Human antibody responses to Trypanosoma cruzi 70-kD heat-shock proteins.

Heat-shock proteins of the 70-kD (hsp70) family are targets of humoral and cellular immune responses following bacterial or parasitic infections, including Chagas' disease. In the present study, we measured antibodies in human sera reactive with hsp70s from the cytoplasm (cy-hsp70), mitochondrion (mt-hsp70), and endoplasmic reticulum (grp78) of Trypanosoma cruzi. Of the three hsp70s tested, only grp78 detected T. cruzi infection in more than 90% of nontreated (NT) patients, with cy-hsp70 and mt-hsp70 detecting only 78% and 25% of NT patients, respectively. Reactivity of leishmanial sera was 77% with cy-hsp70, 13% with grp78, and 5% with mt-hsp70. Therefore, considering sensitivity and specificity, the best candidate for T. cruzi serodiagnosis is grp78. Combination of grp78 with a T. cruzi 24-kD flagellar calcium binding protein (FCaBP) increased the diagnostic sensitivity from 90% to 97% but increased leishmanial reactivity from 3% to 8%. To determine whether hsp70s are useful for discriminating between cured and noncured patients treated with trypanocidal drugs, we tested sera from treated noncured (TNC) patients and cured patients who have positive conventional serology, termed treated dissociated (TD). The cy-hsp70 and grp78 reacted with 74% and 68% of TNC patient sera, respectively, but these antigens did not discriminate TNC from TD patients (52% and 45% positive, respectively). The mt-hsp70 was detected by sera from few TNC patients (18%) and no TD patients. Although individual hsp70s were not useful for determining the effect of trypanocidal drugs on T. cruzi infection in individual patients, the majority of TNC patient sera (70-80%) reacted with two or three of the hsp70s. In contrast, no TD sera reacted with all three hsp70s, and 40% did not react with any of the hsp70s, indicating that the number of hsp70s detected decreases following successful treatment. Considered together, these results show that grp78 has potential as a diagnostic antigen and that absence of reactivity to all three hsp70s may be indicative of effective treatment.

[Soluble antigens released by Trypanosoma cruzi trypomastigotes used in ELISA to detect cure in chagasic patients following specific treatment]. / Antígenos solúveis liberados por tripomastigotas de Trypanosoma cruzi utilizados no teste de ELISA para detectar cura em pacientes chagásicos após tratamento específico.

Two soluble antigens isolated from T. cruzi were evaluated by ELISA for the diagnosis of chronic infection and assessment of cure after specific chemotherapy, namely, supernatants from parasite cell-cultures (SpAg) and trypomastigote excretory/secretory antigens (ESAg). Among the treated patients, a group defined as "dissociated" had been monitored for 3 to 10 years and displayed negative hemocultures and tests of trypomastigote complement-mediated lysis persistently negative although positive by conventional serology (indirect fluorescence with epimastigote, mainly). A negative lysis test indicates parasite elimination by the patient. Our ELISA results showed that among the non-treated chagasic controls the SpAg e ESAg detected 93% and 100%, of the cases, respectively. However, only 28% of sera from the dissociated group, considered as cured, were positive by ELISA using SpAg whereas all of such sera were negative using ESAg. Therefore ELISA with ESAg seems to be ideal to replace the complement-mediated lysis test with the aim to define cure after treatment of the chronic infection by T. cruzi.

The targets of the lytic antibody response against Trypanosoma cruzi.

Trypanosoma cruzi trypomastigotes, but not epimastigotes, are normally resistant to the lytic effects of complement from vertebrate hosts susceptible to infection. This resistance facilitates parasite survival and infectivity. During the course of chronic infections, however, the vertebrate hosts produce antibodies that render the trypomastigotes sensitive to lysis, primarily via the alternative complement cascade and amplified by the classical pathway. Here, Greice Krautz, Jessica Kissinger and Antoniana Krettli summarize research on lytic antibodies, and on their respective target(s) on the T. cruzi surface. These targets are useful in tests aimed at the diagnosis of chronic Chagas disease for control of cure after specific treatment and for vaccine development.

Glycoconjugates of Trypanosoma cruzi: a 74 kD antigen of trypomastigotes specifically reacts with lytic anti-alpha-galactosyl antibodies from patients with chronic Chagas disease.

Protective, lytic antibodies are believed to be correlated with active Trypanosoma cruzi infection. In patients with chronic infection, antibodies lysing trypomastigote forms recognize chiefly alpha-galactosyl structures at the parasite surface. The target molecules on cell-derived trypomastigotes that react with anti-alpha-galactosyl antibodies (anti-Gal) from patients with chronic Chagas disease were investigated. Glycoconjugates were isolated from trypomastigotes and shown to absorb purified Chagasic (Ch) anti-Gel effectively as well as lytic antibodies from Ch sera. Active fractions were F2 (74 kD and 95.6 kD) and F3 (120-200 kD). A differential reactivity with antibodies from untreated Ch patients (trypanolytic) and from treated, presumably cured, individuals (not trypanolytic) was evident using F2 and F3 antigenic fractions. No cross-reactivity with heterologous sera (other infections) was observed. The F2 glycoconjugate (mostly 74 kD) can be used in the diagnosis of active Chagas infection, replacing the quantitative determination of complement-mediated lysis. With the present sample of patients' sera and normal human sera, it showed 100% sensitivity and specificity.

Use of a 24-kilodalton Trypanosoma cruzi recombinant protein to monitor cure of human Chagas' disease.

A 24-kDa recombinant protein from Trypanosoma cruzi (rTc24) was evaluated by enzyme-linked immunosorbent assay (ELISA) and Western blot (immunoblot) tests to identify treated chagasic patients considered parasitologically cured on the basis of persistently negative tests of hemocultures and lytic antibodies. Some of these patients were termed dissociated because their sera, although negative by the complement-mediated lysis test, were positive by conventional serology. The negative lysis test indicates the absence of active infection after specific treatment, but this assay requires live and infectious parasites and cannot be used easily in a laboratory routine. Here we tested rTc24 by ELISA and Western blotting as an alternative for the complement-mediated lysis test. For the group of patients with active infection despite the treatment (uncured patients), all the sera tested recognized rTc24 in both tests. For the dissociated patients, approximately 80% of the sera did not react with rTc24 in the ELISA or in Western blots, in agreement with the negative complement-mediated lysis tests. Thus, the 24-kDa T. cruzi recombinant antigen, when used for initial trials to evaluate cure of chagasic patients submitted to specific treatment, will allow the identification of most, but not all, cases.