Development and Application of an ELISA Assay Using Excretion/Secretion Proteins from Epimastigote Forms ofT. cruzi(ESEA Antigens) for the Diagnosis of Chagas Disease

Abstract

An indirect enzyme-linked immunoabsorbent assay (ELISA) for Trypanosoma cruzi was developed using epimastigote secretion/excretion proteins (ESEA antigens) obtained from axenic culture supernatants. A panel of 120 serum samples from subjects with confirmed Chagas disease (n = 50), healthy controls (n = 50), and patients with other parasitic diseases (n = 20) was used to evaluate the new ESEA-based ELISA (ELISAESEA). This new test had excellent sensitivity (98%) and acceptable specificity (88%). Cross-reactivity was observed largely in sera from subjects with Leishmania and Ascaris infections. Using Western blotting and epimastigotes from two distinct T. cruzi isolates, several polypeptide bands with molecular masses ranging from 50 to 220 kDa were detected in pooled chagasic sera. However, the band pattern for each isolate was different. These data suggest that an inexpensive and technically simple ELISA based on ESEA antigens is a promising new tool for the diagnosis of Chagas disease.

Innovations in diagnosis and post-therapeutic monitoring of Chagas disease: Simultaneous flow cytometric detection of IgG1 antibodies anti-live amastigote, anti-live trypomastigote, and anti-fixed epimastigote forms of Trypanosoma cruzi

“…Despite the high sensitivity of different serological tests for the diagnosis of Chagas disease, the cross-reactivity mainly with sera from the VL and ACL patients is still frequent (Nakazawa et al, 2001;Amato Neto et al, 2005;Berrizbeitia et al, 2006Berrizbeitia et al, , 2012. Berrizbeitia et al (2012) demonstrated that despite the high sensitivity of this test for the diagnosis of Chagas disease, employing secreted/ excreted protein of epimastigotes, its specificity was only 88%, and cross-reactions were observed with Ascaris spp. and Leishmania spp.…”

Comparative serology techniques for the diagnosis of Trypanosoma cruzi infection in a rural population from the state of Querétaro, Mexico

“…The Camargo method was employed for IFA, using complete forms of T. cruzi epimastigotes isolated from strain Y (of Brazilian origin) and strain T3 (of Mexican origin, state of Veracruz, derived from in vitro cultures in Grace medium). Samples with titres > 1:16 were considered positive ( Camargo et al 1986 , Berrizbeitia et al 2012) .…”

“…The extracted enzyme was purified, fractioned and used as an antigen in Western blots to detect anti- T. cruzi antibodies in the 1,033 serum samples. The test was considered to be positive when anti- T. cruzi antibodies were detected at a dilution of 1:80 ( Marín & Sánchez-Moreno 2010 , Berrizbeitia et al 2012) . Evaluations of statistically significant differences between the tests were based on sensitivity, specificity, negative and positive predictive values, concordance percentage and the kappa index.…”

“…Conventional serological tests alone are not sufficient, mainly because antigenic preparations are derived from parasite extracts or semi-purified fractions of epimastigotes (the non-infectious form of the parasite) and procedures differ between laboratories. Therefore, inconclusive results are frequently obtained ( Berrizbeitia et al 2012 ). However, some patients with Chagas disease have false negative results ( Kirchhoff & Neva 1985 , Araújo & Berne 2013) .…”

A Family Cluster of Chagas Disease Detected through Selective Screening of Blood Donors: A Case Report and Brief Review

“…They were referred to the JD MacLean Centre for Tropical Diseases at the McGill University Health Centre (Montreal, Quebec) for assessment. Blood was drawn for confirmatory serological testing using a T cruzi excreted-secreted antigen ELISA (TESA-ELISA) ( 14 , 15 ) and PCR by the National Reference Centre for Parasitology (NRCP) in Montreal. Some samples were also tested using RIPA by Quest Diagnostics (USA).…”