Regulation of hepatic fibrosis and extracellular matrix genes by the th response: new insight into the role of tissue inhibitors of matrix metalloproteinases.

Hepatic fibrosis is the hallmark of Schistosoma mansoni infection and often results in portal hypertension and bleeding from esophageal varices. The fibrotic process is highly dependent on type 2 cytokines, yet their role in the regulation of extracellular matrix remodeling genes remains largely unknown. Here, we examined the expression of matrix metalloproteases (MMP) -2, -3, -9, -12, and -13 and their inhibitors, tissue inhibitor of metalloproteases (TIMP) -1, -2, and -3, in the livers of infected mice and correlated their expression profiles with fibrosis and type 2 cytokine production. Expression of MMP-2, -3, -9, -12, and -13 and of TIMP-1 and -2 mRNA rapidly increased at the onset of egg laying in infected mice, while TIMP-3 was unchanged. Because TIMP are presumed to be important regulators of the extracellular matrix, and their expression correlated with the development of fibrosis, we studied their role in fibrogenesis by infecting TIMP-1- and TIMP-2-deficient mice. Strikingly, our data revealed no role for TIMP-1 or -2 in the fibrotic pathology induced by S. mansoni eggs. Because of these findings, we infected IL-10/IFN-gamma-deficient mice that develop an exaggerated fibrotic response to determine whether changes in type 2 cytokine dominance influence the pattern of MMP and TIMP expression. Fibrosis and type 2 cytokine production correlated with increased MMP-2/MMP-9 vs TIMP-1/TIMP-2 expression. These data, in addition to our knockout studies, demonstrate that TIMP-1/TIMP-2 play no essential role in fibrogenesis in schistosomiasis. Indeed, our findings suggest that inhibiting profibrotic cytokines or specific MMP may be a more effective strategy to ameliorate fibrotic pathology.

Studies of murine schistosomiasis reveal interleukin-13 blockade as a treatment for established and progressive liver fibrosis.

In several allergic, autoimmune, and infectious diseases, fibrosis is a major cause of morbidity and mortality. Here, using a model of infection-induced liver fibrosis, we show that interleukin (IL)-13 is required at all stages of Schistosomiasis mansoni infection to induce fibrosis. IL-4 production was preserved in IL-13-deficient mice, yet failed to significantly contribute to the fibrotic response in either acute or chronic infection. Significant fibrosis develops in all infected mice, although the magnitude of the response varies widely in inbred mice. C3H/HeN, BALB/c, and C57BL/6 mice develop high, intermediate, and low levels of fibrosis, respectively. Despite these differences, IL-13 antagonism resulted in a marked amelioration of fibrosis in all strains. The fibrotic mechanism in the high- and low-responder strains was unrelated to their tissue eosinophil or mast cell responses, but did correlate with their patterns of IL-13, IL-10, and interferon gamma (IFN-gamma) mRNA expression. Indeed, severe fibrosis correlated with a high IL-13 and low IFN-gamma/IL-10 mRNA response. Because fibrotic diseases are typically progressive disorders, an important issue was to determine whether IL-13 inactivation might be used to treat an established and ongoing fibrotic disease. Here, IL-13 antagonism was highly efficacious, even after fibrosis and the Th2 cytokine response were firmly established. These studies demonstrate the central role played by IL-13 in fibrogenesis and suggest that therapeutic approaches aimed at disrupting the IL-13 pathway will be highly effective at preventing fibrotic disease caused by chronic Th2-mediated inflammatory reactions.

CpG oligonucleotides can prophylactically immunize against Th2-mediated schistosome egg-induced pathology by an IL-12-independent mechanism.

Using a Schistosoma mansoni egg-induced granuloma model, we examined the ability of CpG oligodeoxynucleotides (ODN) to suppress Th2-type cytokine expression and to prophylactically immunize against Th2-dependent pulmonary pathology. The mechanism was examined by studying Th2 response regulation in cytokine-deficient mice. Surprisingly, our findings revealed several functions of CpG DNA that were completely IL-12 independent. Most striking was the marked suppression in Th2 cytokine expression and granulomatous inflammation observed in egg/CpG-sensitized IL-12-deficient mice. Immune deviation was not dependent on NK or B cells. However, a role for IL-10, B7.1, and CD40 expression in Th2 response inhibition was suggested. Indeed, CpG ODN up-regulated all three elements in both wild-type and IL-12-deficient mice. The role of IL-10 was demonstrated in mice exhibiting combined deficiencies in IL-12 and IL-10. Here, a marked increase in egg-specific IL-4/IL-5-producing cells confirmed a role for both cytokines in Th2 response inhibition. Nevertheless, the frequency of Th2-producing cells was again reduced by CpG ODN. However, in marked contrast to IL-12-deficient animals, a significant increase in IFN-gamma-producing cells likely explains the reduced Th2 response in IL-10/IL-12-deficient mice. Thus, a novel IL-12-independent type 1-inducing pathway was revealed in the combined absence of IL-12 and IL-10. Together, these data demonstrate 1) that the Th1-promoting activity of CpG DNA is controlled by IL-12 and IL-10, and 2) that Th2 response inhibition by CpG ODN involves IL-12-independent changes in IL-10 and costimulatory molecule expression. These findings illustrate the utility of CpG DNA as adjuvants for vaccines designed to prevent Th2-dependent immunopathology.

An IL-13 inhibitor blocks the development of hepatic fibrosis during a T-helper type 2-dominated inflammatory response.

In schistosomiasis, chronic parasite egg-induced granuloma formation can lead to tissue destruction and fibrosis, which causes much of the morbidity and mortality associated with this disease. Here we show the importance of IL-13 in the pathogenesis of schistosomiasis, and demonstrate, perhaps for the first time, the therapeutic efficacy of an IL-13 inhibitor, sIL-13Ralpha2-Fc, in the control of hepatic fibrosis. T-helper type 2 (Th2) cytokines dominate the immune response in mice infected with Schistosoma mansoni, yet the specific contributions of IL-13 and IL-4 to the development of fibrosis were not previously investigated. Our studies demonstrate that both cytokines play redundant roles in granuloma formation, which explains the ability of IL-4-deficient mice to form granulomas around eggs. More importantly, however, these studies demonstrate that IL-13 is the dominant Th2-type cytokine regulating fibrosis. IL-13 stimulated collagen production in fibroblasts, and procollagen I and procollagen III mRNA expression was decreased in sIL-13Ralpha2-Fc-treated mice. Moreover, the reduction in fibrosis observed in IL-4-deficient mice was much less pronounced than that in sIL-13Ralpha2-Fc-treated animals. Fibrosis is a major pathological manifestation of a number of allergic, autoimmune, and infectious diseases. Thus, our findings provide evidence that IL-13 inhibitors may be of general therapeutic benefit in preventing damaging tissue fibrosis resulting from Th2-dominated inflammatory responses.

Polymerase chain reaction reveals Trypanosoma cruzi infection suspected by serology in cutaneous and mucocutaneous leishmaniasis patients.

The existence of patients suffering a double infection caused by Trypanosoma cruzi and Leishmania spp. has been suggested by several authors. Since the conventional serological tests now available for the diagnosis of Chagas' disease lack specificity due to the cross-reactivity between these two parasites, a serological confirmation of a T. cruzi infection cannot be made unless specific antigens are used. An enzyme linked immunosorbent assay (ELISA) to detect antibodies against a specific T. cruzi antigen, named Ag163B6, and immunoblotting using T. cruzi epimastigotes, are non-conventional serological techniques that could be employed for specific diagnosis of Chagas' disease. Using these two methods 34 cutaneous or mucocutaneous leishmaniasis patients were classified into two groups: (A) patients with serological evidence of T. cruzi infection, i.e. those who tested positive in at least one assay (18/34); and (B) patients with no serological evidence of T. cruzi infection, i.e. those who were negative for both assays (16/34). Taking into account the difficulties of xenodiagnosis and its low sensitivity (less than 50%) for a direct diagnosis in the chronic period of the disease, we used polymerase chain reaction (PCR) to confirm a T. cruzi infection in those leishmaniasis patients who presented positive results with the non-conventional serological techniques. Of the 18 patients with serological evidence of T. cruzi infection, 17 gave positive results when genomic DNA primers were used. Using minicircle primers, 15/18 of that group were positive. Nevertheless, all the patients suspected of being double infected were positive in at least one PCR test. Just one patient with no serological evidence of T. cruzi infection gave a positive PCR result when amplifying the minicircle sequence. The proof of the existence of a T. cruzi infection by PCR in leishmaniasis patients suspected to be chagasic when non-conventional serology was used, strongly supports the use of the specific Ag163B6 and immunoblotting with epimastigotes as specific serological diagnostic tools to determine a T. cruzi infection.

IL-13 is a key regulatory cytokine for Th2 cell-mediated pulmonary granuloma formation and IgE responses induced by Schistosoma mansoni eggs.

Schistosoma mansoni egg-induced pulmonary granuloma formation is a cell-mediated inflammatory response associated with dominant Th2-type cytokine expression, tissue eosinophilia, and high levels of serum IgE. In the present study, we show that in vivo blockade of the Th2 cytokine IL-13, using soluble IL-13R alpha2-Fc fusion protein, significantly reduced the size of pulmonary granulomas in unsensitized as well as egg-sensitized mice. Blocking IL-13 also significantly reduced total serum IgE levels. Interestingly, however, IL-13 blockade did not affect the evolving egg-induced Th2-type cytokine response. IL-4, IL-5, as well as IL-13 responses were indistinguishable in control-Fc- and soluble IL-13R alpha2-Fc fusion protein-treated animals. The smaller granulomas were also phenotypically like the control Fc-treated mice, displaying a similar eosinophil content. Additional studies in IL-4-deficient mice demonstrated that IL-13 was produced, but at much lower levels than in wild-type mice, while IL-4 expression was completely independent of IL-13. Moreover, while granuloma formation was partially reduced in IL-4-deficient mice, blocking IL-13 in these animals almost completely abrogated granuloma development and the pulmonary eosinophilia, while it simultaneously increased IFN-gamma production. Together, these data demonstrate that IL-13 serves as an important mediator of Th2-mediated inflammation and plays a role in eliciting IgE responses triggered by schistosome eggs.

Expression of a recombinant Fab antibody fragment against cruzipain, the major cysteine proteinase of Trypanosoma cruzi.

Cruzipain, the major proteinase of Trypanosoma cruzi, plays an important role in the biology of this parasite. This study reports the development of a recombinant Fab antibody, using RNA isolated from the anti-Ag163B6 hybridoma against cruzipain. This procedure involves the use of cDNAs obtained with the aid of a specific set of primers complementary to the complete light kappa chain (L kappa) and the first two domains of the IgG1 heavy chain (VH/CH1). These products were subsequently cloned in the pComb3 system, from which the gIII gene had been removed, and expressed in Escherichia coli cells. The recombinant Fab molecule recognized cruzipain by ELISA, in a fashion similar to the original mAb anti-Ag163B6. Nucleotide sequence analysis of the recombinant molecule, together with its immunological recognition by specific anti-mouse IgG (Fab)2, indicated the immunoglobulin nature of the recombinant product. Moreover, both the mAb anti-Ag163B6 and the soluble Fab fragment described here react similarly with the intact parasite surface, as observed in an indirect immunofluorescence assay. In conclusion, our recombinant Fab anti-Ag 163B6 allows the possible use of this molecule for diagnosis, antigen purification, and eventually treatment of Chagas-afflicted individuals.

Trypanosoma cruzi and Leishmania spp. human mixed infection.

The occurrence of leishmaniasis patients carrying a double infection with Trypanosoma cruzi has been suspected but not proved. In this study, we analyzed sera of leishmaniasis patients from a region endemic for both parasites by using immunoblotting with epimastigotes and a purified antigen specific for T. cruzi (Ag 163B6). Seven of 12 patients showed a pattern of bands characteristic of chagasic patients reacting with antigens with molecular weights of 131, 125, 116, 111, 51-45, and 43 kD, and positive reactivity with Ag 163B6. Xenodiagnosis for T. cruzi was carried out in all patients; this technique has a positivity rate of 50% in chronic chagasic patients. The presence of T. cruzi trypomastigotes was shown in the blood of three, thus confirming the existence of a double infection in humans. Since the two parasites possess cross-reacting antigens, it may be assumed that previous infection with one of the parasites may affect the course of subsequent infection with the other. Nevertheless, T. cruzi infection did not prevent the appearance of typical leishmaniasis lesions. Therefore, antigenic cross-reactivity is unable to induce a sterilizing immune response against Leishmania.

[Study of cases of leishmaniasis in the Province of Salta: evidences of mixed infection with Trypanosoma cruzi and Leishmania spp]. / Estudio de casos de leishmaniasis en la provincia de Salta. Evidencias de infección mixta por trypanosoma cruzi y leishmania spp.

In many regions of South America there are overlapping endemic areas for American Trypanosomiasis (Chagas' disease) and Leishmaniasis. T. cruzi and Leishmania spp, the causative agents of these parasitoses belong to the Trypanosomatidae family and share various antigens that cause cross-reactivity in serological diagnosis when complex antigenic mixtures are used. We studied patients who sought medical attention because of cutaneous or mucocutaneous lesions typical of leishmaniasis infection. These patients were from the province of Salta where Trypanosomiasis and Leishmaniasis are endemic diseases. Sixty-two patients gave a positive Montenegro skin test and, of these, 53 (85, 48%) showed the presence of amastigotes in Giemsa stained smears of dermal scrapings. Seven patients were not included because they were negative for both assays. We analyzed the leishmaniasic sera against homologous antigens to study the immune response and against complex heterologous antigens from T. cruzi to evaluate cross-reactivity phenomena. We also tested these sera against specific antigens for diagnosis of Chagas' disease in order to search for mixed infections. When complex antigens from leishmania were used, the sera showed an unusually strong antibody response 100% positive by IFA, 88.7% by ELISA and 80.6% by immunoblotting. Furthermore, significant cross-reactivity was found when conventional antigens for the serodiagnosis of Chagas' disease were used: 74.19% by IHA, 91.93% by IFA, and 76.80% by ELISA. We have previously purified by immunoaffinity, using a monoclonal antibody, an antigen termed Ag163B6 which is not present in L. mexicana. This antigen has shown the ability to specifically differentiate sera of chronic chagasic patients from those of leishmaniasic patients in ELISA. Furthermore, recent studies from our laboratory by immunoblotting, have demonstrated that chronic chagasic patients exhibit a specific reactivity pattern against T. cruzi epimastigotes that can be distinguished from those presented by leishmaniasic patients in spite of cross-reactive antigens. According to the results obtained in these assays, we classified the patients in two groups: 1) Patients with evidence of T. cruzi infection, those who tested positive in at least one assay: 2) Patients with no evidence of T. cruzi infection who were negative for both assays. More than 50% (32/62) of the patients showed strong evidence of mixed infection with T. cruzi. On the other hand, high cross-reactivity between these two parasitoses was shown in the second group without any evidence of T. cruzi infection since 18 out of 30 were positive in at least two conventional serological reactions. This implies that they would be misdiagnosed as chagasics if conventional reactions were used. These results emphasize the importance of the use of defined antigens and appropriate techniques for the differential diagnosis of these parasitoses, which is more important in areas endemic for both of them.

Modulation of cardiac physiology by an anti-Trypanosoma cruzi monoclonal antibody after interaction with myocardium.

Circulating antibodies from human and murine chagasic sera are able to interact with myocardium, activating neurotransmitter receptors. Here, we studied the effects of a monoclonal antibody (MAb CAK20.12), which recognizes a 150 kilodalton antigen of Trypanosoma cruzi and reacts with normal human and murine striated muscles and with cardiac tissue. The MAb CAK20.12 binds to purified cardiac membranes and interferes with the binding of beta-adrenergic receptor radioligand ([125I]CYP) and muscarinic cholinergic receptor (mAChR) radioligand ([3H]QNB) in a noncompetitive way. As a consequence of this interaction, beta-adrenergic receptor and mAChR were activated, leading to increased intracellular levels of cyclic AMP as a result of beta-adrenergic receptor-coupled adenylate cyclase triggering. When its sympathetic action was abrogated, it also induced an mAChR-mediated increase in cyclic GMP. Furthermore, cardiac physiology was modified by MAb CAK20.12, as it was able to increase cardiac contractility through beta-adrenoceptor activation and to decrease atrial frequency as a result of mAChR activation. The fact that this MAb modulates and modifies the mechanical and biochemical activity of normal murine heart established an important basis for future research and understanding of how the host's humoral immune response acts on the course and development of the chronic chagasic myocardiopathy.

Cross-reactivity studies and differential serodiagnosis of human infections caused by Trypanosoma cruzi and Leishmania spp; use of immunoblotting and ELISA with a purified antigen (Ag163B6).

Results of our studies on the reactivity of chagasic and leishmaniasic sera with the purified T. cruzi-specific antigen 163B6, as assessed by ELISA, and with complex antigenic mixtures from T. cruzi and Leishmania mexicana, by immunoblotting, are presented here. Our objective was to identify the antigens responsible for the exhibited cross-reactivity between trypanosomiasis and leishmaniasis, and to find a specific reactivity pattern corresponding to each parasitosis. In spite of the high cross-reactivity observed with the immunoblotting, the use of 7.5% A-B gels made it possible to identify a characteristic pattern for each parasitosis, that could be distinguished by the naked eye. The characteristic pattern corresponding to chagasic patients was ascribed to reactivity with T. cruzi bands of mol. wts 131, 125, 116, 111, 51-45 and 43 kD, that were not recognized by leishmaniasic sera. Trypanosoma cruzi antigens of mol. wts 85, 81, 70, 65-60, 37 and 32 kD were considered as crossing antigens, since they were recognized by leishmaniasis sera. With L. mexicana, most of the chagasic patients presented reaction with antigen of mol. wts 124, 107, 92, 59 and 32 kD, while bands of mol. wts 155, 140, 73, 56 and 48 kD were recognized only by leishmaniasic sera. In this study we found 12 out of 45 sera of patients with leishmaniasis, from a region endemic for both parasitoses, which exhibited a pattern of bands very similar to those corresponding to chagasic individuals, strongly suggesting a mixed infection. This hypothesis was verified by using a purified specific antigen of T. cruzi, Ag163B6, which would be the major cysteine proteinase of this specie (cruzipain). By ELISA, these 12 sera showed a positive reaction with this purified antigen, as those of chagasic patients, thus leading to the confirmation of the presence of a mixed infection.

A lytic monoclonal antibody to Trypanosoma cruzi bloodstream trypomastigotes which recognizes an epitope expressed in tissues affected in Chagas' disease.

It has been suggested that molecular mimicry between the antigens of Trypanosoma cruzi and the host could have a role in the onset of the chronic stage of Chagas' disease. In this article, we report on a monoclonal antibody (MAb), CAK20.12 (immunoglobulin G2b), which reacts with a polypeptidic epitope of a 150-kDa antigen expressed on the surface of several strains of T. cruzi. This MAb also causes lysis of bloodstream trypomastigotes. Serum samples from 30 of 30 patients with chronic and 11 of 13 patients with acute Chagas' disease present specific antibodies to this antigen. MAb CAK20.12 reacts, by indirect immunofluorescence, with human and syngeneic murine striated muscle tissue, with the smooth muscle layer of cardiac arteries, with the lamina muscularis mucosae and the external striated muscle layer of the esophagus, and with the smooth muscle cells of the colon from normal syngeneic mice. Reactivity with the small intestine was very weak, and no reactivity with ventricle or atrium tissue was detected. Adsorption with an antigenic fraction from normal murine striated muscle or from T. cruzi epimastigotes confirmed that MAb CAK20.12 recognizes a common epitope present in parasites and host tissues. MAb CAK20.12, lytic for the infective form of T. cruzi, recognizes an epitope expressed in striated and smooth muscle cells of the host tissues affected in the chronic stage of Chagas' disease.

Immune response against Trypanosoma cruzi antigens in Cebus apella monkeys.

The american primate Cebus apella has been used as an experimental model for the study of acute and chronic Chagas' disease. The antibody response elicited by 4 x 10(6) blood trypnomastigotes injected into four monkeys was analysed. Peak titres of IgM and IgG of anti-Trypanosoma cruzi antibodies were found at day 22, and between days 20 and 40 post-infection (p.i.), respectively. The ability of a Mr 37kDa (T37K) glycoprotein purified from T. cruzi epimastigotes to generate IgG anti-T. cruzi antibodies in monkeys, and protect them against a challenge with trypomastigotes, was also studied. Monkeys non-immunized with T37K reached peak values of parasitaemia between days 18 and 21 post-infection, whereas immunized monkeys had lower parasitaemias without important variation. Anti-T37K antibodies in immunized monkeys decreased from day 2 with the lowest titres between days 14 to 22 p.i., coincident with the peak of parasitaemia in control non-immunized monkeys. These results suggest that anti-T37K antibodies could be responsible for the low parasitaemia detected in immunized monkeys.

Identity of the major cysteine proteinase (cruzipain) from Trypanosoma cruzi and an antigen (Ag163B6) isolated with a monoclonal antibody.

Cruzipain, purified by conventional methods, and Ag163B6, isolated by affinity chromatography with a monoclonal antibody raised against a T. cruzi extract, are glycoproteins with a similar electrophoretic mobility, which reacted with sera from most chronic chagasic patients. Their behaviour in SDS-PAGE, Western blotting, isoelectric focusing, two-dimensional electrophoresis (IEF and SDS-PAGE), Ouchterlony's double diffusion, and enzyme activity in SDS-PAGE gels containing 0.1% gelatin suggests that they are identical.

Empleo de epimastigotes formolados para la detección de anticuerpos anti-trypanosoma cruzi mediante técnicas inmunoenzimáticas / Use of formilinized epimastigotes for the detection of anti-Trypanosoma cruzi antibodies by immunoenzyme technics

Se describen dos técnicas, enzimoinmunoensayo (ELISA) y Dot-enzimoinmunoensayo, en las que se emplearon epimastigotes formolados como antígeno para la detección de anticuerpos anti-Trypanosoma cruzi en sueros humanos. Se analizaron 50 sueros positivos y 28 negativos confirmados por inmunofluorescencia indirecta (IFI). El ELISA se realizó en forma cuantitativa, no presentó zona de superposición de resultados entre sueros positivos y negativos y la sensibilidad fue similar a la obtenida con IFI. Por ser un ensayo objetivo que no requiere equipo costosos podría reemplazr con ventajas a la IFI en el diagnóstico de enfermedad de Chagas. El Dot-EIE, una técnica sencilla, de bajo costo con mayor sensibilidad que la IFI podría ser utilizada, por no presentar falsos negativos y sólo un 2,5% de falsos positivos, para el monitoreo en bancos de sangre

Empleo de epimastigotes formolados para la detección de anticuerpos anti-trypanosoma cruzi mediante técnicas inmunoenzimáticas / Use of formilinized epimastigotes for the detection of anti-Trypanosoma cruzi antibodies by immunoenzyme technics

Se describen dos técnicas, enzimoinmunoensayo (ELISA) y Dot-enzimoinmunoensayo, en las que se emplearon epimastigotes formolados como antígeno para la detección de anticuerpos anti-Trypanosoma cruzi en sueros humanos. Se analizaron 50 sueros positivos y 28 negativos confirmados por inmunofluorescencia indirecta (IFI). El ELISA se realizó en forma cuantitativa, no presentó zona de superposición de resultados entre sueros positivos y negativos y la sensibilidad fue similar a la obtenida con IFI. Por ser un ensayo objetivo que no requiere equipo costosos podría reemplazr con ventajas a la IFI en el diagnóstico de enfermedad de Chagas. El Dot-EIE, una técnica sencilla, de bajo costo con mayor sensibilidad que la IFI podría ser utilizada, por no presentar falsos negativos y sólo un 2,5% de falsos positivos, para el monitoreo en bancos de sangre (AU)

[Use of formalinized epimastigotes for the detection of anti-Trypanosoma cruzi antibodies using immunoenzyme technics]. / Empleo de epimastigotes formolados para la detección de anticuerpos anti-Trypanosoma cruzi mediante técnicas inmunoenzimáticas.

Formalin-fixed epimastigotes of T. cruzi were employed to develop an ELISA and a Dot-immunobinding assay (Dot-IA). The results were compared with those obtained by the indirect fluorescent antibody test (IFA). Fifty positive and twenty negative sera for Chagas disease, supplied by the reference center (Institute Mario Fatala Chaben, Buenos Aires, Argentina), were analyzed. When the quantitative indirect ELISA was performed, no overlapping between positive and negative sera was observed and the correlation with IFA was 100%. The sensibility of Dot-IA was greater than that of IFA with 2.5% of false positives. Dot-IA performed with epimastigotes is a simple and qualitative test which could be applied for screening of blood donors. On the other hand, the ELISA presented here is an objective test which does not require specialized equipment and could replace with advantage the IFA test for the serodiagnosis of Chagas' disease.

Empleo de epimastigotes formolados para la detección de anticuerpos anti-Trypanosoma cruzi mediante técnicas inmunoenzimáticas. / [Use of formalinized epimastigotes for the detection of anti-Trypanosoma cruzi antibodies using immunoenzyme technics]

Formalin-fixed epimastigotes of T. cruzi were employed to develop an ELISA and a Dot-immunobinding assay (Dot-IA). The results were compared with those obtained by the indirect fluorescent antibody test (IFA). Fifty positive and twenty negative sera for Chagas disease, supplied by the reference center (Institute Mario Fatala Chaben, Buenos Aires, Argentina), were analyzed. When the quantitative indirect ELISA was performed, no overlapping between positive and negative sera was observed and the correlation with IFA was 100

. The sensibility of Dot-IA was greater than that of IFA with 2.5

of false positives. Dot-IA performed with epimastigotes is a simple and qualitative test which could be applied for screening of blood donors. On the other hand, the ELISA presented here is an objective test which does not require specialized equipment and could replace with advantage the IFA test for the serodiagnosis of Chagas disease.