Farmacovigilancia en enfermedad de chagas / Pharmacovigilance in Chagas disease

INTRODUCCIÓN La enfermedad de Chagas afecta a aproximadamente 1 600 000 personas en Argentina. Las drogas que actualmente tienen efecto tripanocida son el benznidazol (BZN) y nifurtimox (NFX). Sin embargo, la presencia de eventos adversos (EAs) de ambos medicamentos, más predominantes en los pacientes adultos, desalienta a los efectores de salud para el tratamiento etiológico de esta endemia. En consideración a estos antecedentes surge la necesidad de realizar este proyecto de farmacovigilancia activa para ambos tripanocidas. OBJETIVOS Determinar la potencial toxicidad del nuevo benznidazol. MÉTODOS Este estudio de farmacovigilancia se realizó en tres centros, el INP Fatala Chaben, coordinador y eje del mismo, el Hospital Pirovano y el Área de APS de la Dirección de Salud de la Municipalidad de Escobar. Se trataron 107 pacientes con el nuevo BNZ (Abarax®, Laboratorios ELEA, Argentina) a dosis de 5mg/kg/d por 30 días en el área de Escobar y por 60 días por los otros centros. Se realizó tratamiento con BNZ de acuerdo a las Guías de enfermedad de Chagas del Ministerio de Salud. RESULTADOS La farmacodermia, las alteraciones gastrointestinales y las alteraciones del sistema nervioso tuvieron mayor relevancia clínica. A este nivel del análisis, el tratamiento por 60 días del nuevo BZN tuvo mayor impacto clínico negativo en la seguridad que el de 30 días (p<0,0065). Un tratamiento completo se realizó en 70% de los pacientes más allá de la presencia de eventos adversos (EA), 62,7% en esta población. Un 21,5% de los pacientes no completó el tratamiento, ya que fueron excluidos por los EA. La comparación con el estudio TRAENA, efectuado con BNZ de Laboratorio Roche (Radanil®), demostró que el nuevo BNZ sería responsable de mayor frecuencia de farmacodermia, alteraciones gastrointestinales, hepatitis tóxica y neuropatía periférica. Los niveles de intolerancia totales y los niveles de exclusión por los EA no fueron diferentes entre ambos BNZ. DISCUSIÓN En consideración a los límites de esta investigación, el tratamiento de los pacientes durante 30-60 días está en continua revisión. La información proporcionada por este estudio está en consonancia con investigaciones previas realizadas en el INP Fatala Chaben y permitirá generar mayores y más estrictas acciones de prevención en el área de la Salud Pública para mejorar la administración sistemática del tratamiento con BNZ en diferentes poblaciones con enfermedad de Chagas.

Structural and immunological characterization of sulphatides: relevance of sulphate moieties in Trypanosoma cruzi glycoconjugates.

Sulphoglycosphingolipids, present on the surface of diverse cells, participate in the regulation of various cellular events. However, little is known about the structure and the role of sulphoglycosphingolipids in trypanosomatids. Herein, sulphated dihexosylceramide structures - composed mainly of sphingosine as the long chain base acylated with stearic acid - have been determined for the first time in Trypanosoma cruzi epimastigotes by UV-MALDI-TOF-MS analysis. Interestingly, inhibition ELISA assays using cruzipain as antigen and polyclonal rabbit antibodies specific for cruzipain, the major cysteine proteinase of T. cruzi, or for its C-terminal domain, have demonstrated (i) that sulphate epitopes are shared between cruzipain and sulphatides of T. cruzi, (ii) that cross-reactivity maps to the C-terminal domain and (iii) the existence of other antigenic determinants in the glycolipidic structures. These features provide evidence that sulphate groups are antigenic in sulphate-containing parasite glycoconjugates. Furthermore, IgG2 antibody levels inversely correlate with disease severity in chronic Chagas disease patients, suggesting that IgG2 antibodies specific for sulphated epitopes might be associated with protective immunity and might be considered as potential surrogates of the course of chronic Chagas disease.

Immunological and pathological responses in BALB/c mice induced by genetic administration of Tc 13 Tul antigen of Trypanosoma cruzi.

Tc13 is a trans-sialidase family protein of Trypanosoma cruzi, the aetiological agent of Chagas' disease. Recently, in vitro studies had suggested that Tc13 might participate in the pathogenesis of the disease. In order to study the role of Tc13 antigens in an in vivo model, we administered plasmid DNA encoding a Tc13 antigen from the Tulahuén strain (Tc13 Tul) to BALB/c mice and evaluated the immunological and pathological manifestations as well as the capacity of this antigen to confer protection against T. cruzi infection. Tc13 Tul immunization did not elicit a detectable humoral immune response but induced specific memory T-cells with no capacity to produce IFN-gamma. Five months after DNA-immunization with Tc13 Tul, signs of hepatotoxicity and reactive changes in the heart, liver and spleen were observed in 40-80% of mice. When Tc13 Tul DNA-immunized animals were challenged with trypomastigotes, a significant decrease in parasitaemia in early and late acute phase was observed without modification in the survival rate. Surprisingly, Tc13 Tul-immunized mice chronically infected with T. cruzi showed a decrease in the severity of heart damage. We conclude that, in BALB/c mice, genetic immunization with Tc13 Tul mainly induces immune responses associated with pathology.

Chagas disease in bone marrow transplantation: an approach to preemptive therapy.

The efficacy of preemptive therapy was evaluated in bone marrow transplantation (BMT) recipients associated with Chagas disease (CD). The criterion to include patients in the protocol was the serological reactivity for CD in recipients and/or donors before transplant. After BMT, the monitoring was performed using the direct Strout method (SM), which detects clinical levels of Trypanosome cruzi parasitemia, and CD conventional serological tests. Monitoring took place during 60 days in ABMT and throughout the immunosuppressive period in allogeneic BMT. Reactivation of CD was diagnosed by detecting T. cruzi parasites in blood or tissues. In primary T. cruzi infection, an additional diagnostic criterion was the serological conversion. A total of 25 CD-BMT patients were included. Two ABMT and four allogeneic BMT recipients showed CD recurrences diagnosed by SM. One patient also showed skin lesions with T. cruzi amastigotes. Benznidazole treatment (Roche Lab), an antiparasitic drug, was prescribed at a dose of 5 mg/kg/day during 4-8 weeks with recovery of patients. Primary T. cruzi infection was not observed. This report proves the relevance of monitoring CD in BMT patients and demonstrates that preemptive therapy was able to abrogate the development of clinical and systemic disease.

Humoral immune response to cruzipain and cardiac dysfunction in chronic Chagas disease.

The humoral immune response to epitopes expressed on cruzipain was evaluated in 31 Chagas disease patients (CDP) with different degrees of cardiac dysfunction. We took advantage of the availability of anti-Trypanosoma cruzi microsomal fraction monoclonal antibodies (MoAbs) reactive with epitopes that are recognized (5A9B11) or not recognized (1A10C11) by CDP sera. The 5A9B11- and 1A10C11-like epitopes are expressed on cruzipain. The reactivity of 5A9B11 against cruzipain was completely inhibited by sera of severe cardiopathy patients while a partial inhibition was found with sera from chagasic patients with mild disease. CDP sera did not block cruzipain recognition by 1A10C11. The antigenic determinants recognized by CDP sera appeared to be linear and carbohydrate free. When the overall anti-cruzipain immune response was evaluated, 70% of CDP with severe disease showed cruzipain titers higher than 1/800 while none of them displayed titers lower that 1/400. This report shows for the first time that the humoral immune response against epitopes expressed on cruzipain appeared to be related with the severity of chronic Chagas disease.

alpha 4 Integrins and sialyl Lewis x modulation in chronic Chagas disease: further evidence of persistent immune activation.

We have previously shown that titers of soluble platelet selectin (s-P-selectin) and soluble vascular cell adhesion molecule-1 (s-VCAM-1) were increased in sera of patients with chronic Trypanosoma cruzi infection. In this study, we analyzed the expression of CD49d-integrins, that bind to VCAM-1, and sialyl Lewis x (SLe(x)), which binds selectins, in peripheral blood lymphocytes of 27 patients with Chagas' disease at different levels of disease severity. Patients with a mild form of Chagas' disease showed a lower number of CD49d(+) cells, in comparison with those with severe chronic cardiopathy. Decreased levels of CD49d(+) cells were detected in CD3(-) cell populations. Conversely, SLe(x) expression was found to be decreased in patients with severe Chagas' disease. Levels of soluble platelet endothelial cell adhesion molecule-1 (s-PECAM-1) were significantly increased in the plasma of patients with severe Chagas' disease while unaltered levels of MCP-1 were recorded. These data show that VCAM-1 and P-Selectin ligands are differentially expressed during the chronic phase of the Trypanosoma cruzi infection. These findings also reinforce a role of the P-selectin/SLe(x) adhesion pathway rather than very late antigen-4 (VLA-4)/VCAM-1, in the pathogenesis of Chagas' disease.

Soluble platelet selectin (sP-selectin) and soluble vascular cell adhesion molecule-1 (sVCAM-1) decrease during therapy with benznidazole in children with indeterminate form of Chagas' disease.

The immune response against Trypanosoma cruzi infection has been associated with both protection and pathogenesis. Central events in host defence system- and immune-mediated damage are tightly regulated by cell adhesion molecules (CAM). Levels of sP-selectin and sVCAM-1 were measured in sera from 41 children with the indeterminate phase of Chagas' disease. Simultaneously, levels of soluble adhesion molecule were also quantified in Chagas' disease children undergoing specific chemotherapy with benznidazole. Levels of sP-selectin and sVCAM-1 were found to be elevated in children with indeterminate Chagas' disease before aetiologic therapy was started. However, a small group of patients showed sP-selectin and sVCAM-1 levels comparable to those of non-infected children. A positive correlation between levels of sVCAM-1 and sP-selectin in sera from Chagas' disease patients was found. There was a significantly greater decrease in the titres of sP-selectin and sVCAM-1 in those children receiving benznidazole therapy compared with those children receiving placebo. Measurement of soluble adhesion molecules revealed differences in the activation of the immune system in children with the indeterminate form of Chagas' disease. The early decrease of sP-selectin and sVCAM-1 levels after anti-parasitic treatment suggests that these molecules might be valuable indicators of effective parasitologic clearance.

Chagas' disease in patients with kidney transplants: 7 years of experience 1989-1996.

Chagas' disease was present in 17.22% of persons undergoing kidney transplantation in an Argentine Hospital. The criterion for attributing reactivation of chronic Chagas' disease and transmission of Trypanosoma cruzi to grafts was detection of parasites in blood (patent parasitemia) or tissues. Reactivation was diagnosed in 5 (21.7%) of 23 recipients. Ten (43.4%) of 23 chagasic recipients without reactivation of chronic Chagas' disease had abrogation of serological reactivity. T. cruzi infection was transmitted to 3 (18.7%) of 16 non-chagasic recipients. Reactivation and infection were diagnosed by patent parasitemia or cutaneous panniculitis. For diagnosis, detection of parasites in blood and tissues had more relevance than serology. Sequential monitoring detected early reactivation and infection, permitting application of preemptive or therapeutic therapy with benznidazole, thus inhibiting, in all patients, severe clinical disease produced by a progressive and systemic replication of the parasite.

Reactivation of chronic Chagas' disease following allogeneic bone marrow transplantation and successful pre-emptive therapy with benznidazole.

This report shows the early detection of reactivation of chronic Chagas' disease (CCd) in a 27-year-old man with chronic myelogenous leukemia undergoing allogeneic bone marrow transplantation (ABMT). Pre-emptive therapy with benznidazole during a period of 7 weeks led to a rapid recovery of the patient, who remains free of parasitemia 2 years after the bone marrow transplantation.

Recipients and donors of bone marrow transplants suffering from Chagas' disease: management and preemptive therapy of parasitemia.

We report the clinical course of five adult patients with chronic Chagas' disease (Cd) who underwent BMT. Two patients with non-Hodgkin's lymphoma and one with ALL received an ABMT. Allogeneic BMT was performed in two patients with AML and CML respectively. One donor had chronic Cd. Samples of peripheral blood for parasite investigation by the Strout method, blood culture, and immunological studies by indirect immunofluorescent assay, ELISA and indirect hemagglutination tests were performed weekly from the start of chemotherapy until day +60 for ABMT and during the period of immunosuppression for allogeneic BMT. No prophylaxis was given to any of these patients. In only one ABMT patient were trypomastigotes detected early by blood culture without symptoms of reactivation. Benznidazole as preemptive treatment was administered at 5-8 mg/kg/daily for 30 days. Parasitemia was rapidly cleared and at the end of therapy xenodiagnosis was negative. The other Cd patients showed no evidence of relapse of parasitemia or signs and symptoms of reactivation. In brief, evidence of Cd should be sought in all BMT patients coming from endemic areas because parasitemia and reactivation are potential complications during the period of neutropenia and immunosuppression. The strategy used for early detection and treatment of parasitemia and reactivation was safe and effective.

[Clinical features and diagnosis of mucocutaneous leishmaniasis in patients of an endemic area in Salta]. / Caracteristícas clínicas y diagnóstico de la leishmaniasis mucocutanea en pacientes de un área endémica de Salta.

A total of 39 patients with a clinical diagnosis of mucocutaneous leishmaniasis, in an endemic area for leishmaniasis in Salta, Argentina, were examined between June 1990 and December 1992. Of these cases, 87% (34/39) presented the cutaneous simple form, 10.3% the cutaneous multiple form and 2.6% the mucosal form. Lesions were more frequently located in legs and arms (71.8%), followed by trunk and multiple location (10.3%). Of the patients, 43% were housewives, students or children, suggesting that the infection could be contracted in the domestic or peridomestic environment. Of 39 patients diagnosed, in 22 (56.4%) the parasite was found. Direct microscopy (smear) permitted a diagnosis in 13 (59.4%) of these 22 patients. Among these, 5 (22.7%) had positive diagnosis by culture, and 9 (40.9%) by inoculation in hamsters. Ten parasite isolates (45.4%) were obtained. The smear is recommended as a diagnostic method for epidemiological surveillance due to the sensibility demonstrated herein and its easy application in the endemic area. The time of clinical evolution, from the appearance of the lesion up to the detection of the patient by Sanitary Agents, was approximately 90 days. This would be related to the frequency of the visits, usually every 3 months. Only one of 30 treated patients had a relapse at 6 months, due to non fulfillment of the treatment.

Leishmania (Viannia) braziliensis: biological behavior in golden hamsters of isolates from Argentine patients.

This study reports intraspecific variations of native isolates of Leishmania (Viannia) braziliensis from patients with leishmaniasis from Salta, Argentina. These isolates induced skin lesions in golden hamsters, initially showing rapid development, reaching their largest size between 28 and 35 days postinfection (PI). Thereafter, the infections were self-limiting and total regression was observed at 80-150 days PI. The majority of the native isolates were characterized by low infectivity in the experimental animals, and a classic pattern of dissemination to systemic organs was established. However, unusual features for L. braziliensis were displayed by two isolates; one showed evidence of high infectivity in hamsters characterized by a short prepatent period and larger, severe and persistent lesions at the inoculation site. The other isolate, of low infectivity, showed cutaneous metastasis and recurrent systemic dissemination in the same animals, suggesting dissociation between infectivity and pathogenicity. Metastasis has been frequently described in hamsters infected with L. (V) guyanensis and L. (V) panamensis, but not in infections induced by L. (V) braziliensis, as was observed in this study. Active and/or regressive histopathologic lesions were observed, depending on the stage of the infection. An exudative and mixed inflammatory pattern with microabscesses and necrotic areas was observed during early infection, while well-defined granulomas and collagen formation were the predominant features detected at a later time. Amastigotes were easily detected in the tissues, although in low numbers. Schaumann bodies were always detected. The characterization of the unique features of these native isolates, and the verification of their reproducibility in vitro and in vivo will be useful tools in tests related to immunoprophylaxis and chemotherapy.

Increased expression and secretion of ICAM-1 during experimental infection with Trypanosoma cruzi.

In the present study we demonstrate that spleens and hearts from BALB/c mice infected with the virulent Tulahuén or the low virulent CA-I strains of Trypanosoma cruzi, contain substantially higher ICAM-1 transcripts than uninfected controls. ICAM-1 expression in heart cells was also increased in the protein level, as measured by flow cytometry, ELISA and immunohistochemistry. The adhesive receptor was observed not only on inflammatory cells but also on sarcolemma of cardiac myocytes from T. cruzi infected mice. ICAM-1 expression was higher during the acute phase than in the chronic phase of infection, and paralleled the density of inflammatory leukocytes. Elevated titres of soluble ICAM-1 (s-ICAM-1) were detected in sera from mice during the acute phase of infection with CA-I or Tulahuén parasites. Cytokines, including IFN-gamma, IL-1 alpha, IL-6 and TNF-alpha have been shown to modulate expression of ICAM-1. Spleens and hearts from mice infected with CA-I or Tulahuen strains showed increased accumulation of mRNAs specific for these cytokines, which peaked during the acute phase of infection. However, IFN-gamma activity was not necessary for ICAM-1 induction, as its levels were also increased during infection in IFN-gamma receptor knock-out (IFN-gamma R- ) mice. Upregulation of ICAM-1 expression might be a direct consequence of parasite infection, since its density on cell lines of different lineages was enhanced after 24 or 48 h of infection with T. cruzi.

Chronic experimental infection by Trypanosoma cruzi in Cebus apella monkeys

Twenty young male Cebus apella monkeys were infected with CAl Trypanosoma cruzi strain and reinfected with CA l or Tulahuen T.cruzi strains, with different doses and parasite source. Subpatent parasitemia was usually demonstrated in acute and chronic phases. Patent parasitemia was evident in one monkey in the acute phase and in four of them in the chronic phase after re-inoculations with high doses of CAl strain. Serological conversion was observed in all monkeys; titers were low, regardless of the methods used to investigate anti-T. cruzi specific antibodies. Higher titers were induced only when re-inoculations were perfomed with the virulent Tulahuén strain or high doses of CAl strain. Clinical electrocardiographic and ajmaline test evaluations did not reveal changes between infected and control monkeys. Histopathologically, cardiac lesions were always characterized by focal or multifocal mononuclear infiltrates and/or isolated fibrosis, as seen during the acute and chronic phases; neither amastigote nests nor active inflammation and fibrogenic processes characteristic of human acute and chronic myocarditis respectively, were observed. These morphological aspects more closely resemble those found in the "indeterminate phase" and contrast with the more diffuse and progressive pattern of the human chagasic myocarditis. All monkeys survived and no mortality was observed.

Chronic experimental infection by Trypanosoma cruzi in Cebus apella monkeys.

Twenty young male Cebus apella monkeys were infected with CA1 Trypanosoma cruzi strain and reinfected with CA1 or Tulahuen T. cruzi strains, with different doses and parasite source. Subpatent parasitemia was usually demonstrated in acute and chronic phases. Patent parasitemia was evident in one monkey in the acute phase and in four of them in the chronic phase after re-inoculations with high doses of CA1 strain. Serological conversion was observed in all monkeys; titers were low, regardless of the methods used to investigate anti-T. cruzi specific antibodies. Higher titers were induced only when re-inoculations were performed with the virulent Tulahuén strain or high doses of CA1 strain. Clinical, electrocardiographic and ajmaline test evaluations did not reveal changes between infected and control monkeys. Histopathologically, cardiac lesions were always characterized by focal or multifocal mononuclear infiltrates and/or isolated fibrosis, as seen during the acute and chronic phases; neither amastigote nests nor active inflammation and fibrogenic processes characteristic of human acute and chronic myocarditis respectively, were observed. These morphological aspects more closely resemble those found in the "indeterminate phase" and contrast with the more diffuse and progressive pattern of the human chagasic chronic myocarditis. All monkeys survived and no mortality was observed.

Outcome of infection with different strains of Trypanosoma cruzi in mice lacking CD4 and/or CD8.

Mice lacking CD4 and/or CD8 gene expression, generated by embryonic stem-cell technology, were used to study the role of CD4+ and CD8+ cells in the resistance to the acute infection with virulent (Tulahuén and RA) or mild (CA-I) strains of Trypanosoma cruzi. The presence of both CD4+ and CD8+ cells contributed to the survival of mice infected with T. cruzi, and each T-cell subtype was able to sustain protective functions in the absence of the other one. However, in certain host-parasite combinations, CD8+ cell-independent mechanisms were able to control the parasite load. Moreover, CD8- mice chronically infected with a low virulent strain of T. cruzi were protected from an otherwise lethal challenge with the parasite. A different organ distribution of parasite nests was observed when mutant (but not wild type) animals infected with different parasite strains were compared. CD4- mice produced high levels of IgG antibodies against peptide antigens or a whole homogenate from the parasite after infection with CA-I strain. A dramatic enhancement of IgG1- and IgG2a-specific antibodies was observed.

Cognitive impairment in human chronic Chagas' disease.

UNLABELLED: We proposed to investigate subclinical cognitive impairment secondary to chronic Chagas' disease (CCD). No similar study was previously done. The neuropsychological performance of 45 chronic Chagasic patients and 26 matched controls (age, education place and years of residency in endemic area) was compared using the Mini Mental State Exam (MMSE), Weschler Memory Scale (WMS) and the Weschler Adult Intelligent Scale (WAIS). Non-parametric tests and Chi2 were used to compare group means and multivariate statistics in two way frequency tables for measures of independence and association of categorical variables with the disease. RESULTS: Chagasic patients showed lower MMSE scores (p < .004), poor orientation (p < .004), and attention (p < .007). Lower WMS MQ were associated with CCD (Chi2 5.9; p < .01; Fisher test p < .02). Lower WAIS IQ were associated with CCD (Chi2 6.3, p < .01; Fisher test p < .01) being the digit symbol (p < .03), picture completion (p < .03), picture arrangement (p < .01) and object assembly (p < .03) subtests the most affected. The impairment in non-verbal reasoning, speed of information processing, problem solving, learning and sequencing observed in chronic Chagas disease patients resembles the cognitive dysfunction associated with white matter disease.

Cognitive impairment in human chronic Chagas'disease

El objetivo de esta investigación fue determinar compromiso cognitivo en pacientes con enfermedad de Chagas en estadio cronico. Se estudio el perfil cognitivo de 45 pacientes chagasicos cronicos (CC) y 26 controles apareados por edad, educación, lugar y tiempo de residencia en area endemica. El Minimental State (MMSE), la escala de memoria de Weschlelr (WMS) y el test de Inteligencia de Weschler (WAIS) han dio utilizados para evaluar ambos grupos. Para el estudio estadistico de los datos se utilizaron pruebas no parametricas, Chi2 y estadistica multivariada en tabla de 2 x 2 para medir la association o independencia de variables categoriales con la presencia de enfermedad. Los resultados mostraron que los pacientes alcanzaban score menor que los controles en el MMSE (p < 0.004) debido basicamente a una mas pobre orientacion (P < 0.004) y atencion (p < 0.007). Cocientes bajos de memoria en el WMS se asociaron a la presencia de enfermedad (Chi25.9, p < 0.01; test de Fisher p < 0.02). Cocientes bajos de inteligencia en el WAIS se asociaron con la presencia de enfermedad (Chi26.3, p < 0.01; test de Fisher p < 0.01). Los subtests simbolos digitos (p < 0.03), completamientos de figuras (p < 0.03), ordenamiento de laminas (p < 0.01) y rompecabezas (p < 0.03) mostraron mayor compromiso. Estos resultados sugieren disfuncion del razonamiento no verbal, disminucion de la velocidad de procesado de la informacion y dificultad en la resolucion de problemas nuevos, en la habilidad de secuenciacion y en el aprendizaje. Este conjunto de hallazgos sugiere posible compromiso de la sustancia blanca subcortical en estos enfermos