Serodiagnosis of Chronic Chagas Infection by Using EIE-Recombinant-Chagas-Biomanguinhos Kit
Vol. 96(4): 497-501, May 2001
Yara M Gomes/+, Valéria RA Pereira, Mineo Nakazawa, Daniela S Rosa, Maria das Neves DS Barros*, Antonio GP Ferreira**, Edimilson D Silva**, Sueli F Yamada Ogatta***, Marco Aurélio Krieger****, Samuel Goldenberg****
Departamento de Imunologia, Centro de Pesquisas Aggeu Magalhães-Fiocruz, Av. Moraes Rego s/no, Cidade Universitária, 50670-420 Recife, PE, Brasil *Ambulatório de Doença de Chagas, Hospital Universitário Oswaldo Cruz-UPE, Recife, PE, Brasil **Laboratório de Reativos do Instituto de Tecnologia em Imunobiológicos, Bio-Manguinhos-Fiocruz, Rio de Janeiro, RJ, Brasil ***Departamento de Microbiologia, Universidade Estadual de Londrina, Londrina, PR, Brasil ****Departamento de Bioquímica e Biologia Molecular, Instituto Oswaldo Cruz- Fiocruz, Rio de Janeiro, RJ, Brasil
A kit based on an enzyme immunoassay, EIE-Recombinant-Chagas-Biomanguinhos, developed by the Oswaldo Cruz Foundation, was evaluated for the serodiagnosis of chronic Chagas disease. Evaluation was performed with 368 serum samples collected from individuals living in an endemic area for Chagas disease: 131 patients in the chronic phase with confirmed clinical, epidemiological, and serological diagnosis (indirect immunofluorescence, indirect hemagglutination or enzyme-linked immunosorbent assay) and 237 nonchagasic seronegative individuals were considered negative control. The EIE-Recombinant-Chagas-Biomanguinhos kit showed high sensitivity, 100% (CI 95%: 96.4-100%) and high specificity, 100% (CI 95%: 98-100%). The data obtained were in full agreement with clinical and conventional serology data. In addition, no cross-reaction was observed with sera from patients with cutaneous (n=14) and visceral (n=3) leishmaniasis. However, when these sera were tested by conventional serological assays for Chagas disease, cross-reactions were detected in 14.3% and 33.3% of the patients with cutaneous and visceral leishmaniasis, respectively. No cross-reactions were observed when sera from nonchagasic seronegative patients bearing other infectious disease (syphilis, n=8; HTLV, n=8; HCV, n=7 and HBV, n=12) were tested. In addition, sera of patients with inconclusive results for Chagas disease by conventional serology showed results in agreement with clinical evaluation, when tested by the kit. These results are relevant and indicate that the refered kit provides a safe immunodiagnosis of Chagas disease and could be used in blood bank screening.
Key words: serodiagnosis - recombinant antigens - Trypanosoma cruzi - Chagas disease
Chagas disease is still a major health problem in Latin America where 16-18 million individuals are infected with the causative agent Trypanosoma cruzi and at least 90 million people are estimated to be at risk of infection (WHO 1996). Under natural conditions, infected reduviid bugs transmit the T. cruzi to humans when broken skin or mucous membranes contact metacyclics trypomastigotes from insect excreta. However, T. cruzi may bypass the vector bugs and be transmitted to man by a number of alternative mechanisms: blood transfusion, congenital transmission, accidental laboratory contamination, organ transplantation from infected donors and transmission by oral route (Umezawa et al. 1996, Gomes 1997). Blood transfusion is the second most common means of infection and the human migration from endemic areas to urban centers is proving a rising risk of transfusional Chagas disease in all Latin America and in non endemic countries (Schmuñis 1991).
Diagnosis of chronic Chagas disease is based on the detection of parasite by indirect parasitological methods (xenodiagnosis and hemoculture) or more usually on the detection of IgG antibodies against T. cruzi in the sera of patients by immunological methods (complement fixation-CF, indirect immunofluorescence-IIF, direct agglutination-DA, indirect hemagglutination-IHA and enzyme-linked immunosorbent assay-ELISA). The methods based on detection of the parasite, although highly specific, are of limited sensitivity, because parasites are detected in only 20-50% of individuals known to be infected, resulting in many false negative results (Gomes 1997). On the other hand, the methods based on the detection of an immune response to the parasite in the mamalian host lack specificity since they use crude or partially purified parasite extracts. Cross-reaction to T. cruzi has been observed with related protozoan diseases, particularly leishmaniasis. The problems with conventional assays (CF, IHA, DA, IIF and ELISA) may be overcome by using recombinant polypeptides containing specific T. cruzi epitopes that elicit an immune response in the majority of chagasic patients.
Several T. cruzi genes have been cloned and some of recombinant antigens have been assayed for their use in diagnosis (Affranchino et al. 1989, Levin et al. 1989, Almeida et al. 1990, Paranhos et al. 1990, Cotrin et al. 1990, Zingales et al. 1990, Goldenberg et al. 1991, Umezawa et al. 1999). Two recombinant antigens, CRA and FRA, expressed in the bacterium Escherichia coli were analyzed by Krieger et al. (1992) in a diagnostic test for Chagas disease. The data indicated that recombinant antigens displayed better results when used in combination than separately. These authors developed a direct ELISA which involves the use of peroxidase-labeled antigens to detect the immune-complexes. The results indicate that the recombinant (CRA+FRA) ELISA was better than the conventional ELISA in the diagnosis of Chagas disease, providing 100% specificity and sensitivity in all sera tested (Almeida et al. 1990, Krieger et al. 1992). These antigens were characterized and shown to display a repetitive epitope structure (Lafaille et al. 1989, Krieger et al. 1990). FRA (flagellar repetitive antigen) is located in the flagellum of the parasite and displays a 68-amino acid repeat, while CRA (cytoplasmic repetitive antigen) is distributed throughout the cytoplasm and has a 14-amino acid repeat (Lafaille et al. 1989).
Recently, a kit for diagnosis of chronic Chagas disease, using CRA+FRA antigens was developed by Oswaldo Cruz Foundation (Fiocruz), Rio de Janeiro, Brazil. The kit is based on enzyme immunoassay, the direct ELISA.
In the present work we report the evaluation of the EIE-Recombinant-Chagas-Biomanguinhos kit for the diagnosis of T. cruzi infection using characterized serum samples from individuals living in Chagas disease endemic areas and from individuals with other infectious diseases.
ACKNOWLEDGEMENTS
To Edileuza Brito for providing sera of leishmaniasis and Wayner Souza for performing the statistical analysis. To Genilda Medeiros and Ana Cristina B Souza of the blood center Fundação Hemope/Hemocentro, Pernambuco for performing several serological assays for Chagas disease and providing sera with other infectious diseases.
This work was partially supported by a PADCT-Finep to MAK. SG is recipient of a research fellowship from CNPq.
+Corresponding author. Fax: +55-81-453.2449. E-mail:yara@cpqam.fiocruz.br
Received 16 June 2000
Accepted 20 December 2000
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