Characterization of an Immunodominant Antigenic Epitope from Trypanosoma cruzi as a Biomarker of Chronic Chagas' Disease Pathology

ABSTRACTNowadays, the techniques available for chronic Chagas' disease diagnosis are very sensitive; however, they do not allow discrimination of the patient's clinical stages of the disease. The present paper describes that three out of the five different repeats contained in theTrypanosoma cruziTcCA-2 membrane protein (3972-FGQAAAGDKPPP, 6303-FGQAAAGDKPAP, and 3973-FGQAAAGDKPSL) are recognized with high sensitivity (>90%) by sera from chronic Chagas' disease patients and that they are not recognized by sera from patients in the acute phase of the disease. A total of 133 serum samples from chagasic patients and 50 serum samples from healthy donors were tested. In addition, sera from 15 patients with different autoimmune diseases, 43 serum samples from patients suffering an infectious disease other than Chagas' disease, and 38 serum samples from patients with nonchagasic cardiac disorders were also included in this study. The residue 3973 peptide shows a specificity of >98%, as it is not recognized by individuals with autoimmune and inflammatory processes or by patients with a nonchagasic cardiomyopathy. Remarkably, the levels of antibody against the 3973 epitope detected by the sera from Chagas' disease patients in the symptomatic chronic phase, involving cardiac or digestive alterations, are higher than those detected by the sera from Chagas' disease patients in the indeterminate phase of the disease. It is suggested that the diagnostic technique described could also be used to indicate the degree of pathology. The amino acids F, Q, and DKP located in the peptide at positions 1, 3, and 8 to 10, respectively, are essential to conform to the immunodominant antigenic epitope.

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Serological Diagnosis of Chronic Chagas Disease: Is It Time for a Change?

Journal of Clinical Microbiology ◽

10.1128/jcm.00142-16 ◽

2016 ◽

Vol 54 (6) ◽

pp. 1566-1572 ◽

Cited By ~ 32

Author(s):

Alba Abras ◽

Montserrat Gállego ◽

Teresa Llovet ◽

Silvia Tebar ◽

Mercedes Herrero ◽

...

Keyword(s):

Chagas Disease ◽

False Positive ◽

Disease Diagnosis ◽

Cross Reactivity ◽

Human Migration ◽

Serological Diagnosis ◽

Content Type ◽

New Generation ◽

Single Technique ◽

Chronic Chagas Disease

Chagas disease has spread to areas that are nonendemic for the disease with human migration. Since no single reference standard test is available, serological diagnosis of chronic Chagas disease requires at least two tests. New-generation techniques have significantly improved the accuracy of Chagas disease diagnosis by the use of a large mixture of recombinant antigens with different detection systems, such as chemiluminescence. The aim of the present study was to assess the overall accuracy of a new-generation kit, the Architect Chagas (cutoff, ≥1 sample relative light units/cutoff value [S/CO]), as a single technique for the diagnosis of chronic Chagas disease. The Architect Chagas showed a sensitivity of 100% (95% confidence interval [CI], 99.5 to 100%) and a specificity of 97.6% (95% CI, 95.2 to 99.9%). Five out of six false-positive serum samples were a consequence of cross-reactivity withLeishmaniaspp., and all of them achieved results of <5 S/CO. We propose the Architect Chagas as a single technique for screening in blood banks and for routine diagnosis in clinical laboratories. Only gray-zone and positive sera with a result of ≤6 S/CO would need to be confirmed by a second serological assay, thus avoiding false-positive sera and the problem of cross-reactivity withLeishmaniaspecies. The application of this proposal would result in important savings in the cost of Chagas disease diagnosis and therefore in the management and control of the disease.

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Immunoblotting analyses using two-dimensional gel electrophoresis of Trypanosoma cruzi excreted-secreted antigens

Revista da Sociedade Brasileira de Medicina Tropical ◽

10.1590/s0037-86822004000600005 ◽

2004 ◽

Vol 37 (6) ◽

pp. 454-459

Author(s):

Adriano Gomes Silva ◽

Elisangela Paula Silveira-Lacerda ◽

Jair Pereira Cunha-Júnior ◽

Maria Aparecida de Souza ◽

Silvio Favoreto Junior

Keyword(s):

Trypanosoma Cruzi ◽

Chagas Disease ◽

Gel Electrophoresis ◽

Complex Mixture ◽

Chronic Phase ◽

Disease Diagnosis ◽

Basic Knowledge ◽

Two Dimensional ◽

Two Dimensional Gel Electrophoresis ◽

Secreted Antigens

Trypanosoma cruzi trypomastigotes excrete-secrete a complex mixture of antigenic molecules. This antigenic mixture denominated trypomastigote excreted-secreted antigens contains a 150-160 kDa band that shows excellent performance in Chagas' disease diagnosis by immunoblotting. The present study partially characterized by two-dimensional gel electrophoresis the immunoreactivity against the 150-160kDa protein using sera samples from chagasic patients in different phases of the disease. Trypomastigote excreted-secreted antigen preparations were subjected to high-resolution two-dimensional (2D) gel electrophoresis followed by immunoblotting with sera from chagasic and non-chagasic patients. The 150-160kDa protein presented four isoforms with isoelectric focusing ranging from 6.2 to 6.7. The four isoforms were recognized by IgM from acute phase and IgG from chronic phase sera of chagasic patients. The 150-160kDa isoform with IF of approximately 6.4 became the immunodominant spot with the progression of the disease. No cross-reactivity was observed with non-chagasic or patients infected with Leishmania sp. In this study we provide basic knowledge that supports the validation of trypomastigote excreted-secreted antigens for serological diagnosis of Chagas' disease.

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Towards a Paradigm Shift in the Treatment of Chronic Chagas Disease

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01662-13 ◽

2013 ◽

Vol 58 (2) ◽

pp. 635-639 ◽

Cited By ~ 142

Author(s):

R. Viotti ◽

B. Alarcón de Noya ◽

T. Araujo-Jorge ◽

M. J. Grijalva ◽

F. Guhl ◽

...

Keyword(s):

Chagas Disease ◽

Paradigm Shift ◽

Chronic Phase ◽

World Health ◽

Chronic Patients ◽

Content Type ◽

Current Medical Practice ◽

Health Organization ◽

Criteria For Evaluation ◽

Chronic Chagas Disease

ABSTRACTTreatment for Chagas disease with currently available medications is recommended universally only for acute cases (all ages) and for children up to 14 years old. The World Health Organization, however, also recommends specific antiparasite treatment for all chronic-phaseTrypanosoma cruzi-infected individuals, even though in current medical practice this remains controversial, and most physicians only prescribe palliative treatment for adult Chagas patients with dilated cardiomyopathy. The present opinion, prepared by members of the NHEPACHA network (Nuevas Herramientas para el Diagnóstico y la Evaluación del Paciente con Enfermedad de Chagas/New Tools for the Diagnosis and Evaluation of Chagas Disease Patients), reviews the paradigm shift based on clinical and immunological evidence and argues in favor of antiparasitic treatment for all chronic patients. We review the tools needed to monitor therapeutic efficacy and the potential criteria for evaluation of treatment efficacy beyond parasitological cure. Etiological treatment should now be mandatory for all adult chronic Chagas disease patients.

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Evaluation of the rabbit as a model for chagas' disease: I. Parasitological studies

Memórias do Instituto Oswaldo Cruz ◽

10.1590/s0074-02761987000400010 ◽

1987 ◽

Vol 82 (4) ◽

pp. 531-536 ◽

Cited By ~ 7

Author(s):

Luiz Eduardo Ramirez ◽

Zigman Brener

Keyword(s):

Tissue Culture ◽

Trypanosoma Cruzi ◽

Chagas Disease ◽

Chronic Phase ◽

Suitable Model ◽

Parasite Strain ◽

Fresh Blood ◽

Blood Examination ◽

Chronic Chagas Disease ◽

Selective Interactions

In order to investigate the value of the rabbit as an experimental model for Chagas' disease, 72 animals have been inoculated by intraperitoneal and conjunctival route with bloodstream forms, vector-derived metacyclic trypomastigotes and tissue culture trypomastigotes of Trypanosoma cruzi strains Y, CL and Ernane. In 95.6% of the animals trypomastigotes had been detected at the early stages of infection by fresh blood examination. The course of parasitemia at the acute phase was strongly influenced by the parasite strain and route of inoculation. At the chronic phase parasites had been recovered by xenodiagnosis and/or hemoculture in 40% of the examined animals. The xenodiagnosis studies have shown selective interactions between the T. cruzi strains and the four species of vectors used, inducing significant variability in the results. The data herein present are consistent with the parasitological requirements established for a suitable model for chronic Chagas' disease.

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Cross-Reactivity Using Chimeric Trypanosoma cruzi Antigens: Diagnostic Performance in Settings Where Chagas Disease and American Cutaneous or Visceral Leishmaniasis Are Coendemic

Journal of Clinical Microbiology ◽

10.1128/jcm.00762-19 ◽

2019 ◽

Vol 57 (8) ◽

Cited By ~ 2

Author(s):

Ramona Tavares Daltro ◽

Leonardo Maia Leony ◽

Natália Erdens Maron Freitas ◽

Ângelo Antônio Oliveira Silva ◽

Emily Ferreira Santos ◽

...

Keyword(s):

Visceral Leishmaniasis ◽

Chagas Disease ◽

Diagnostic Performance ◽

Latent Class ◽

Cross Reactivity ◽

Chimeric Proteins ◽

Serum Samples ◽

Perfect Agreement ◽

Content Type ◽

Chronic Chagas Disease

ABSTRACT Chimeric T. cruzi antigens have been proposed as a diagnostic tool for chronic Chagas disease (CD) in both settings where Chagas disease is endemic and those where it is not endemic. Antibody response varies in accordance to each T. cruzi strain, presenting challenges to the use of antigens lacking demonstrated cross-reactivity with Leishmania spp. Our group expressed four chimeric proteins (IBMP-8.1, IBMP-8.2, IBMP-8.3, and IBMP-8.4) and previously assessed their diagnostic performance to determine cross-reactivity with Leishmania spp. Here, we validated our findings using serum samples from different Brazilian geographic areas reporting endemic Chagas disease, endemic visceral or American cutaneous leishmaniasis (ACL), or both. Overall, 829 serum samples were evaluated using commercial and IBMP enzyme-linked immunosorbent assays. Due to the absence of a reference assay to diagnosis CD, latent class analysis (LCA) was performed through the use of a statistical model. The incidence of cross-reactivity for ACL-positive samples varied from 0.35% (IBMP-8.3) to 0.70% (IBMP-8.1 and IBMP-8.2). Regarding visceral leishmaniasis (VL)-positive samples, the IBMP-8.2 and IBMP-8.3 antigens cross-reacted with six (3.49%) and with only one sample (0.58%), respectively. No cross-reactivity with either ACL or VL was observed for the IBMP-8.4 antigen. Similarly, no cross-reactions were found when VL-positive samples were assayed with IBMP-8.1. The agreement among the results obtained using IBMP antigens ranged from 97.3% for IBMP-8.2 and 99% for IBMP-8.1 and IBMP-8.3 to 100% for IBMP-8.4, demonstrating almost perfect agreement with LCA. Accordingly, in light of the negligible cross-reactivity with both ACL and VL, we suggest the use of IBMP antigens in regions where T. cruzi and Leishmania spp. are coendemic.

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Validation of a particle gel immunoassay for Trypanosoma cruzi antibody detection using plasma samples collected with capillary tubes

The Journal of Infection in Developing Countries ◽

10.3855/jidc.784 ◽

2010 ◽

Vol 4 (09) ◽

pp. 590-592

Author(s):

Marlene MY Kawai ◽

Jose AP Salas ◽

Luiz AP Tavares ◽

Cor JF Fontes

Keyword(s):

Trypanosoma Cruzi ◽

Chagas Disease ◽

Antibody Detection ◽

Plasma Samples ◽

Reference Standard ◽

Kappa Index ◽

Capillary Tubes ◽

Serum Samples ◽

Practical Test ◽

Chronic Chagas Disease

Introduction: The Chagas disease particle gel immunoassay (PaGIA-Chagas) is a simple, fast and practical test used for the diagnosis of the chronic Chagas disease and is based on anti-Trypanosoma cruzi antibody detection in serum. This study aimed to validate the PaGIA-Chagas on plasma collected with capillary tubes. Methodology: Serum samples from 74 T. cruzi-infected and 26 non-infected individuals were tested by conventional indirect immunofluorescence and PaGIA-Chagas. Later, plasma specimens collected with capillary tubes from these same individuals were tested by PaGIA-Chagas. Results from serum samples tested by IFA and PAGIA- Chagas were considered as the reference standard to determine the accuracy parameters of the particle gel tested on plasma samples. The inter-test agreement of T. cruzi antibody detection by PaGIA-Chagas on serum and plasma was calculated using Kappa index. Results: The PaGIA-Chagas performed on plasma collected with capillary tubes had sensitivity and specificity of 99% and 100%, respectively. The crude agreement observed with the results of the PaGIA-Chagas on plasma and serum was 99% and the Kappa index was 0.975 (CI95%: 0.782 - 1.000). Conclusion: PaGIA-Chagas for anti-T. cruzi detection on plasma collected with capillary tubes is accurate and might be indicated to reduce fieldwork time and materials in epidemiological screening of chronic Chagas disease.

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Excretory-Secretory Antigens of Trypanosoma cruzi Are Potentially Useful for Serodiagnosis of Chronic Chagas' Disease

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.8.5.1024-1027.2001 ◽

2001 ◽

Vol 8 (5) ◽

pp. 1024-1027 ◽

Cited By ~ 16

Author(s):

Mineo Nakazawa ◽

Daniela S. Rosa ◽

Valéria R. A. Pereira ◽

Milena O. Moura ◽

Veridiana C. Furtado ◽

...

Keyword(s):

Trypanosoma Cruzi ◽

Chagas Disease ◽

Sensitivity And Specificity ◽

Immunosorbent Assay ◽

High Sensitivity ◽

Enzyme Linked Immunosorbent Assay ◽

Chronic Chagas Disease ◽

Secreted Antigens

ABSTRACT The reactivities of sera from chronic chagasic patients against the trypomastigote excreted-secreted antigens (TESA) of Trypanosoma cruzi strains with different biodemes were analyzed by TESA-blot and TESA–enzyme-linked immunosorbent assay (ELISA). Although both tests presented high sensitivity and specificity, TESA-ELISA is more appropriate for screening a larger number of samples.

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Alkaline soluble Trypanosoma cruzi epimastigote antigen (ASEA) applied to dot-ELISA

Revista do Instituto de Medicina Tropical de São Paulo ◽

10.1590/s0036-46651994000200012 ◽

1994 ◽

Vol 36 (2) ◽

pp. 163-166 ◽

Cited By ~ 7

Author(s):

Ana Maria Lissaldo ◽

Sumie Hoshino-Shimizu ◽

Eufrosina Setsu Umezawa ◽

Anna Maria Simonsen Stolf

Keyword(s):

Trypanosoma Cruzi ◽

Chagas Disease ◽

Control Group ◽

Igg Antibodies ◽

Serum Samples ◽

Endemic Areas ◽

Dot Elisa ◽

Chronic Chagas Disease

The alkaline soluble Trypanosoma cruzi epimastigote antigen (ASEA) was assessed in dot-ELISA for the diagnosis of Chagas' disease. Serum samples (355) from chagasic and non-chagasic patients were studied, and IgG antibodies to ASEA were found in all patients with chronic Chagas' disease. In non-chagasic patients 95.6% were negative, except for those with leishmaniasis (visceral and mucocutaneous), and some patients from control group reacted in low titers. The data indicate that dot-ELISA using ASEA is suitable for seroepidemiologic surveys to be employed in endemic areas for Chagas' disease.

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Lychnopholide in Poly(D,L-Lactide)-Block-Polyethylene Glycol Nanocapsules Cures Infection with a Drug-Resistant Trypanosoma cruzi Strain at Acute and Chronic Phases

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01937-19 ◽

2020 ◽

Vol 64 (4) ◽

Cited By ~ 1

Author(s):

Renata Tupinambá Branquinho ◽

Carlos Geraldo Campos de Mello ◽

Maykon Tavares Oliveira ◽

Levi Eduardo Soares Reis ◽

Paula Mello de Abreu Vieira ◽

...

Keyword(s):

Polyethylene Glycol ◽

Trypanosoma Cruzi ◽

Chagas Disease ◽

Chronic Phase ◽

Heart Tissue ◽

Oral Route ◽

Enzyme Linked Immunosorbent Assay ◽

Drug Resistant ◽

Content Type ◽

Negative Results

ABSTRACT Chagas disease remains neglected, and current chemotherapeutics present severe limitations. Lychnopholide (LYC) at low doses loaded in polymeric poly(d,l-lactide)-block-polyethylene glycol (PLA-PEG) nanocapsules (LYC-PLA-PEG-NC) exhibits anti-Trypanosoma cruzi efficacy in mice infected with a partially drug-resistant strain. This study reports the efficacy of LYC-PLA-PEG-NC at higher doses in mice infected with a T. cruzi strain resistant to benznidazole (BZ) and nifurtimox (NF) treated at both the acute phase (AP) and the chronic phase (CP) of infection by the oral route. Mice infected with the T. cruzi VL-10 strain were treated by the oral route with free LYC (12 mg/kg of body weight/day), LYC-PLA-PEG-NC (8 or 12 mg/kg/day), or BZ at 100 mg/kg/day or were not treated (controls). Treatment efficacy was assessed by hemoculture (HC), PCR, enzyme-linked immunosorbent assay (ELISA), heart tissue quantitative PCR (qPCR), and histopathology. According to classical cure criteria, treatment with LYC-PLA-PEG-NC at 12 mg/kg/day cured 75% (AP) and 88% (CP) of the animals, while at a dose of 8 mg/kg/day, 43% (AP) and 43% (CP) were cured, showing dose-dependent efficacy. The negative qPCR results for heart tissue and the absence of inflammation/fibrosis agreed with the negative results obtained by HC and PCR. Thus, the mice treated with the highest dose could be considered 100% cured, in spite of a low ELISA reactivity in some animals. No cure was observed in animals treated with free LYC or BZ or the controls. These results are exceptional in terms of experimental Chagas disease chemotherapy and provide evidence of the outstanding contribution of nanotechnology in mice infected with a T. cruzi strain totally resistant to BZ and NF at both phases of infection. Therefore, LYC-PLA-PEG-NC has great potential as a new treatment for Chagas disease and deserves further investigations in clinical trials.

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A Fluorinated Phenylbenzothiazole Arrests the Trypanosoma cruzi Cell Cycle and Diminishes the Infection of Mammalian Host Cells

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01742-19 ◽

2019 ◽

Vol 64 (2) ◽

Author(s):

Roberto I. Cuevas-Hernández ◽

Richard M. B. M. Girard ◽

Sarai Martínez-Cerón ◽

Marcelo Santos da Silva ◽

Maria Carolina Elias ◽

...

Keyword(s):

Trypanosoma Cruzi ◽

Chagas Disease ◽

Structural Characteristics ◽

Chronic Phase ◽

Human Infection ◽

Host Cells ◽

Mammalian Host ◽

Content Type ◽

Trypanocidal Drugs

ABSTRACT Chagas disease (CD) is a human infection caused by Trypanosoma cruzi. CD was traditionally endemic to the Americas; however, due to migration it has spread to countries where it is not endemic. The current chemotherapy to treat CD induces several side effects, and its effectiveness in the chronic phase of the disease is controversial. In this contribution, substituted phenylbenzothiazole derivatives were synthesized and biologically evaluated as trypanocidal agents against Trypanosoma cruzi. The trypanocidal activities of the most promising compounds were determined through systematic in vitro screening, and their modes of action were determined as well. The physicochemical-structural characteristics responsible for the trypanocidal effects were identified, and their possible therapeutic application in Chagas disease is discussed. Our results show that the fluorinated compound 2-methoxy-4-[5-(trifluoromethyl)-1,3-benzothiazol-2-yl] phenol (BT10) has the ability to inhibit the proliferation of epimastigotes [IC50(Epi) = 23.1 ± 1.75 μM] and intracellular forms of trypomastigotes [IC50(Tryp) = 8.5 ± 2.9 μM] and diminishes the infection index by more than 80%. In addition, BT10 has the ability to selectively fragment 68% of the kinetoplastid DNA compared with 5% of nucleus DNA. The mode of action for BT10 on T. cruzi suggests that the development of fluorinated phenylbenzothiazole with electron-withdrawing substituent is a promising strategy for the design of trypanocidal drugs.

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