through STAT3 and STAT5 and induction of the downstream genes TBX21, EOMES and CXCL9 in response to IL-27. In vitro treatment of PBMCs with IL-27 and IL-7 improved monofunctional and polyfunctional Th1 responses, accompanied by the induction of IL-10 and Bcl-2 expression in subjects without heart disease but did not improve those in subjects with cardiomyopathy. Our findings support the process of desensitization of the IL-27/IL-27R pathway along with disease severity and that the pro-inflammatory and immunomodulatory mechanisms of IL-27 might be interconnected.

Changes in Trypanosoma cruzi-specific immune responses after treatment: surrogate markers of treatment efficacy.

BACKGROUND: As many as 20 million people are living with Trypanosoma cruzi infection in Latin American, yet few receive any treatment. The major limitation in developing and evaluating potential new drugs for their efficacy is the lack of reliable tests to assess parasite burden and elimination. METHODS: Adults volunteers with chronic T. cruzi infection were evaluated clinically and stratified according to the Kuschnir classification. Individuals with group 0 and group 1 clinical status were treated with benznidazole (5 mg/kg per day for 30 days). The changes in T. cruzi-specific T cell and antibody responses, as well as in clinical status, were measured periodically over the 3-5-year follow-up period and were compared with pretreatment conditions and with values in an untreated control group. RESULTS: The frequency of peripheral interferon (IFN)-gamma-producing T cells specific for T. cruzi declined as early as 12 months after benznidazole treatment and subsequently became undetectable in a substantial proportion of treated subjects. In addition, decreases in antibody responses to a pool of recombinant T. cruzi proteins also decreased in many of these same subjects. The shift to negative IFN-gamma T cell responses was highly associated with an early increase in IFN-gamma-producing T cells with phenotypic features of effector/effector memory cells in a subset of subjects. Benznidazole treatment also resulted in an increase in naive and early differentiated memory-like CD8(+) T cells in a majority of subjects. CONCLUSIONS: Benznidazole treatment during chronic Chagas disease has a substantial impact on parasite-specific immune response that is likely indicative of treatment efficacy and cure.

Correction: Trypanosoma cruzi-specific IFN-γ-producing cells in chronic Chagas disease associate with a functional IL-7/IL-7R axis.

[This corrects the article DOI: 10.1371/journal.pntd.0006998.].

Impaired frequencies and function of platelets and tissue remodeling in chronic Chagas disease.

Chronic inflammation, as a consequence of the persistent infection with Trypanosoma cruzi, leads to continuous activation of the immune system in patients with chronic Chagas disease. We have previously shown that increased sera levels of soluble P-selectin are associated with the severity of the cardiomyopathy distinctive of chronic Chagas disease. In this study, we explored the expression of biomarkers of platelet and endothelial activation, tissue remodeling, and mediators of the coagulation cascade in patients at different clinical stages of chronic Chagas heart disease. The frequencies of activated platelets, measured by the expression of CD41a and CD62P were decreased in patients with chronic Chagas disease compared with those in uninfected subjects, with an inverse association with disease severity. Platelet activation in response to adenosine diphosphate was also decreased in T. cruzi-infected subjects. A major proportion of T. cruzi infected subjects showed increased serum levels of fibrinogen. Patients with severe cardiac dysfunction showed increased levels of endothelin-1 and normal values of procollagen I. In conclusion, chronic infection with T. cruzi induced hemostatic alterations, even in those patients who do not yet present cardiac symptoms.

Trypanosoma cruzi-specific IFN-γ-producing cells in chronic Chagas disease associate with a functional IL-7/IL-7R axis.

BACKGROUND: The severity of cardiac disease in chronic Chagas disease patients is associated with different features of T-cell exhaustion. Here, we assessed whether the ability of T cells to secrete IFN-γ in response to T. cruzi was linked to disruption in immune homeostasis and inflammation in patients with chronic Chagas disease. METHODOLOGY/PRINCIPAL FINDINGS: PBMCs from chronic Chagas disease patients and uninfected controls were examined for frequencies of T. cruzi-responsive IFN-γ-producing cells by ELISPOT and cellular expression and function of IL-7R using flow cytometry. Serum levels of IL-7, IL-21, IL-27, soluble IL-7R, and inflammatory cytokines were also evaluated by ELISA or CBA techniques. Patients possessing T. cruzi-specific IFN-γ-producing cells (i.e. IFN-γ producers) had higher levels of memory T cells capable of modulating the alpha chain of IL-7R and an efficient response to IL-7 compared to that in patients lacking (i.e. IFN-γ nonproducers) parasite-specific T-cell responses. IFN-γ producers also showed low levels of soluble IL-7R, high basal expression of Bcl-2 in T cells and low basal frequencies of activated CD25+ T cells. Modulation of IL-7R was inversely associated with serum IL-6 levels and positively associated with serum IL-8 levels. Circulating IL-21 and IL-27 levels were not associated with the frequency of IFN-γ producing cells but were reduced in less severe clinical forms of the disease. In vitro stimulation of PBMCs with IL-7 or IL-27 enhanced IFN-γ production in IFN-γ producers but not in IFN-γ nonproducers. CONCLUSIONS/SIGNIFICANCE: Alterations of the IL-7/IL-7R axis and in the levels of inflammatory cytokines were linked to impaired T. cruzi-specific IFN-γ production. These alterations might be responsible of the process of immune exhaustion observed in chronic Chagas disease.

Distinct monocyte subset phenotypes in patients with different clinical forms of chronic Chagas disease and seronegative dilated cardiomyopathy.

BACKGROUND: Chronic infection with Trypanosoma cruzi leads to a constant stimulation of the host immune system. Monocytes, which are recruited in response to inflammatory signals, are divided into classical CD14hiCD16-, non-classical CD14loCD16+ and intermediate CD14hiCD16+ subsets. In this study, we evaluated the frequencies of monocyte subsets in the different clinical stages of chronic Chagas disease in comparison with the monocyte profile of seronegative heart failure subjects and seronegative healthy controls. The effect of the anti-parasite drug therapy benznidazole on monocyte subsets was also explored. METHODOLOGY/PRINCIPAL FINDINGS: The frequencies of the different monocyte subsets and their phenotypes were measured by flow cytometry. Trypanosoma cruzi-specific antibodies were quantified by conventional serological tests. T. cruzi-infected subjects with mild or no signs of cardiac disease and patients suffering from dilated cardiomyopathy unrelated to T. cruzi infection showed increased levels of non-classical CD14loCD16+ monocytes compared with healthy controls. In contrast, the monocyte profile in T. cruzi-infected subjects with severe cardiomyopathy was skewed towards the classical and intermediate subsets. After benznidazole treatment, non-classical monocytes CD14loCD16+ decreased while classical monocytes CD14hiCD16-increased. CONCLUSIONS/SIGNIFICANCE: The different clinical stages of chronic Chagas disease display distinct monocyte profiles that are restored after anti-parasite drug therapy. T. cruzi-infected subjects with severe cardiac disease displayed a profile of monocytes subsets suggestive of a more pronounced inflammatory environment compared with subjects suffering from heart failure not related to T. cruzi infection, supporting that parasite persistence might also alter cell components of the innate immune system.

Long-term cardiac outcomes of treating chronic Chagas disease with benznidazole versus no treatment: a nonrandomized trial.

BACKGROUND: Benznidazole is effective for treating acute-stage Chagas disease, but its effectiveness for treating indeterminate and chronic stages remains uncertain. OBJECTIVE: To compare long-term outcomes of patients with nonacute Chagas disease treated with benznidazole versus outcomes of those who did not receive treatment. DESIGN: Clinical trial with unblinded, nonrandom assignment of patients to intervention or control groups. SETTING: Chagas disease center in Buenos Aires, Argentina. PATIENTS: 566 patients 30 to 50 years of age with 3 positive results on serologic tests and without heart failure. MEASUREMENTS: The primary outcome was disease progression, defined as a change to a more advanced Kuschnir group or death. Secondary outcomes included new abnormalities on electrocardiography and serologic reactivity. INTERVENTION: Oral benznidazole, 5 mg/kg of body weight per day for 30 days (283 patients), or no treatment (283 patients). RESULTS: Fewer treated patients had progression of disease (12 of 283 [4%] vs. 40 of 283 [14%]; adjusted hazard ratio, 0.24 [95% CI, 0.10 to 0.59]; P = 0.002) or developed abnormalities on electrocardiography (15 of 283 [5%] vs. 45 of 283 [16%]; adjusted hazard ratio, 0.27 [CI, 0.13 to 0.57]; P = 0.001) compared with untreated patients. Left ventricular ejection fraction (hazard ratio, 0.97 [CI, 0.94 to 0.99]; P < 0.002) and left ventricular diastolic diameter (hazard ratio, 2.45 [CI, 1.53 to 3.95]; P < 0.001) were also associated with disease progression. Conversion to negative results on serologic testing was more frequent in treated patients than in untreated patients (32 of 218 [15%] vs. 12 of 212 [6%]; adjusted hazard ratio, 2.1 [CI, 1.06 to 4.06]; P = 0.034). LIMITATIONS: Nonrandom, unblinded treatment assignment was used, and follow-up data were missing for 20% of patients. Loss to follow-up was more common among patients who were less sick. Two uncontrolled interim analyses were conducted. CONCLUSIONS: Compared with no treatment, benznidazole treatment was associated with reduced progression of Chagas disease and increased negative seroconversion for patients presenting with nonacute disease and no heart failure. These observations indicate that a randomized, controlled trial should now be conducted.

Prevention of congenital Chagas disease by Benznidazole treatment in reproductive-age women. An observational study.

Since the decline in new cases of infection by insect/vector, congenital Chagas disease has become more relevant in the transmission of Chagas disease. Treatment with benznidazole significantly reduces the parasitemia, which constitutes an important factor linked to vertical transmission. The objective of this study was to evaluate whether treatment with benznidazole previously administered to women of childbearing age can prevent or reduce the incidence of new cases of congenital Chagas disease. An historical cohort study that included all women in reproductive age (15-45 years) assisted in our center was designed. We included 67 mothers with chronic Chagas disease; 35 women had not been treated prior to pregnancy, 15 had been treated prior to pregnancy and 17 gave birth prior and after treatment with benznidazole. Eight mothers gave birth to 16 children with congenital Chagas disease (8/67, 12%). The prevalence of congenital Chagas was 16/114 (14%) children born to untreated mothers and 0/42 (0%) children born to benznidazole- treated mothers, p=0.01. No significant differences were observed in clinical, serologic, epidemiological or socioeconomic baseline variables between mothers with and without children born with congenital Chagas. A 32% conversion rate to negative serology was observed in benznidazole-treated women after long-term follow up. Antiparasitic treatment administered to women in reproductive age can prevent the occurrence of congenital 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 predictors of chronic chagasic myocarditis progression]. / Indicadores clínicos de progresión de la miocarditis chagásica crónica.

INTRODUCTION AND OBJECTIVES: Previous prognostic studies of Chagas' disease have focused on mortality associated with end-stage cardiopathy (i.e., heart failure). Our aim was to identify indicators of progression in early-stage Chagas' heart disease. MATERIAL AND METHOD: The study included 856 patients with 3 positive anti-Trypanosoma cruzi test results. Those with heart failure were excluded. Patients were divided into 3 clinical groups: those without heart disease (Group I); those with heart disease but without left ventricular enlargement (Group II); and those with left ventricular enlargement but without heart failure (Group III). The endpoint was progression to a more severe clinical stage or death due to cardiovascular disease. A Cox regression model was used to derive a clinical risk score from clinical, electrocardiographic and echocardiographic variables. RESULTS: At study entry, the patients' mean age was 43.7 years. They were followed up for a mean of 8 years. The following were predictors of heart disease progression: age at entry (HR=1.05; 95% CI, 1.02-1.07; P<.001), left ventricular systolic diameter (HR=1.06; 95% CI, 1.04-1.09; P<.001), intraventricular conduction abnormalities (HR=1.85; 95% CI, 1.02-3.36; P=.04), and sustained ventricular tachycardia (HR=3.97; 95% CI, 1.65-9.58; P=.002). Treatment with benznidazole reduced the risk of progression (HR=0.40; 95% CI, 0.23-0.72; P=.002). The devised clinical risk score was effective in stratifying the likelihood of cardiopathy progression. CONCLUSIONS: Specific clinical indicators and a derived clinical risk score can be used to predict the progression of chronic chagasic myocarditis in patients without heart failure.

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.

Inhibitory receptors are expressed by Trypanosoma cruzi-specific effector T cells and in hearts of subjects with chronic Chagas disease.

We had formerly demonstrated that subjects chronically infected with Trypanosoma cruzi show impaired T cell responses closely linked with a process of T cell exhaustion. Recently, the expression of several inhibitory receptors has been associated with T cell dysfunction and exhaustion. In this study, we have examined the expression of the cytotoxic T lymphocyte antigen 4 (CTLA-4) and the leukocyte immunoglobulin like receptor 1 (LIR-1) by peripheral T. cruzi antigen-responsive IFN-gamma (IFN-γ)-producing and total T cells from chronically T. cruzi-infected subjects with different clinical forms of the disease. CTAL-4 expression was also evaluated in heart tissue sections from subjects with severe myocarditis. The majority of IFN-γ-producing CD4(+) T cells responsive to a parasite lysate preparation were found to express CTLA-4 but considerably lower frequencies express LIR-1, irrespective of the clinical status of the donor. Conversely, few IFN-γ-producing T cells responsive to tetanus and diphtheria toxoids expressed CTLA-4 and LIR-1. Polyclonal stimulation with anti-CD3 antibodies induced higher frequencies of CD4(+)CTAL-4(+) T cells in patients with severe heart disease than in asymptomatic subjects. Ligation of CTLA-4 and LIR-1 with their agonistic antibodies, in vitro, reduces IFN-γ production. Conversely, CTLA-4 blockade did not improved IFN-γ production in response to T. cruzi antigens. Subjects with chronic T. cruzi infection had increased numbers of CD4(+)LIR-1(+) among total peripheral blood mononuclear cells, relative to uninfected individuals and these numbers decreased after treatment with benznidazole. CTLA-4 was also expressed by CD3(+) T lymphocytes infiltrating heart tissues from chronically infected subjects with severe myocarditis. These findings support the conclusion that persistent infection with T. cruzi leads to the upregulation of inhibitory receptors which could alter parasite specific T cell responses in the chronic phase of Chagas disease.

Impact of aetiological treatment on conventional and multiplex serology in chronic Chagas disease.

BACKGROUND: The main criterion for treatment effectiveness in Chagas Disease has been the seronegative conversion, achieved many years post-treatment. One of the main limitations in evaluating treatment for chronic Chagas disease is the lack of reliable tests to ensure parasite clearance and to examine the effects of treatment. However, declines in conventional serological titers and a new multiplex assay can be useful tools to monitor early the treatment impact. METHODOLOGY/PRINCIPAL FINDINGS: Changes in antibody levels, including seronegative conversion as well as declines in titers, were serially measured in 53 benznidazole-treated and 89 untreated chronic patients in Buenos Aires, Argentina with a median follow-up of 36 months. Decrease of titers (34/53 [64%] treated vs. 19/89 [21%] untreated, p<0.001) and seronegative conversion (21/53, [40%] treated vs. 6/89, [7%] untreated, p<0.001) in at least one conventional serological test were significantly higher in the benznidazole-treated group compare with the untreated group. When not only complete seronegative conversion but also seronegative conversion on 2 tests and the decreases of titers on 2 or 3 tests were considered, the impact of treatment on conventional serology increased from 21% (11/53 subjects) to 45% (24/53 subjects). A strong concordance was found between the combination of conventional serologic tests and multiplex assay (kappa index 0.60) to detect a decrease in antibody levels pos-treatment. CONCLUSIONS/SIGNIFICANCE: Treatment with benznidazole in subjects with chronic Chagas disease has a major impact on the serology specific for T. cruzi infection in a shorter follow-up period than previously considered, reflected either by a complete or partial seronegative conversion or by a significant decrease in the levels of T. cruzi antibodies, consistent with a possible elimination or reduction of parasite load.