Evaluation of In-House ELISA Using Trypanosoma cruzi Lysate and Recombinant Antigens for Diagnosis of Chagas Disease and Discrimination of Its Clinical Forms

Abstract

Abstract. The aim of this work was to investigate the potential usefulness of Trypanosoma cruzi lysate, recombinant protein JL7, and peptides P013, R13, JL18, JL19, and P0β as serological markers for human Chagas disease. We analyzed 228 sera from Brazilian Chagas disease patients classified into four clinical groups and 108 from non-chagasic patients. We defined the diagnostic sensitivity, specificity, and Kappa index measured by enzyme-linked immunosorbent assay (ELISA). As previously described, the highest values of diagnostic parameters were achieved for T. cruzi lysate and JL7; peptide P013 showed high specificity but low sensitivity. The other peptides resulted in lower sensitivity and specificity in our ELISA than T. cruzi lysate and JL7 protein. Antibodies against JL7 protein were mainly detected in sera from patients with severe chagasic cardiomyopathy, compared with those from the indeterminate form, whereas peptides failed to discriminate between the clinical forms of the disease.Chagas disease, caused by the hemoflagellate Trypanosoma cruzi, is a widespread tropical disease affecting at least 8 million people primarily in Latin American countries.1 The acute phase lasts 1 or 2 months and is usually symptomless, 2 however most of the infected individuals enter a life-long chronic phase with its asymptomatic and symptomatic forms. The asymptomatic, also called indeterminate form is characterized by the absence of clinical symptoms. However,~40% of persons with chronic T. cruzi infection develop symptoms of visceral damage, which may include cardiac lesions, digestive alterations, or both clinical manifestations (cardiac plus digestive). 2In the chronic phase, the primary method for diagnosis is the search of antibodies by serological testing, whereas the secondary diagnostic techniques are parasitological tests.1 So far, conventional serological tests include complement fixation, 3,4 indirect immunofluorescence assay (IFA), 5 indirect hemagglutination assay (IHA), 6 direct agglutination with 2-mercaptoethanol (DA-2ME), 7 and enzyme-linked immunosorbent assay (ELISA). [8][9][10] These tests usually use crude or semi-purified parasite preparations, often derived from a stage present only in the insect and in cultures at 26 C (epimastigote) but absent in the human host. Other assays incorporate more defined parasite components, like multiple fusion proteins containing epitopes from various T. cruzi antigens.11-15 Moreover, a rapid immunochromatographic assay (Chagas Stat-Pak, Chembio Diagnostic Systems, Medford, NY) has also been developed by employing a mixture of T. cruzi recombinant antigens, presenting a different degree of performance. [16][17][18] However, in the absence of a true gold standard, it is still necessary to carry out at least two different serological tests to establish a reliable diagnosis of Chagas disease. Indeed, the World Health Organization (WHO) consensus guidelines recommend to perform a third assay or repeat sampling to confirm or exclude the diagnosis if two serological tests ...

Evaluation of a chemiluminescent enzyme-linked immunosorbent assay for the diagnosis of Trypanosoma cruzi infection in a nonendemic setting

“…These values are comparable to those achieved using T. cruzi crude lysates (Riera et al 2009), as observed in Figure (B), for mixtures of certain specific parasite recombinant antigens (Umezawa et al 2003, Hernández et al 2010, Longhi et al 2012) and for other sensitive and specific confirmatory tests for Chagas disease, such as the trypomastigote excreted-secreted antigen blotting test (Umezawa et al 1996). The high stability of tGPI-mucin antigen gives us the opportunity to use this unconventional technique in a nonendemic setting without any significant decrease in reactivity.…”

Chronic Chagas Disease Diagnosis: A Comparative Performance of Commercial Enzyme Immunoassay Tests

“…14 Indeed, an Argentine study in a co-endemic area for Chagas disease and leishmaniasis showed 81.8% (9/11) cross-reactivity by using conventional ELISA 27 and a relative large number of false-positive or inconclusive results have been reported using conventional serologic testing. 13,28,29 Imuno-ELISA Chagas and Pathozyme Chagas use recombinant proteins as antigen. As has already been shown, this approach proved to be successful, with improvement of Sp and Se.…”

Chagas Disease Diagnostic Applications

“…using full-length or partially deleted recombinant, fusion proteins or synthetic peptides functionalized in different ways) and antigen display strategies (i.e. using mono- or multi-epitopic molecules) were evaluated before them being incorporated either individually or in defined mixtures into in-house and/or commercial kits (Godsel et al, 1995; Hernandez et al, 2010; Houghton et al, 2000; Krieger et al, 1992; Longhi et al, 2012; Oelemann et al, 1999; Pastini et al, 1994). Some of these tests have shown very good performances, and were thus extensively used in basic research and/or as confirmatory tests in clinical practice up to this day.…”