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Vol.2 , No. 3, Publication Date: May 6, 2015, Page: 26-37
 were isolated, purified, characterized and antigenically validated to be proposed as candidates to serologically diagnose Chagas disease. The partition Triton-114 protein fractioning method allowed recovering nearly 7% T. cruzi-GPI-AMP from the total parasite proteins. The immunogenic capability of T. cruzi-GPI-AMP was demonstrated in experimental hosts. Validation of T. cruzi-GPI-AMP, as an antigenic product to make up an alternative test for serologic diagnosis of Chagas disease, was performed in 174 sera samples: 112 from unquestionable chagasic patients and 62 from non-chagasic to estimate its sensitivity and specificity, respectively. Statistical analysis revealed 100% sensitivity, specificity, positive and negative predictive values, with P<0.005. In addition, estimation of the Kappa index revealed 100% agreement, indicating a perfect concordance between the T. cruzi-GPI-AMP test and the condition of the chagasic patients following clinical, parasitological and serological criteria. Separation by electroelution revealed the presence of several T. cruzi-GPI-AMP fractions, ranging from 23 to 123 kDa, specifically recognized by anti-T. cruzi antibodies contained in sera from acute and chronic chagasic patients. These results lead us to conclude that T. cruzi-GPI-AMP antigen is an alternative diagnostic test sensitive and specific enough to warrant a reliable sero-diagnosis of Chagas disease.&picture=http://www.aascit.org/aascit/decorators/img/journal/919.jpg)
| [1] | Gladys Crisante, Center for Parasitological Research “J. F. Torrealba”, Department of Biology, Faculty of Sciences, University of Los Andes, Merida, 5101, Venezuela. |
| [2] | Pablo García, Laboratory of Biotechnology of Microorganisms (BIOMI), Faculty of Sciences, University of Los Andes, Merida, 5101, Venezuela. |
| [3] | Agustina Rojas, Center for Parasitological Research “J. F. Torrealba”, Department of Biology, Faculty of Sciences, University of Los Andes, Merida, 5101, Venezuela. |
| [4] | Diana Graterol, Laboratory of Protozoology, Institute of Molecular Biology of Parasites, Faculty of Health Sciences, University of Carabobo, Valencia, Venezuela. |
| [5] | Víctor Contreras, Laboratory of Protozoology, Institute of Molecular Biology of Parasites, Faculty of Health Sciences, University of Carabobo, Valencia, Venezuela. |
| [6] | Néstor Añez, Center for Parasitological Research “J. F. Torrealba”, Department of Biology, Faculty of Sciences, University of Los Andes, Merida, 5101, Venezuela. |
Trypanosoma cruzi-GPI anchored membrane proteins (T. cruzi-GPI-AMP) were isolated, purified, characterized and antigenically validated to be proposed as candidates to serologically diagnose Chagas disease. The partition Triton-114 protein fractioning method allowed recovering nearly 7% T. cruzi-GPI-AMP from the total parasite proteins. The immunogenic capability of T. cruzi-GPI-AMP was demonstrated in experimental hosts. Validation of T. cruzi-GPI-AMP, as an antigenic product to make up an alternative test for serologic diagnosis of Chagas disease, was performed in 174 sera samples: 112 from unquestionable chagasic patients and 62 from non-chagasic to estimate its sensitivity and specificity, respectively. Statistical analysis revealed 100% sensitivity, specificity, positive and negative predictive values, with P<0.005. In addition, estimation of the Kappa index revealed 100% agreement, indicating a perfect concordance between the T. cruzi-GPI-AMP test and the condition of the chagasic patients following clinical, parasitological and serological criteria. Separation by electroelution revealed the presence of several T. cruzi-GPI-AMP fractions, ranging from 23 to 123 kDa, specifically recognized by anti-T. cruzi antibodies contained in sera from acute and chronic chagasic patients. These results lead us to conclude that T. cruzi-GPI-AMP antigen is an alternative diagnostic test sensitive and specific enough to warrant a reliable sero-diagnosis of Chagas disease.
Keywords
Trypanosoma cruzi, GPI-Anchored Proteins, Diagnosis, Chagas Disease
Reference
| [01] | Lopez-Antuano, F. J., Rangel-Flores, H., Ramos, C. (2000). Diagnosis of Chagas’ disease. Rev. Latin. Microbiol. 42(3), 121-129. |
| [02] | Luquetti, A. O., Rassi, A. (2000). Diagnóstico laboratorial da infecçao pelo Trypanosoma cruzi. In: Trypanosoma cruzi e doença de Chagas. (2ª ed.). Ed. Brener, Andrade & Barral-Neto; Guanabara Koogan, Rio de Janeiro, pp:344-378. |
| [03] | Ferreira, M. S., Lopes, E. R., Chapadeiro, E., Dias, J. C. P., Luquetti, A. O. (1996). Doença de Chagas. Em R. Veronesi, & R. Focaccia (Ed.). Tratado de infectologia. (pp. 1175-1213). São Paulo, Brasil: Editora Atheneus. |
| [04] | Schechter, M., Nogueira, N. (1988). Variations induced by different methodologies in Trypanosoma cruzi surface antigen profiles. Mol. Biochem. Parasitol. 29(1), 37-46. |
| [05] | Silva, A. M., Brodskyn, C. I., Takehara, H. A., Mota, I. (1989). Differences in the antigenic profile of bloodstream and cell culture derived trypomastigotes of Trypanosoma cruzi. Rev. Inst. Med. Trop. São Paulo. 31(3), 146-150. |
| [06] | Ferguson, M. A. J. (1997). The surface glycoconjugates of Trypanosomatid parasites. Phil. Trans. Roy. Soc. London. Series B: Biological Sciences. 352(1359), 1295-1302. |
| [07] | Añez-Rojas, N., García–Lugo, P., Crisante, G., Rojas, A., Añez, N. (2006). Isolation, purification and characterization of GPI anchored membrane protein from Trypanosoma rangeli and Trypanosoma cruzi. Acta Tropica. 97(2), 140-145. |
| [08] | Englund, P. (1993). The structure and biosynthesis of glycosyl phosphatidyl inositol protein anchors. Ann. Rev. Biochem. 62(1), 121-138. |
| [09] | McConville, M., Ferguson, M. A. (1993). The structure, biosynthesis and function of glycosylated phosphatidyl inositol in the parasitic protozoa and higher eukaryotes. Biochem. J. 294(2), 305-324. |
| [10] | Snary, D., Hudson, L. (1979). Trypanosoma cruzi cell surface proteins identification of major glycoprotein. FEBS letters. 100(1), 166-170. |
| [11] | Añez, N., Crisante, G., Rojas, A. (2004). Update on Chagas disease in Venezuela: A review. Mem. Inst. Oswaldo Cruz, 99(8), 781-787. |
| [12] | Añez, N., Crisante, G., Añez-Rojas, N., Rojas, A., Moreno, G., Da Silva, F., Teixeira, M. (2009). Genetic typing of Trypanosoma cruzi isolated from different hosts and geographical areas of western Venezuela. Bol. Malariol. Sal. Amb. 49(2), 55-62. |
| [13] | Ko, Y.G., Thompson, J. R. (1995). Purification of Glycosyl-phosphatidyl-inositol-anchored proteins by modified Triton X-114 partitioning and preparative gel electrophoresis. Anal. Biochem. 224(1), 166-172. |
| [14] | Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193(1), 265-269. |
| [15] | Laemmli, U. K. (1970). SDS-polyacrylamide gel electrophoresis. Nature. 277, 680. |
| [16] | Añez, N., Crisante, G., Rojas, A., Carrasco, H., Parada, H., Yepez, Y., Borges, R., Guevara, P., Ramírez, J.L. (2001). Detection and significance of inapparent infection in Chagas disease in western Venezuela. Am. J. Trop. Med. Hyg. 65(3), 227-232. |
| [17] | Añez, N., Crisante, G., Caraballo, F., Delgado, W., Parada, H. (2011) Trypanosoma cruzi persistence at oral inflammatory foci in chronic chagasic patients. Acta Tropica. 117, 207-211 |
| [18] | Añez, N., Romero, M., Crisante, G., Bianchi, G., Parada, H. (2010). Valoración comparativa de pruebas serodiagnósticas utilizadas para detectar enfermedad de Chagas en Venezuela. Bol. Malariol. Sal. Amb. 50(1), 17-27. |
| [19] | Añez, N., Crisante, G., Rojas, G., Dávila, D. (2013). Brotes de enfermedad de Chagas agudo de posible transmisión oral en Mérida, Venezuela. Bol. Malariol. Sal. Amb. 53(1), 1-11. |
| [20] | Begg, C. B. (2005). Diagnostic Test, Evaluation. Armitage, P. & Colton, T. (Ed.). In Encyclopedia of Biostatistics. Second edition. Chichester, Inglaterra: John Wiley and Sons, Inc. (pp. 1-6). |
| [21] | Dean, A. G., Dean J. A., Coulombier, D., Brendel, K. A., Smith, D. C., Burton, A. H., … Arner, T. G. (1995). Epi Info, Version 6: A Word-Processing, Database, and Statistical Program for Public Health on IBM-Compatible Microcomputers. Center for Disease Control and Prevention, Atlanta, GA, USA |
| [22] | Rojas, A., García-Lugo, P., Crisante, G., Añez-Rojas, N., Añez, N. (2008). Isolation, purification, characterization and antigenic evaluation of GPI-anchored membrane proteins from Leishmania (Viannia) braziliensis. Acta Tropica. 105, 139-144. |
| [23] | Schijman, A.G., Bisio, M., …, Crisante, G., Añez, N., ….,et al., (2011). International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients. PLOS Negl. Trop. Dis. 5(1), 1-13. |
| [24] | Añez, N., Carrasco, H., Parada, H., Crisante, G., Rojas, A., Gonzalez, N., Ramirez, J. L., Guevara, P., Rivero, C., Borges, R., Scorza, J. V. (1999). Acute Chagas’ disease in western Venezuela: a Clinical, seroparasitologic and epidemiologic study. Am. J. Trop. Med. Hyg. 60, 215-222. |
| [25] | Rojas, A. (2013) Purificación, caracterización y evaluación antigénica de proteínas ancladas mediante GPI a la membrana de Leishmania (Viannia) braziliensis y su utilidad en el diagnóstico de casos activos de leishmaniasis cutánea americana. (Doctoral Thesis. University of Los Andes, Mérida-Venezuela. |
