Enzyme-linked immunoassay using recombinant trans-sialidase of Trypanosoma cruzi can be employed for monitoring of patients with Chagas' disease after drug treatment

Abstract

trans-Sialidase is an enzyme present on the surface of Trypanosoma cruzi and is an important antigen recognized by sera from patients with Chagas' disease. In the present study we investigated whether the benznidazole treatment of patients with Chagas' disease induced changes in the reactivity of serum toward a recombinant form of trans-sialidase in order to develop an assay for monitoring of patients after treatment for Chagas' disease, which is needed at Chagas' disease control centers. By using an enzyme-linked immunosorbent assay containing a recombinant protein corresponding to the catalytic domain of trans-sialidase, we found that the antigen had a high specificity for sera from untreated patients with Chagas' disease. Sera from healthy individuals or patients with active visceral leishmaniasis minimally cross-reacted with the antigen. Anti-trans-sialidase immunoglobulin was detected in 98% of 151 untreated patients with Chagas' disease. Of these, 124 patients were treated for 60 days with benznidazole (5 mg/kg of body weight/day), and their sera were assayed for reactivity with the recombinant trans-sialidase. By using this methodology, three groups of patients could be established. The first group (60 patients), which was considered to have been successfully treated, showed no reactivity after treatment. The second group (46 patients) still showed signs of infection, and after treatment their sera recognized trans-sialidase, but with reduced titers. The third group (18 patients) was considered to be resistant to drug treatment, and their sera presented identical reactivities before and after treatment. These results suggest that determination of the absence of antibodies to recombinant trans-sialidase in treated patients by the present assay is indicative of treatment success, while the presence of antibodies may indicate the persistence of infection. Therefore, this method may be useful for the diagnosis and monitoring of patients undergoing benznidazole treatment.

FIG. 1.

Reactivities of sera from healthy individuals (squares), patients with Chagas' disease (circles), and patients with leishmaniasis (triangles) against recombinant TS by the TS ELISA. The results were calculated as the ratio of the absorbance of each serum sample at an optical density of 492 nm (A₄₉₂) to the cutoff value. Values greater than 1.0 were considered reactive. The horizontal lines represent the arithmetic means.

FIG. 2.

Treatment follow-up. The reactivities of sera from chronic Chagas' disease patients with successful therapy (A), a decrease in titers after treatment (C), and resistance to treatment (E) determined before benznidazole treatment (○) and 1 year (□) and 2 years (▵) after benznidazole treatment are shown. The values are the ratio of the absorbance of each serum sample at 492 nm to the reaction cutoff. Values greater than 1.0 were considered reactive. The horizontal lines represent the arithmetic means. Immunoblots for serum samples from the three groups of patients before treatment (Pre) and after treatment (Post-T) are shown in panels B, D, and F, respectively.

FIG. 3.

Distribution of reactivities of sera from each group of patients. Each point represents the reactivity of each serum sample before and after benznidazole treatment (second serum sample). (A) Chronic Chagas' disease patients with successful therapy; (B) chronic Chagas' disease patients with decreases in titers after treatment; (C) chronic Chagas' disease patients with resistance to treatment.