Este estudio fue realizado para evaluar las características de sensibilidad y especificidad de una formulación antigénica (AgA), producida a bajo costo, para detectar anticuerpos dirigidos a Trypanosoma cruzi, en muestras de suero de pacientes con enfermedad de Chagas. El AgA fue producido por el efecto combinado de estrés nutricional y autoclave de los parásitos. La especificidad y sensibilidad fueron evaluadas en dos estudios separados, con 152 sueros de individuos sanos y 175 de pacientes Chagásicos. La reactividad cruzada con 289 sueros de pacientes con diagnóstico parasitológico de enfermedades con anticuerpos que reaccionan con antígenos de T. cruzi. Estos sueros fueron evaluados con AgA-ELISA y con tres estuches comerciales. 566 muestras de suero provenientes de un área endémica, fueron empleadas para estudiar la concordancia entre nuestro diagnostico y una prueba designada por nosotros como patrón oro estándar. Resultados: la sensibilidad y especificidad fue de 100%. El AgA presento el más bajo porcentaje de reactividad cruzada, respecto a los estuches comerciales evaluados. La concordancia con la prueba patrón oro, en Venezuela, fue excelente (kappa=0,92). En conclusión, Aga-ELISA, presentó niveles de sensibilidad, especificidad y de reactividad cruzada, comparables o superiores a los obtenidos por los tres estuches comerciales mas empleados en el país, pero con costos de producción al menos 10 veces menor. Este balance conveniente, favorece su potencial uso para el despiste en los bancos de sangre de los países pobres y endémicos para la enfermedad de Chagas. Futuras validaciones de esta formulación en otros países es necesaria.

In this prospective study we evaluated the performance characteristics of a specific and sensitive antigen preparation (AgA) used in an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-Trypanosoma cruzi antibodies in serum samples, for Chagas’ disease diagnosis. The antigen production was achieved by combination of nutritional stress and autoclaving the parasites. Specificity and sensitivity were evaluated in two separate tests, using 152 sera from healthy individuals and 175 sera from Chagas’ patients (70 by xenodiagnosis). Cross-reactivity was tested using 289 sera from patients who had a parasitological diagnosis of a disease known to induce antigenic responses towards T. cruzi. All of these sera were tested with our AgA-ELISA and with 3 commercial diagnosis kits. To evaluate the agreement of results between our AgA-ELISA and a “gold standard” test for Chagas, we tested 566 sera from an endemic area. Results: sensitivity and specificity were 100%; cross-reactivity was the lowest compared with commercial kits. Overall agreement with the gold standard test was excellent (kappa=0.92). AgA-ELISA exhibits levels of sensitivity, specificity and cross-reactivity comparable or superior to those shown, obtained with the commercial kits used in our country, while being at least 10 times less expensive. This balance between diagnostic accuracy and cost makes AgA-ELISA useful for blood bank screening in poor regions of the world suffering from Chagas’ disease. Further validations of this antigenic formulation in other countries are necessary.

In spite of evident progress in the serology of Chagas disease, the requirement for new diagnostic antigens persists. We have evaluated different antigens obtained from Trypanosoma cruzi grown in medium rich in nutrients or under nutrient stress, autoclaved or sonicated and fractionated by differential centrifugation. The resulting antigens were evaluated for diagnosis of Chagas disease using ELISA. Immunofluorescence of the parasites demonstrated that nutrient stress induced changes in the distribution and density of antigens recognised by a pool of sera from experimentally infected mice. When evaluated using ELISA, it was evident that most fractions had good sensitivity but poor specificity. Surprisingly, the best specificity and sensitivity was observed with parasites cultured under nutrient stress and autoclaved. Furthermore this antigen had low cross reactivity with sera from other parasitic diseases, Leishmaniasis in particular. Western blot analysis demonstrated that autoclaving seems to non-specifically eliminate cross-reactive antigens. In conclusion, autoclaving epimastigotes of T. cruzi, after nutrient stress, allowed us to obtain an antigen that could be used in the serological diagnosis of Chagas disease.

Hemos estudiado, in vitro, la respuesta proliferativa de células mononucleares a antígenos de Flagelo (FE) y Membrana (ME) de epimastigotes de T. cruzi, así como su efecto regulador en la respuesta proliferativa a PPD (Protein Purified derivative). Fue evaluada tanto la respuesta a Flagelo crudo como a las bandas de Western blots de FE y ME. El flagelo crudo indujo una proliferación de las células mononucleares más intensa en los pacientes con cardiomiopatía (CDM) que en los pacientes sin evidencia de patología cardiaca (INF). Los antígenos fraccionados causaron una menor respuesta proliferativa, tanto en intensidad como en frecuencia, que los antígenos crudos. Las bandas de FE, entre 150 y 24,3 kDa (B3 a B18 con la excepción de B4 y B13), indujeron CPM mas altas en CDM. En INF solo las bandas B7 (87,3 a 80,1 kDa), 9 (69,8 a 64,6 kDa) y 13 (45,4 a 41,5 kDa) causaron CPM (Cuentas Por Minuto) altas. Las bandas de ME también indujeron una proliferación mayor en CDM. Sin embargo, solo 5 de 14 bandas tuvieron CPM promedio mayores de 1000 en INF. La mayor parte de las bandas causaron una baja regulación (BR) que fue similar en ambos grupos. Sin embargo, la frecuencia de BR relevante producida por FE fue significativamente mayor en CDM. Por el contrario la sobre regulación (SB) fue mayor en INF. Las bandas 13 y 14 de ME no indujeron BR en la mayor parte de los INF. La discordancia entre la frecuencia de BR relevante en CDM e INF fue mas evidente con ME que con FE. La frecuencia de SB fue 50% o mas con todas las bandas de ME en INF, pero menor que 12% en los CDM. El predominio de SB en INF y de BR en CDM sugiere la presencia, en los INF, de algún tipo de equilibrio o interacción que esta ausente en los CDM. En ME pudieran estar presente antígenos relevantes para la inmunoprofilaxis de la enfermedad de Chagas. En conjunto nuestros resultados sugieren que las diferencias en los cuadros clínicos pudieran tener relación con la respuesta observada a los diferentes antígenos tanto a nivel de regulación como de proliferación.

We have studied, in vitro, proliferation induced by flagella (FE) and membrane (ME) antigenic fractions of T. cruzi epimastigotes, as well as their regulatory effect on the proliferative response to PPD (Protein Purified Derivative). Crude flagella as well as bands from Western blots of flagella and membrane of epimastigotes were tested. Crude flagella elicited higher proliferation in mononuclear cells from patients with Chagasic cardiomyopathy (CDM) than in patients with no evidence of cardiac pathology (INF). Fractionated antigens induced a lower proliferative response, in intensity as well as in frequency, than the crude extracts. With FE, bands between 150 and 24.3 kDa (B3 to B18 with the exception of B4 and B13) induced higher CPM (Counts Per Minute) in CDM. In INF only bands B7 (87.3 to 80.1 kDa), 9 (69.8 to 64.6 kDa) and 13 (45.4 to 41.5 kDa) had high CPM. ME bands also elicited higher proliferation in CDM. However, only 5 out of 14 bands gave CPM higher than 1000 in CDM and none in INF. The mean down regulation (DR) of most bands was similar in both groups. But the frequency of relevant DR elicited by FE was significantly higher in CDM. In contrast the frequency of up regulation (UR) was higher in INF. Bands 13 and 14 of ME did not induce DR in most INF. The discordance between the frequency of relevant DR in CDM and INF was more evident with ME than with FE. The frequency of (UR) was 50% or higher with all ME bands in INF, but, lower than 12% in CDM. The higher UR in INF and of DR in CDM, suggest the presence of some balance or interaction in INF that is lost in CDM. In ME there might be antigens that could be relevant for the immunoprofilaxis of Chagas' disease. The difference in the clinical status of the two groups seems to be associated with the recognition of different groups of antigens together with variations in the nature of the regulation of the response of mononuclear cells to these antigens.

Se estudió tanto la respuesta proliferativa inducida por antígenos solubles de epimastigotes y tripomastigotes de T. cruzi, como su efecto regulador en la respuesta proliferativa a PPD. Se evaluaron tanto los extractos crudos del parásito como las bandas de “Western blots” de antígenos solubles de epimastigotes y tripomastigotes. La proliferación de las células mononucleares, en respuesta a los extractos crudos, fue mas intensa en los pacientes con cardiomiopatía chagásica (CDM) que en los pacientes sin evidencia de patología cardiaca (INF). La respuesta a los antígenos fraccionados fue menor, en frecuencia e intensidad, a la observada con los antígenos crudos. Las bandas de bajo peso molecular (17 a 30 kDa) de los antígenos solubles de epimastigotes dieron una respuesta mas alta en pacientes CDM y las de peso molecular intermedio (31 a 62 kDa). Este patrón se invirtió con las bandas de antígenos solubles de tripomastigotes. Ambos extractos solubles indujeron sobre regulación o baja regulación de la respuesta a PPD en un número variable de pacientes en ambos grupos. Con los antígenos semi purificados la intensidad de la baja regulación fue mas intensa en pacientes sin evidencia de enfermedad cardiaca, sin embargo la frecuencia fue mayor en los pacientes con cardiomiopatía chagásica (CDM). Seis bandas del Western blot de antígenos solubles de tripomastigotes (B1, 4-7, y 9) se asociaron con una baja regulación en 100% de los pacientes CDM. La sobre regulación observada con la mayoría de las bandas de la fracciones crudas fue significativamente mas alta, y mas frecuente en pacientes con patología cardíaca. La mayoría de las bandas de antígeno soluble de tripomastigotes (10/15; 66,6%) no indujeron sobre regulación en pacientes con cardiomiopatía. Estos datos sugieren que la diferencia en la clínica de los dos grupos pueden deberse a que los pacientes de cada grupo reconocen diferentes antígenos conjuntamente con variaciones en la regulación asociada con esto.

We have studied in vitro proliferation induced by soluble antigenic fractions of T. cruzi epimastigotes and trypomastigotes, as well as their regulatory effect on the proliferative response to PPD. Both crude extracts of the parasite as well as bands from Western blots of soluble epimastigotes and trypomastigotes antigens were tested. Crude extracts elicited higher proliferation in mononuclear cells from patients with chagasic cardiomyopathy (CDM) than in those from patients with no evidence of cardiac pathology (INF). Fractionated antigens induced a lower proliferative response, in intensity as well as in frequency, than the crude extracts. With the soluble antigenic fractions of epimastigotes, cells from CDM patients gave higher responses to low molecular weight (MW) bands (17 to 30 kDa), and from INF patients, to bands of intermediate MW (31 to 62 kDa); this pattern was inverted with soluble antigenic fractions of trypomastigotes. The two crude preparations induced either up-regulation or down-regulation of the PPD response in variable numbers of patients from both groups. With fractionated antigens, down-regulation intensity was stronger in patients without evidence of heart disease, but frequency was greater in patients with Chagasic cardiomyopathy (CDM). Six bands of western-blot of soluble trypomastigote antigens (B1, 4-7, and 9) induced significant down-regulation in 100% of CDM patients. The up-regulation elicited by most bands of the antigenic fractions was significantly higher, and more frequent in patients without heart pathology. Most bands of soluble trypomastigote antigens (10/15; 66.6%) did not induce up-regulation in patients with cardiomyopathy. These data suggest that differences in the clinical status of the two groups may reflect the recognition of different groups of antigens together with variations in the nature of the regulatory response.

The production of interleukin 2 (IL-2) by peripheral blood mononuclear cells, from patients with different clinical forms of Chagas disease and healthy controls, was evaluated after stimulation with Trypanosoma cruzi antigen, PPD and PHA. PHA induced higher production of IL-2 in infected patients than healthy controls. No diferences were found between infected groups. With PPD the trend was similar, the only difference was that asymptomatic infected patients (INF) showed higher levels of IL-2 production than patients with cardiomyopathy (CDM). With T. cruzi antigen, most patients showed little or no IL-2 production at 24 hr, a peak at 48 hr and an abrupt fall at 72 hr. A similar pattern of IL- 2 production was observed in INF and CDM. To evaluate the physiologic relevance of the deficit in IL-2 production, we studied the effect of non-mitogenic concentratios of IL-2 in the proliferative response to specific antigens. The addition of IL-2 only enhanced the proliferative response of CDM patients. These observations suggest that patients suffering Chagas' disease, particularly CDM, have a significant reduction in the capacity to produce IL-2. These findings could be of importance in the pathogenesis of Chagas' disease.

The partial suppression of the cell-mediated immune response by Trypanosoma cruzi antigens in patients with Chagas' disease is demonstrated in a costimulation assay with T. cruzi antigens and Mycobacterium tuberculosis purified protein derivative (PPD) or Tetanus toxoid (TT). ononuclear cells from 13 patients with chagasic infection without evidence of heart disease, 10 patients with chagasic cardiomyopathy and 7 healthy blood donors were stimulated with antigen A (autoclaved epimastigotes), PPD, TT, PPD + A, PPD + TT and TT + A. The average percentage of suppression induced by costimulation of mononuclear cells with PPD and antigen A was 47.1% in patients with chagasic infection without heart disease (INF), 38.8% in patients with chagasic cardiomyopathy (CDM) and 23.3% in healthy controls. Similar values were observed when living trypomastigotes were used. A costimulatory study with PPD and TT, PPD and A and TT and A was carried out in 8 patients with chagasic infection, in order to evaluate the possibility that this difference could be due to a nonspecific inhibitory effect. The mean suppression induced by TT + PPD was -8.9, with TT + A was 52.7 and with PPD + A was 50.1. The data reported show that T. cruzi antigens induce a specific suppression of the proliferative responseof mononuclear cells, that might be relevant to the persistence of the parasite in the host.