Serological diagnosis of Chagas disease with purified and defined Trypanosoma cruzi antigens

Umezawa, Eufrosina Setsu; Silveira, José Franco da

doi:10.1590/S0074-02761999000700051

Chagas disease; immunodiagnosis; recombinant peptides; ELISA; shed antigens; TESA-blot

Serological Diagnosis of Chagas Disease with Purified and Defined Trypanosoma cruzi Antigens

Vol. 94, Suppl. I: 285-288, 1999

Eufrosina Setsu Umezawa/⁺, José Franco da Silveira*

Instituto de Medicina Tropical de São Paulo, FMUSP, Av. Dr. Enéas de Carvalho Aguiar 470, 05403-000

São Paulo, SP, Brasil *Departamento de Micro, Imuno e Parasitologia da Escola Paulista de Medicina, Unifesp,

Rua Botucatu 862, 0423-062 São Paulo, SP, Brasil

Key words: Chagas disease - immunodiagnosis - recombinant peptides - ELISA - shed antigens - TESA-blot

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Chagas disease is caused by the protozoan parasite Trypanosoma cruzi, widespread in the American Continent. A recent report of the World Health Organization estimated that 16-18 million people are currently infected and that about 90 million individuals living in endemic areas are at risk of contracting T. cruzi infection. The high prevalence and continental extension of Chagas disease in Latin America, as well as the constant migration of T. cruzi infected individuals to non-endemic areas, require efficient and practical diagnostic procedures (Schmunis 1991,Wendel & Gonzaga 1993).

Chagas disease is characterized by the chronological appearance of specific classes of antibodies during the development of the infection. Antibodies from IgM class first appears as a typical sign of the acute phase of the disease, and there are some reports of an increase of total and specific IgA antibody class at this early phase (Lorca et al. 1995, Umezawa et al. 1996a). Antibodies of the IgG class, already present in the acute phase, accompanies the infection until the chronic phase.

Detection of antibodies against T. cruzi antigens by serological methods is still the main support for diagnosis of Chagas disease. The commercial available diagnostic tests are based on the whole or semi-purified antigenic fractions from T. cruzi epimastigote (the non-infective forms of the parasite). Considerable variation in the reproducibility and reliability of these tests have been reported by different laboratories, mainly to cross-reactivity with other pathogens and standardization of the reagents (Camargo et al. 1986). Diagnostic tests employing epimastigote antigenic extract have a limited specificity, associated to the fact of they do not possess highly reactive epitopes for IgG/IgM antibodies present in patients with acute or congenital Chagas disease (Umezawa et al. 1996a-c). The epimastigote antigenic fraction is constituted by complex molecules, that favors the appearance of false positive reactions and cross reaction with sera from patients with another infections, mainly visceral leishmaniasis (Chiller et al. 1990). This antigenic heterogeneity does not allow the differential diagnosis between the acute and chronic phases, and also among the clinical manifestations of Chagas disease.

EVALUATION OF RECOMBINANT ANTIGENS FOR CHAGAS DISEASE SERODIAGNOSIS

As discussed above the commercial immuno-diagnostic tests are based on the whole or semi-purified extracts of T. cruzi. The lack of specific and well characterized antigens prepared under quality-control conditions have introduced a source of variability in the final reagent and controversial results have been obtained with these reagents (Camargo et al. 1986). T. cruzi recombinant antigens may provide a convenient tool to improve current methods of serological diagnosis of Chagas disease (Moncayo & Luquetti 1990, Levin et al. 1991, Krieger et al. 1992, Franco da Silveira 1992, Paranhos et al. 1994, Pastini et al. 1994). Cloned peptides carrying well defined epitopes can be produced on a large scale and with acceptable degree of purity. For this reason, we have decided to evaluate the diagnostic efficiencies of different T. cruzi recombinant antigens in an ELISA format (Umezawa et al. 1999). Six T. cruzi recombinant antigens were expressed in fusion with glutathione S-transferase (GST) in Escherichia coli and purified by affinity chromatography. The selected antigens (H49, JL7, B13 and JL8, see Table) are composed by tandem amino acid repeats and showed high sensitivity, specificity, and positive and negative predictive values in previous studies (Moncayo & Luquetti 1990, Levin et al. 1991). Two non repetitive antigens A13 (Paranhos et al. 1990) and 1F8 (Gonzalez et al. 1985) were also included in this evaluation. The diagnostic efficiency of the ELISA using the six recombinant antigens was carried out with 541 serum samples (304 samples from chagasic patients, and 237 samples from nonchagasic patients) from nine South and Central America countries (Argentina, Brazil, Bolivia, Chile, Colombia, El Salvador, Guatemala, Honduras and Venezuela). The development and evaluation of the recombinant ELISA was carried out by a central laboratory (E. Umezawa, Instituto de Medicina Tropical da USP, SP, Brasil). The results were compared to those obtained with the whole extract of epimastigote forms.

Four recombinant antigens (1F8, H49, JL7 and B13) showed high sensitivity varying from 93.4% to 99%. A large number of individuals living in endemic areas produce specific antibodies against repetitive amino-acid antigens (H49, JL7 and B13). Interestingly, the sensitivity (99%) and specificity (99.6%) of 1F8 antigen is comparable to that other repetitive antigens indicating that chronic chagasic patients also display antibodies against non repetitive antigens. Serum samples from chagasic patients reacted with, at least, one recombinant antigen, suggesting that a mixture of recombinant antigens may detect anti-T. cruzi antibodies in all of serum samples used in this study. The positivity of a hypothetical antigenic mixture composed by the recombinant peptides H49 or JL7, B13 and 1F8 was calculated as being 100%. Available recombinant antigens react with 87-99% of the chronic chagasic sera, suggesting that the combination of two or more antigens to build up a multi-antigen immunoassay may result in a truly reliable T. cruzi serodiagnostic test.

In this study the epimastigote antigen showed 100% of sensitivity. However, the specificity of the epimastigote antigenic extract was lower (84%) than recombinant proteins which displayed specificity values between 96.2% (JL8) to 99.6% (A13, B13 and 1F8). The lower specificity of the epimastigote antigen is mainly due to cross-reacting epitopes between T. cruzi and Leishmania sp. Our results indicate that one of the major advantage of recombinant ELISA for the serodiagnosis of Chagas disease is the lack of cross-reaction with other parasitic diseases such as leishmaniasis. As it has been suggested by several authors (Levin et al. 1991, Krieger et al. 1992, Pastini et al. 1994, Umezawa et al. 1999), the use of a cocktail of recombinant antigens should provide a reliable T. cruzi serodiagnostic test. Different complementary antigens could be combined in a relatively simple immunoassay, and lead to the development of a multi-antigen diagnostic kit standardization for routine diagnosis of Chagas disease.

DIFFERENTIAL DIAGNOSIS OF ACUTE, CONGENITAL AND CHRONIC CHAGAS DISEASE BY WESTERN BLOT WITH TRYPOMASTIGOTE EXCRETED/SECRETED ANTIGENS (TESA-BLOT)

As it was discussed above, the diagnosis of acute and congenital Chagas disease, and the follow up of treated chagasic patients are of paramount importance. So far, the unique test described to control the parasitological cure of treated chagasic patients is the lysis of bloodstream trypomastigotes mediated by complement (Krettli et al. 1982). However, several groups point many methodological difficulties for the inclusion of that test in the routine of clinical laboratories (Pereira et al. 1989). For this reason, the search of specific antigens and/or alternative tests remains important for the follow up of the chagasic patients after drug treatment (Almeida et al. 1991, Norris et al. 1994, Krautz et al. 1994, Levy et al. 1996).

We have developed an immunodiagnostic test for Chagas disease using the TESA liberated, as exoantigens, in the supernatant of mammalian cell cultures infected with T. cruzi (Umezawa et al. 1996c). Exoantigens were collected from supernatants of LLC-MK2 cell cultures, separated by SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose filters. The test was developed by immunoblotting and termed TESA-blot. The main components of TESA antigens are a 150-160-kDa protein and several components (130-200 kDa) of SAPA antigen (Shed Acute Phase Antigen), the last previously described by Frasch and co-workers (Affranchino et al. 1989). The presence of SAPA/trans-sialidase in TESA-blot was confirmed by reactivity with a specific antiserum against SAPA as well as with IgG/IgM antibodies from acute and congenital human sera (Umezawa et al. 1996c).

The evaluation of TESA-blot was carried out with serum samples from 512 patients, 111 non-chagasic including cases of leishmaniasis and other pathologies, and 401 chagasic patients includind acute, congenital and chronic cases. TESA-blot showed 100% of sensitivity and 100% of specificity. Furthermore, this assay allows the discrimination between acute and chronic phases of Chagas disease. We have found that IgGs from chronic chagasic patients reacted with the 150-160 kDa exoantigen whereas IgGs/IgMs from acute patients reacted with the components of SAPA/trans-sialidase. This test, until now, have been demonstrated to be useful as a confirmatory test, since it has been used to confirm several inconclusive results obtained by conventional serology. Preliminary results from our laboratory (Matsumoto & Umezawa) showed that a 160 kDa antigen, obtained from DNA recombinant technology, may correspond to the CRP (Complement Regulatory Protein), previously described by Norris et al. (1994). Martins at al. (1985) had previously demonstrated that reactivity with a 160-kDa trypomastigote surface protein could discriminate between cured treated patients from the non cured ones. For these reasons, we started to use these antigens in the follow up of chagasic patients submitted to chemotherapy.

^REFERENCES

Table

This work was supported by grants from Fapesp (96/06736-1), CYTED (Ibero American Project of Biotechnology), International Atomic Energy Agency (IAEA), FMUSP-LIM49 and CNPq.

⁺Corresponding author. Fax: +55-11-852.3622. Email: eumezawa@usp.br

Received 9 June 1999

Accepted 9 August 1999

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Table

Affranchino JL, Ibańez CF, Luquetti AO, Rassi A, Reyes MB, Macina RA, Aslund L, Pettersson U, Frasch AC 1989. Identification of a Trypanosoma cruzi antigen that is shed during the acute phase of Chagas disease. Mol Biochem Parasitol 34: 221-228.
Almeida IC, Milani S, Gorin PAJ, Travassos LR 1991. Complement mediated lysis of Trypanosoma cruzi trypomastigotes by human anti-a-galactosyl antibodies. J Immunol 146: 2394-2400.
Camargo ME, Segura EL, Kagan, IG, Souza JMP, Carvalheiro JR, Yanovsky JF, Guimarăes MCS 1986. Collaboration on the standardization of Chagas disease in the Americas: an appraisal. PAHO Bul 20: 233-244.
Chiller TM, Samudio M and Zoulek G 1990. IgG reactivity with Trypanosoma cruzi and Leishmania antigens in sera of patients with Chagas disease and leishmaniasis. Am J Trop Med Hyg 43: 650-656.
Cotrim PC, Paranhos GP, Mortara RA, Wanderley J, Rassi A, Camargo ME, Franco da Silveira J 1990. Expression in Escherichia coli of a dominant immunogen of Trypanosoma cruzi recognized by human sera. J Clin Microbiol 28: 519-524.
Franco da Silveira J 1992. Trypanosoma cruzi recombinant antigens for serodiagnosis, p. 207-218. In S Wendel, Z Brener, ME Camargo & A Rassi (eds), Chagas'Disease (American Trypanosomiasis): its Impact on Transfusion and Clinical Medicine, Sociedade Brasileira de Hematologia e Hemoterapia, Săo Paulo.
Gonzalez A, Lerner TJ, Huecas M, Sosa-Pineda B, Nogueira N, Lizardi PM 1985. Apparent generation of a segmented mRNA from two separate tandem gene families in Trypanosoma cruzi. Nucleic Acids Res 13: 5789-5803.
Godsel LM, Tibbetts RS, Olson CL, Chaudoir BM, Engman DM 1995. Utility of recombinant flagelar calcium-binding protein for serodiagnosis of Trypanosoma cruzi infection. J Clin Microbiol 33: 2082-2085.
Gruber A, Zingales B 1993. Trypanosoma cruzi: characterization of two recombinant antigens with potential aplication in the diagnosis of Chagas disease. Exp Parasitol 76: 1-12.
Krautz GM, Micheli GC, Galvăo LMC, Cançado JR, Krettli A 1994. Antígenos solúveis liberados por tripomastigotas de Trypanosoma cruzi utilizados no teste de ELISA para detectar cura em pacientes chagásicos após tratamento. Rev Soc Bras Med Trop 27: 199-207.
Kretli A, Cançado, JR, Brener Z 1982. Effect of specific chemotherapy on the levels of lytic antibodies in Chagas disease. Trans R Soc Trop Med Hyg 76: 334-340.
Krieger MA, Almeida E, Oelemann W, Lafaille JJ, Pereira JB, Krieger H, Carvalho MR, Goldenberg S 1992. Use of recombinant antigens for the accurate immunodiagnosis of Chagas disease. Am J Trop Med Hyg 46: 427-434.
Levin MJ, Mesti EA, Benarous R, Levitus G, Schijman A, Levy-Yeyati P, Ruiz A, Kahan A, Rosembaum MB, Torres HN, Segura EL 1989. Identification of major Trypanosoma cruzi antigenic determinants in chronic Chagas heart disease. Am J Trop Med Hyg 41: 530-539.
Levin MJ, Franco da Silveira J, Frasch AC, Camargo ME, Lafon S, Degrave W, Rangel-Aldao R 1991. Recombinant antigens and Chagas disease diagnosis: analysis of a workshop. FEMS Microbiol Immunol 89: 11-20.
Levy AMA, Boainain E, Kloetzel JK 1996. In situ indirect fluorescent antibody: a new specific test to detect ongoing chagasic infections. J Clin Lab Anal 10: 98-103.
Lorca M,Veloso C, Munoz P, Bahamonde MI, Garcia A 1995. Diagnostic value of detecting specific IgA and IgM with recombinant T. cruzi antigens in congenital Chagas disease. Am J Trop Med Hyg 52: 512-515.
Martins MS, Hudson L, Krettli AU, Cançado JR, Brener Z 1985. Human and mouse sera recognize the same polypeptide associated with immunological resistance to T.cruzi infection. Clin exp Immunol 61: 343-350.
Norris KA, Galvăo LMC, Schrimpf JE, Cançado JR, Krettli AU 1994. Humoral immune response to the Trypanosoma cruzi complement regulatory protein as an indicator of a parasitologic clearance in human Chagas disease. Infect Immun 62: 4072-4074.
Moncayo A, Luquetti AO 1990. Multicentre double blind study for evaluation of Trypanosoma cruzi defined antigens as diagnostic reagents. Mem Inst Oswaldo Cruz 85: 489-495.
Paranhos GS, Cotrim PC, Mortara RA, Rassi A, Corral R, Freilij HL, Grinstein S, Wanderley J, Camargo ME, Franco da Silveira J 1990. Trypanosoma cruzi: cloning and expression of an antigen recognized by acute and chronic human chagasic sera. Exp Parasitol 71: 284-293.
Paranhos GS, Santos MRM, Cotrim PC, Rassi A, Jolivet M, Camargo ME, Franco da Silveira J 1994. Detection of antibodies in sera from Chagas disease patients using a Trypanosoma cruzi immunodominant recombinant antigen. Parasite Immunol 16: 165-169.
Pastini AC, Iglesias SR, Carricarte VC, Guerin ME, Sanchez DO, Frasch AC 1994. Immunoassay with recombinant Trypanosoma cruzi antigens potentially useful for screening donated blood and diagnosing Chagas' disease. Clin Chem 40: 1893-1897.
Pereira VL, Lvy AM, Boainain E 1989. Xeno-diagnostico, hemocultura e lise mediada por complemento como citérios de seleçăo de pacientes chagásicos cronicos para quimioterapia. Rev Inst Med Trop Săo Paulo 31: 301-307.
Schmunis GA 1991. Trypanosoma cruzi, the etiologic agent of Chagas disease: status in the blood supply in endemic and nonendemic countries. Transfusion 31: 547-557.
Umezawa ES, Shikanai-Yasuda MA, Stolf AMS 1996a. Changes in isotype composition and antigen of anti-Trypanosoma cruzi antibodies from acute to chronic Chagas disease. J Clin Lab Anal 10: 407-413.
Umezawa ES, Shikanai-Yasuda MA, Gruber A, Pereira-Chiocolla VL, Zingales B 1996b. Trypanosoma cruzi defined antigens in the serological evaluation of an outbreak of acute Chagas disease in Brazil (Catolé do Rocha, Paraíba). Mem Inst Oswaldo Cruz 91: 87-93.
Umezawa ES, Nascimento MS, Kesper Jr N, Coura JR, Borges-Pereira J, Junqueira CV, Camargo ME 1996c. Immunoblot assay using excreted-secreted antigens of Trypanosoma cruzi in serodiagnosis of congenital, acute, and chronic Chagas disease. J Clin Microbiol 34: 2143-2147.
Umezawa ES, Bastos SF, Camargo ME, Yamauchi LM, Santos MR, Gonzalez A, Zingales B, Levin MJ, Sousa O, Rangel-Aldao R, Franco da Silveira J 1999. Evaluation of recombinant antigens for Chagas disease serodiagnosis in South and Central America. J Clin Microbiol 37: 1554-1560.
Wendel S, Gonzaga AL 1993. Chagas disease and blood transfusion: a new world problem? Vox Sang 64: 1-12.

Publication Dates

Publication in this collection
18 Aug 2004
Date of issue
Sept 1999

History

Received
09 June 1999
Accepted
09 Aug 1999

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

[1] Affranchino JL, Ibańez CF, Luquetti AO, Rassi A, Reyes MB, Macina RA, Aslund L, Pettersson U, Frasch AC 1989. Identification of a Trypanosoma cruzi antigen that is shed during the acute phase of Chagas disease. Mol Biochem Parasitol 34: 221-228.

[2] Almeida IC, Milani S, Gorin PAJ, Travassos LR 1991. Complement mediated lysis of Trypanosoma cruzi trypomastigotes by human anti-a-galactosyl antibodies. J Immunol 146: 2394-2400.

[3] Camargo ME, Segura EL, Kagan, IG, Souza JMP, Carvalheiro JR, Yanovsky JF, Guimarăes MCS 1986. Collaboration on the standardization of Chagas disease in the Americas: an appraisal. PAHO Bul 20: 233-244.

[4] Chiller TM, Samudio M and Zoulek G 1990. IgG reactivity with Trypanosoma cruzi and Leishmania antigens in sera of patients with Chagas disease and leishmaniasis. Am J Trop Med Hyg 43: 650-656.

[5] Cotrim PC, Paranhos GP, Mortara RA, Wanderley J, Rassi A, Camargo ME, Franco da Silveira J 1990. Expression in Escherichia coli of a dominant immunogen of Trypanosoma cruzi recognized by human sera. J Clin Microbiol 28: 519-524.

[6] Franco da Silveira J 1992. Trypanosoma cruzi recombinant antigens for serodiagnosis, p. 207-218. In S Wendel, Z Brener, ME Camargo & A Rassi (eds), Chagas'Disease (American Trypanosomiasis): its Impact on Transfusion and Clinical Medicine, Sociedade Brasileira de Hematologia e Hemoterapia, Săo Paulo.

[7] Gonzalez A, Lerner TJ, Huecas M, Sosa-Pineda B, Nogueira N, Lizardi PM 1985. Apparent generation of a segmented mRNA from two separate tandem gene families in Trypanosoma cruzi. Nucleic Acids Res 13: 5789-5803.

[8] Godsel LM, Tibbetts RS, Olson CL, Chaudoir BM, Engman DM 1995. Utility of recombinant flagelar calcium-binding protein for serodiagnosis of Trypanosoma cruzi infection. J Clin Microbiol 33: 2082-2085.

[9] Gruber A, Zingales B 1993. Trypanosoma cruzi: characterization of two recombinant antigens with potential aplication in the diagnosis of Chagas disease. Exp Parasitol 76: 1-12.

[10] Krautz GM, Micheli GC, Galvăo LMC, Cançado JR, Krettli A 1994. Antígenos solúveis liberados por tripomastigotas de Trypanosoma cruzi utilizados no teste de ELISA para detectar cura em pacientes chagásicos após tratamento. Rev Soc Bras Med Trop 27: 199-207.

[11] Kretli A, Cançado, JR, Brener Z 1982. Effect of specific chemotherapy on the levels of lytic antibodies in Chagas disease. Trans R Soc Trop Med Hyg 76: 334-340.

[12] Krieger MA, Almeida E, Oelemann W, Lafaille JJ, Pereira JB, Krieger H, Carvalho MR, Goldenberg S 1992. Use of recombinant antigens for the accurate immunodiagnosis of Chagas disease. Am J Trop Med Hyg 46: 427-434.

[13] Levin MJ, Mesti EA, Benarous R, Levitus G, Schijman A, Levy-Yeyati P, Ruiz A, Kahan A, Rosembaum MB, Torres HN, Segura EL 1989. Identification of major Trypanosoma cruzi antigenic determinants in chronic Chagas heart disease. Am J Trop Med Hyg 41: 530-539.

[14] Levin MJ, Franco da Silveira J, Frasch AC, Camargo ME, Lafon S, Degrave W, Rangel-Aldao R 1991. Recombinant antigens and Chagas disease diagnosis: analysis of a workshop. FEMS Microbiol Immunol 89: 11-20.

[15] Levy AMA, Boainain E, Kloetzel JK 1996. In situ indirect fluorescent antibody: a new specific test to detect ongoing chagasic infections. J Clin Lab Anal 10: 98-103.

[16] Lorca M,Veloso C, Munoz P, Bahamonde MI, Garcia A 1995. Diagnostic value of detecting specific IgA and IgM with recombinant T. cruzi antigens in congenital Chagas disease. Am J Trop Med Hyg 52: 512-515.

[17] Martins MS, Hudson L, Krettli AU, Cançado JR, Brener Z 1985. Human and mouse sera recognize the same polypeptide associated with immunological resistance to T.cruzi infection. Clin exp Immunol 61: 343-350.

[18] Norris KA, Galvăo LMC, Schrimpf JE, Cançado JR, Krettli AU 1994. Humoral immune response to the Trypanosoma cruzi complement regulatory protein as an indicator of a parasitologic clearance in human Chagas disease. Infect Immun 62: 4072-4074.

[19] Moncayo A, Luquetti AO 1990. Multicentre double blind study for evaluation of Trypanosoma cruzi defined antigens as diagnostic reagents. Mem Inst Oswaldo Cruz 85: 489-495.

[20] Paranhos GS, Cotrim PC, Mortara RA, Rassi A, Corral R, Freilij HL, Grinstein S, Wanderley J, Camargo ME, Franco da Silveira J 1990. Trypanosoma cruzi: cloning and expression of an antigen recognized by acute and chronic human chagasic sera. Exp Parasitol 71: 284-293.

[21] Paranhos GS, Santos MRM, Cotrim PC, Rassi A, Jolivet M, Camargo ME, Franco da Silveira J 1994. Detection of antibodies in sera from Chagas disease patients using a Trypanosoma cruzi immunodominant recombinant antigen. Parasite Immunol 16: 165-169.

[22] Pastini AC, Iglesias SR, Carricarte VC, Guerin ME, Sanchez DO, Frasch AC 1994. Immunoassay with recombinant Trypanosoma cruzi antigens potentially useful for screening donated blood and diagnosing Chagas' disease. Clin Chem 40: 1893-1897.

[23] Pereira VL, Lvy AM, Boainain E 1989. Xeno-diagnostico, hemocultura e lise mediada por complemento como citérios de seleçăo de pacientes chagásicos cronicos para quimioterapia. Rev Inst Med Trop Săo Paulo 31: 301-307.

[24] Schmunis GA 1991. Trypanosoma cruzi, the etiologic agent of Chagas disease: status in the blood supply in endemic and nonendemic countries. Transfusion 31: 547-557.

[25] Umezawa ES, Shikanai-Yasuda MA, Stolf AMS 1996a. Changes in isotype composition and antigen of anti-Trypanosoma cruzi antibodies from acute to chronic Chagas disease. J Clin Lab Anal 10: 407-413.

[26] Umezawa ES, Shikanai-Yasuda MA, Gruber A, Pereira-Chiocolla VL, Zingales B 1996b. Trypanosoma cruzi defined antigens in the serological evaluation of an outbreak of acute Chagas disease in Brazil (Catolé do Rocha, Paraíba). Mem Inst Oswaldo Cruz 91: 87-93.

[27] Umezawa ES, Nascimento MS, Kesper Jr N, Coura JR, Borges-Pereira J, Junqueira CV, Camargo ME 1996c. Immunoblot assay using excreted-secreted antigens of Trypanosoma cruzi in serodiagnosis of congenital, acute, and chronic Chagas disease. J Clin Microbiol 34: 2143-2147.

[28] Umezawa ES, Bastos SF, Camargo ME, Yamauchi LM, Santos MR, Gonzalez A, Zingales B, Levin MJ, Sousa O, Rangel-Aldao R, Franco da Silveira J 1999. Evaluation of recombinant antigens for Chagas disease serodiagnosis in South and Central America. J Clin Microbiol 37: 1554-1560.

[29] Wendel S, Gonzaga AL 1993. Chagas disease and blood transfusion: a new world problem? Vox Sang 64: 1-12.