ParasitologyEnzyme-linked immunosorbent assay with Trypanosoma cruzi excreted-secreted antigens (TESA-ELISA) for serodiagnosis of acute and chronic Chagas’ disease
Introduction
Serological methods are widely accepted for the diagnosis of Chagas’ disease (CD). These methods usually employ antigens derived from the non-infective epimastigote forms that react with anti-T. cruzi IgG antibodies from most patients in the chronic phase of CD. However, IgG from sera of acute phase patients can result in low reactivity with these antigens Schattschneider et al 1992, Umezawa et al 1996a, Umezawa et al 1996b. Moreover, epimastigote antigens show cross-reactivity with sera from patients infected with Leishmania sp Schattschneider et al 1992, Umezawa et al 1996a.
Besides the characteristic IgM antibodies, anti-T. cruzi IgG antibodies are produced since the beginning of the acute phase Camargo and Amato-Neto 1974, Israelski et al 1988. These antibodies react mainly with surface molecules of the infective trypomastigote forms whereas most epitopes shared by trypomastigote and epimastigote forms belong to internal antigens Rangel-Aldao et al 1986, Umezawa et al 1996a, Umezawa et al 1996b. This fact may explain why epimastigote antigens do not result in high IgG reactivity with acute phase sera.
T. cruzi recombinant antigens reduce the cross-reactivity of serogical assays but reduce also their sensitivity Levin et al 1991, Umezawa et al 1999, unless a mixture of recombinant antigens is used (Pastini et al., 1994). Inclusion of shed-acute-phase antigen (SAPA) recombinant protein (Affranchino et al., 1989) results in IgG positive results with acute sera (Pastini et al., 1994). However, recombinant proteins are not widely commercially available and their high costs impair their utilization in most CD endemic countries. A mucin-like antigen purified from tissue culture-derived trypomastigote forms shows high sensitivity and specificity but requires several purification steps besides amplification of the ELISA signal by chemiluminescence Almeida et al 1994, Almeida et al 1997.
The above data indicate the need for an assay employing an easy to prepare antigen which results in a strong IgG positive signals for sera from acute and chronic chagasic patients and presents high specificity, eliminating cross-reactivity of sera from patients infected with Leishmania sp.
Exoantigens of trypomastigote forms react with anti-T. cruzi IgG antibodies from acute and chronic patient sera Jazı́n et al 1991, Gazzinelli et al 1993, Umezawa et al 1996a, Umezawa et al 1996b. Immunoblotting of this fraction, termed by us TESA-blot (from Trypomastigote Excreted-Secreted Antigens) showed excellent sensitivity and specificity in CD serodiagnosis Umezawa et al 1996b, Umezawa et al 1996a. However, immunoblotting is not useful for assaying a large number of sera.
In the present report, we describe the use of TESA in ELISA. The assay showed high specificity and sensitivity with sera from patients in both the acute and chronic phases of CD. The antigen preparation does not require any purification step and does not present the cross-reactivity of leishmaniasis sera observed with epimastigote extracts. ELISA competition assays confirmed that anti-T. cruzi antibodies of chagasic sera that react with TESA are different from those that react with whole epimastigote form extract. Besides, partial characterization of TESA showed that several epitopes present in this fraction are absent in EAE.
Section snippets
Human sera
Serum samples were collected from 120 chagasic seropositive cases and 164 nonchagasic seronegative cases from Brazil. Among the 120 chagasic cases, 67 were in the chronic-phase, with positive serology for Chagas’ disease and with a clinical diagnosis confirmed by electrocardiography and radiology. The acute cases of Chagas’ disease (n = 53) included: 15 infected by oral transmission (Shikanai-Yasuda et al., 1991), 1 by blood transfusion, 5 by accidental laboratory contamination, and 32 by
TESA-ELISA detects anti-T.cruzi antibodies from both acute and chronic chagasic sera with high sensitivity
Fig. 1 shows the ELISA OD492nm data and Table 1 the positivity indexes obtai ned with TESA and EAE in the IgG (Fig. 1A) and IgM (Fig. 1B) anti-T. cruzi survey of the 120 chagasic (acute and chronic phases) and the 164 nonchagasic patient sera.
The mean OD492nm of TESA reactivity of IgG antibodies from the 53 acute chagasic sera was 1.25 ± 0.28. A much lower mean was obtained with EAE (0.61 ± 0.32, Fig. 1A). The difference between the mean values was statistically significant (P < 0.0001).
Discussion
We had previously shown that exoantigens shed by T. cruzi (TESA) to the media of in vitro cultured infected cells presented 100% sensitivity and 100% specificity in Chagas’ disease immunodiagnosis by immunoblotting Umezawa et al 1996a, Umezawa et al 1996b. Here we show that this fraction can be employed in ELISA, provided that serum-free media is used in the culture. TESA-ELISA detected IgG antibodies from 100% of acute and chronic chagasic sera (Fig. 1A, Table 1), indicating that the TESA
Acknowledgments
This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), CNPq and LIM49-FMUSP.
References (32)
- et al.
Identification of a Trypanosoma cruzi antigen that is shed during the acute phase of Chagas’ disease
Mol Biochem Parasitol
(1989) - et al.
Stage-specific surface antigens expressed during the morphogenesis of vertebrate forms of Trypanosoma cruzi
Exp Parasitol
(1987) - et al.
Galactofuranose-containing glycoconjugates of epimastigote and trypomastigote forms of Trypanosoma cruzi
Mol Biochem Parasitol
(1993) - et al.
Nitrocellulose-based assays for the detection of glycolipids and other antigensmechanism of binding to nitrocellulose
J Immunol Meth
(1985) - et al.
A novel cell surface trans-sialidase of Trypanosoma cruzi generates a stage-specific epitope required for invasion of mammalian cells
Cell
(1991) - et al.
Glycoconjugates of Trypanosoma cruzia 74 kD antigen of trypomastigotes specifically reacts with lytic anti-α-galactosyl antibodies from patients with chronic Chagas’ disease
J Clin Lab Analysis
(1993) - et al.
Lytic anti-α-galactosyl antibodies from patients with chronic Chagas’ disease recognize novel O-linked oligosaccharides on mucin-like glycosyl-phosphatidylinositol-anchored glycoproteins of Trypanosoma cruzi
Biochem J
(1994) - et al.
A highly sensitive chemiluminescence enzyme-linked immunosorbent assay for diagnosis of active Trypanosoma cruzi infection
Transfusion
(1997) - et al.
Persistence of elevated levels of galactosyl-α(1-3) galactose antibodies in sera from patients cured of visceral leishmaniasis
J Clin Microbiol
(1988) - Camargo, M. E., & Amato-Neto, V. (1974). Anti-Trypanosoma cruzi IgM antibodies as serological evidence of recent...
Anti-Trypanosoma cruzi and anti-laminin antibodies in chagasic patients after specific treatment
J Clin Microbiol
Use of Trypanosoma cruzi purified glycoprotein (GP57/51) of trypomastigote-shed antigens to assess cure for human Chagas’ disease
Am J Trop Med Hyg
Immunological characterization of antigens released by Trypanosoma cruzi-infected cells
J Parasitol
Antibody response and antigen recognition in human infection with Trypanosoma cruzi
Am J Trop Med Hyg
Shift of excretory-secretory immunogens of Trypanosoma cruzi during human Chagas’ disease
Infect Immunol
Cited by (46)
Conventional serological performance in diagnosis of Chagas' disease in southern Brazil
2013, Brazilian Journal of Infectious DiseasesCitation Excerpt :Although there are several tests available to indirect diagnosis (especially enzyme-linked immunosorbent assay (ELISA), indirect hemagglutination (IHA) and indirect immunofluorescense (IIF)), there is not a gold-standard technique, and the results obtained by different techniques show disagreement, with varying levels of sensitivity and specificity.6–8 These results may be related to the parasitic form used to obtain the antigens; there are antigenic differences between epimastigote and amastigote forms, accepting that their immunodominant fractions are not the same.9 Trypomastigotes-based ELISA showed higher specificity10,11 and, in some cases, higher sensitivity too.9
Cavia porcellus as a model for experimental infection by trypanosoma cruzi
2011, American Journal of PathologyCitation Excerpt :The number of parasites encountered was expressed in terms of parasites per milliliter of blood. The kinetics for the production of IgM and IgG to T. cruzi were measured by trypomastigote excreted-secreted antigens (TESA)–ELISA, as described previously.37 Briefly, ELISA 96-well plates (Immulon 2; Thermo Labsystems, Franklin, MA) were coated with 2 μg/mL of TESA and were incubated with guinea pig serum, 1:100 dilution (IgG detection) or 1:50 dilution (IgM detection).
Anti-triatomine saliva immunoassays for the evaluation of impregnated netting trials against Chagas disease transmission
2011, International Journal for ParasitologyImmunoglobulin M antibodies against CRA and FRA recombinant antigens of Trypanosoma cruzi in chronic chagasic patients
2011, Human ImmunologyCitation Excerpt :Using cruzipain from epimastigotes as antigen, Zúñiga et al. [18] demonstrated 9.5% positivity among chronic chagasic patients, whereas Umezawa et al. [19], using trypomastigote excreted-secreted antigens, demonstrated only 3.0% positivity between chronic chagasic individuals. In addition to our study, these cited data [17–19] indicated lower positivity to anti-T. cruzi antibodies of the IgM class in the chronic phase of Chagas disease.