Evaluation of TcH2AF-R and S35-S36 primers in PCR tests for the detection of Trypanosoma cruzi in mouse cardiac tissue
Keywords:
Trypanosoma cruzi, Chagas disease, Chagas cardiomyopathy, polymerase chain reaction
Abstract
Introduction. Heart transplant is a therapeutic option in the treatment of chagasic cardiomyopathy. For early detection of Chagas reactivation cases, the use of PCR tests using endomyocardial biopsies has been proposed. Development of an animal model will be the first step in evaluating the applicability of this approach.Objective. PCR tests based on the TcH2AF-R and S35-S36 primers were evaluated for the detection of T. cruzi in heart tissue of mice experimentally infected with the parasite.
Materials and methods. Two groups of ICR mice of 15 and 10 individuals were infected by intraperitoneal injection with 0.3 ml of PBS containing 1x106 trypomastigotes of the MHOM/CO/ 2001/D.A. (T. cruzi I) strain or 1x104 trypomastigotes of MHOM/BR/00/Y (T. cruzi II) strain. Parasitemia and cardiac parasitic infection were determined at 30, 60 (acute model), 100 and 150 (chronic model) days by means of histopathological examination and by PCR, using the TcH2AF-R and S35-S36 primers.
Results. The histopathological findings revealed alterations in the heart and the presence of intracellular amastigotes in acute and chronic models. In contrast to parasitemia levels and histopathological analyses, S35-S36 PCR detected infections in mice that were infected with either parasite strain. TcH2AF-R PCR detected T. cruzi I-infected mice earlier and more frequently than inspection for parasitemia or histopathological examination. Conclusions. Applying PCR tests with both primers proved superior for Chagas disease confirmation over currently standard detection methods.
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References
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2. Moncayo A, Ortiz MI. An update on Chagas disease (Human American trypanosomiasis). Ann Trop Med Parasitol. 2006;100:663-77.
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5. Teixeira AR, Nitz N, Guimaro MC, Gomes C, Santos- Buch CA. Chagas disease. Postgrad Med J. 2006;82:788-98.
6. Bértoli M, Andó MH, De Ornelas Toledo MJ, De Araújo SM, Gomes ML. Infectivity for mice of Trypanosoma cruzi I and II strains isolated from different hosts. Parasitol Res. 2006;99:7-13.
7. Schijman AG, Vigliano CA, Viotti RJ, Burgos JM, Brandariz S, Lococo BE, et al. Trypanosoma cruzi DNA in cardiac lesions of Argentinian patients with endstage chronic Chagas heart disease. Am J Trop Med Hyg. 2004;70:210-20.
8. Andrade Z, Andrade S. Chagas disease, pathology and pathogenesis. En: Gilles HM, editor. Protozoal diseases. New York, USA: Arnold Publishers; 1999. p. 323-35.
9. Kirchhoff LV, Votava JR, Ochs DE, Moser DR. Comparison of PCR and microscopic methods for detecting Trypanosoma cruzi. J Clin Microbiol. 1996;34:1171-5.
10. Pavía P, Cuervo C, Montilla M, Nicholls S, Puerta C. Estandarización de una prueba de PCR específica para la detección de Trypanosoma cruzi. Infectio. 2003;7:129-35.
11. Pavia PX, Vallejo GA, Montilla M, Nicholls RS, Puerta CJ. Detection of Trypanosoma cruzi and Trypanosoma rangeli infection in triatomine vectors by amplification of the histone H2A/SIRE and the sno-RNA-C11 genes. Rev Inst Med Trop Sao Paulo. 2007;49:23-30.
12. Gil J, Pavía P, Montilla M, Florez AC, Quintero C, Mercado M, et al. Comparación de una prueba de PCR basada en los genes codificantes para la histona H2A/SIRE con pruebas serológicas convencionales para el diagnóstico de la enfermedad de Chagas crónica en pacientes colombianos. Biomédica. 2007;27(Suppl.1):83-91.
13. Sturm NR, Degrave W, Morel C, Simpson L. Sensitive detection and schizodeme classification of Trypanosoma cruzi cells by amplification of kinetoplast minicircle DNA sequences: use in diagnosis of Chagas’ disease. Mol Biochem Parasitol. 1989;33:205-14.
14. Montilla MM, Guhl F, Jaramillo C, Nicholls S, Barnabe C, Bosseno MF, et al. Isoenzyme clustering of Trypanosomatidae Colombian populations. Am J Trop Med Hyg. 2002;66:394-400.
15. Vallejo GA, Guhl F, Chiari E, Macedo AM. Species specific detection of Trypanosoma cruzi and Trypano soma rangeli in vector and mammalian hosts by polymerase chain reaction amplification of kinetoplast minicircle DNA. Acta Trop. 1999;72:203-12.
16. de Arias AR, Ferro EA. Quantification of Trypanosoma cruzi parasitemia by direct micromethod. Trans R Soc Trop Med Hyg. 1988;82:248.
17. Martin DL, Postan M, Lucas P, Gress R, Tarleton RL. TGF-beta regulates pathology but not tissue CD8+ T cell dysfunction during experimental Trypanosoma cruzi infection. Eur J Immunol. 2007;37:2764-71.
18. Freitas JM, Lages-Silva E, Crema E, Pena SD, Macedo AM. Real time PCR strategy for the identification of major lineages of Trypanosoma cruzi directly in chronically infected human tissues. Int J Parasitol. 2005;35:411-7.
19. Puerta C, Urueña C. Electroforesis en geles de agarosa. En: Puerta CJ, editor. Prácticas de biología molecular. Colección Biblioteca del Profesional. Bogotá D.C.: Editorial Pontificia Universidad Javeriana; 2005. p. 24-30.
20. Vallejo GA. Estudios comparativos entre las secuencias de kDNA de Trypanosoma cruzi y Trypanosoma rangeli y su aplicación en el diagnóstico molecular de la tripanosomiasis americana. Actual Biol. 1998;20:43-56.
21. Guarner J, Bartlett J, Zaki SR, Colley DG, Grijalva MJ, Powell MR. Mouse model for Chagas disease: inmunohistochemical distribution of different stages of Trypanosoma cruzi in tissues throughout infection. Am J Trop Med Hyg. 2001;65:152-8.
22. Marinho CR, Bucci DZ, Dagli ML, Bastos KR, Grisotto MG, Sardinha LR, et al. Pathology affects different organs in two mouse strains chronically infected by a Trypanosoma cruzi clone: a model for genetic studies of Chagas’ disease. Infect Immun. 2004;72:2350-7.
23. Andersson J, Orn A, Sunnemark D. Chronic murine Chagas’ disease: the impact of host and parasite genotypes. Immunol Lett. 2003;86:207-12.
24. Cummings KL, Tarleton RL. Rapid quantitation of Trypanosoma cruzi in host tissue by real-time PCR. Mol Biochem Parasitol. 2003;129:53-9.
25. Melo RC, Brener Z. Tissue tropism of different Trypanosoma cruzi strains. J Parasitol. 1978;64:475-82.
26. Pinto PL, Takami R, Nunes EV, Guilherme CS, Oliveira OC Jr, Gama-Rodrigues J, et al. Life cycle of Trypanosoma cruzi (Y strain) in mice. Rev Hosp Clin Fac Med Sao Paulo. 1999;54:141-6.
27. Soares MB, Pontes-De-Carvalho L, Ribeiro-Dos- Santos R. The pathogenesis of Chagas’ disease: when autoimmune and parasite-specific immune responses meet. An Acad Bras Cienc. 2001;73:547-59.
28. Lane JE, Olivares-Villagomez D, Vnencak-Jones CL, Mccurley TL, Carter CE. Detection of Trypanosoma cruzi with the polymerase chain reaction and in situ hybridization in infected murine cardiac tissue. Am J Trop Med Hyg. 1997;56:588-95.
29. Añez N, Carrasco HA, Parada H, Crisante G, Rojas A, Fuenmayor C, et al. Myocardial parasite persistence in chronic chagasic patients. Am J Trop Med Hyg. 1999;60:726-32.
30. Lane JE, Ribeiro-Rodrigues R, Olivares-Villagomez D, Vnencak-Jones CL, McCurley TL, Carter CE. Detection of Trypanosoma cruzi DNA within murine cardiac tissue sections by in situ polymerase chain reaction. Mem Inst Oswaldo Cruz. 2003;98:373-6.
31. Elias FE, Vigliano CA, Laguens RP, Levin MJ, Berek C. Analysis of the presence of Trypanosoma cruzi in the heart tissue of three patients with chronic Chagas’ heart disease. Am J Trop Med Hyg. 2003;68:242-7.
32. Thomas MC, Olivares M, Escalante M, Marañón C, Montilla M, Nicholls S, et al. Plasticity of the histone H2A genes in a Brazilian and six Colombian strains of Trypanosoma cruzi. Acta Trop. 2000;75:203-10.
33. Saravia NG, Holguín AF, Cibulskis RE, D’ Alessandro A. Divergent isoenzyme profiles of sylvatic and domiciliary Trypanosoma cruzi in the eastern plains, piedmont, and highlands of Colombia. Am J Trop Med Hyg. 1987;36:59-69.
34. Triana O, Jaramillo N, Moreno J. Genetic variability of Colombian populations of Trypanosoma cruzi and Trypanosoma rangeli. Biol Res. 1999; 32:1-10.
35. Nitz N, Gomes C, de Cássia Rosa A, D’souza-Ault MR, Moreno F, Lauria-Pires L, et al. Heritable integration of kDNA minicircle sequences from Trypanosoma cruzi into the avian genome: insights into human Chagas disease. Cell. 2004;118:175-86.
36. Diez M, Favaloro L, Bertolotti A, Burgos JM, Vigliano C, Lastra MP, et al. Usefulness of PCR strategies for early diagnosis of Chagas’ disease reactivation and treatment follow-up in heart transplantation. Am J Transplant. 2007;7:1633-40.
37. Maldonado C, Albano S, Vettorazzi L, Salomone O, Zlocowski JC, Abiega C, et al. Using polymerase chain reaction in early diagnosis of re-activated Trypanosoma cruzi infection after heart transplantation. J Heart Lung Transplant. 2004;23:1345-8.
38. Benvenuti LA, Roggério A, Sambiase NV, Fiorelli A, Higuchi M. Polymerase chain reaction in endomyocardial biopsies for monitoring reactivation of Chagas’ disease in heart transplantation: a case report and review of the literature. Cardiovasc Pathol. 2005;14:265-8.
2. Moncayo A, Ortiz MI. An update on Chagas disease (Human American trypanosomiasis). Ann Trop Med Parasitol. 2006;100:663-77.
3. Urinovsky FM, Salomone OA, Córdoba R, Zazu AJ, Martínez A, Zlocowsky JC, et al. Morbimortalidad de los pacientes con cardiomiopatía chagásica y trasplante cardiaco. Experiencia inicial. Rev Arg Cardiol. 2003;71:325-31.
4. Castro AM, Luquetti AO, Rassi A, Rassi GG, Chiari E, Galvao LM. Blood culture and polymerase chain reaction for the diagnosis of the chronic phase of human infection with Trypanosoma cruzi. Parasitol Res. 2002;88:894-900.
5. Teixeira AR, Nitz N, Guimaro MC, Gomes C, Santos- Buch CA. Chagas disease. Postgrad Med J. 2006;82:788-98.
6. Bértoli M, Andó MH, De Ornelas Toledo MJ, De Araújo SM, Gomes ML. Infectivity for mice of Trypanosoma cruzi I and II strains isolated from different hosts. Parasitol Res. 2006;99:7-13.
7. Schijman AG, Vigliano CA, Viotti RJ, Burgos JM, Brandariz S, Lococo BE, et al. Trypanosoma cruzi DNA in cardiac lesions of Argentinian patients with endstage chronic Chagas heart disease. Am J Trop Med Hyg. 2004;70:210-20.
8. Andrade Z, Andrade S. Chagas disease, pathology and pathogenesis. En: Gilles HM, editor. Protozoal diseases. New York, USA: Arnold Publishers; 1999. p. 323-35.
9. Kirchhoff LV, Votava JR, Ochs DE, Moser DR. Comparison of PCR and microscopic methods for detecting Trypanosoma cruzi. J Clin Microbiol. 1996;34:1171-5.
10. Pavía P, Cuervo C, Montilla M, Nicholls S, Puerta C. Estandarización de una prueba de PCR específica para la detección de Trypanosoma cruzi. Infectio. 2003;7:129-35.
11. Pavia PX, Vallejo GA, Montilla M, Nicholls RS, Puerta CJ. Detection of Trypanosoma cruzi and Trypanosoma rangeli infection in triatomine vectors by amplification of the histone H2A/SIRE and the sno-RNA-C11 genes. Rev Inst Med Trop Sao Paulo. 2007;49:23-30.
12. Gil J, Pavía P, Montilla M, Florez AC, Quintero C, Mercado M, et al. Comparación de una prueba de PCR basada en los genes codificantes para la histona H2A/SIRE con pruebas serológicas convencionales para el diagnóstico de la enfermedad de Chagas crónica en pacientes colombianos. Biomédica. 2007;27(Suppl.1):83-91.
13. Sturm NR, Degrave W, Morel C, Simpson L. Sensitive detection and schizodeme classification of Trypanosoma cruzi cells by amplification of kinetoplast minicircle DNA sequences: use in diagnosis of Chagas’ disease. Mol Biochem Parasitol. 1989;33:205-14.
14. Montilla MM, Guhl F, Jaramillo C, Nicholls S, Barnabe C, Bosseno MF, et al. Isoenzyme clustering of Trypanosomatidae Colombian populations. Am J Trop Med Hyg. 2002;66:394-400.
15. Vallejo GA, Guhl F, Chiari E, Macedo AM. Species specific detection of Trypanosoma cruzi and Trypano soma rangeli in vector and mammalian hosts by polymerase chain reaction amplification of kinetoplast minicircle DNA. Acta Trop. 1999;72:203-12.
16. de Arias AR, Ferro EA. Quantification of Trypanosoma cruzi parasitemia by direct micromethod. Trans R Soc Trop Med Hyg. 1988;82:248.
17. Martin DL, Postan M, Lucas P, Gress R, Tarleton RL. TGF-beta regulates pathology but not tissue CD8+ T cell dysfunction during experimental Trypanosoma cruzi infection. Eur J Immunol. 2007;37:2764-71.
18. Freitas JM, Lages-Silva E, Crema E, Pena SD, Macedo AM. Real time PCR strategy for the identification of major lineages of Trypanosoma cruzi directly in chronically infected human tissues. Int J Parasitol. 2005;35:411-7.
19. Puerta C, Urueña C. Electroforesis en geles de agarosa. En: Puerta CJ, editor. Prácticas de biología molecular. Colección Biblioteca del Profesional. Bogotá D.C.: Editorial Pontificia Universidad Javeriana; 2005. p. 24-30.
20. Vallejo GA. Estudios comparativos entre las secuencias de kDNA de Trypanosoma cruzi y Trypanosoma rangeli y su aplicación en el diagnóstico molecular de la tripanosomiasis americana. Actual Biol. 1998;20:43-56.
21. Guarner J, Bartlett J, Zaki SR, Colley DG, Grijalva MJ, Powell MR. Mouse model for Chagas disease: inmunohistochemical distribution of different stages of Trypanosoma cruzi in tissues throughout infection. Am J Trop Med Hyg. 2001;65:152-8.
22. Marinho CR, Bucci DZ, Dagli ML, Bastos KR, Grisotto MG, Sardinha LR, et al. Pathology affects different organs in two mouse strains chronically infected by a Trypanosoma cruzi clone: a model for genetic studies of Chagas’ disease. Infect Immun. 2004;72:2350-7.
23. Andersson J, Orn A, Sunnemark D. Chronic murine Chagas’ disease: the impact of host and parasite genotypes. Immunol Lett. 2003;86:207-12.
24. Cummings KL, Tarleton RL. Rapid quantitation of Trypanosoma cruzi in host tissue by real-time PCR. Mol Biochem Parasitol. 2003;129:53-9.
25. Melo RC, Brener Z. Tissue tropism of different Trypanosoma cruzi strains. J Parasitol. 1978;64:475-82.
26. Pinto PL, Takami R, Nunes EV, Guilherme CS, Oliveira OC Jr, Gama-Rodrigues J, et al. Life cycle of Trypanosoma cruzi (Y strain) in mice. Rev Hosp Clin Fac Med Sao Paulo. 1999;54:141-6.
27. Soares MB, Pontes-De-Carvalho L, Ribeiro-Dos- Santos R. The pathogenesis of Chagas’ disease: when autoimmune and parasite-specific immune responses meet. An Acad Bras Cienc. 2001;73:547-59.
28. Lane JE, Olivares-Villagomez D, Vnencak-Jones CL, Mccurley TL, Carter CE. Detection of Trypanosoma cruzi with the polymerase chain reaction and in situ hybridization in infected murine cardiac tissue. Am J Trop Med Hyg. 1997;56:588-95.
29. Añez N, Carrasco HA, Parada H, Crisante G, Rojas A, Fuenmayor C, et al. Myocardial parasite persistence in chronic chagasic patients. Am J Trop Med Hyg. 1999;60:726-32.
30. Lane JE, Ribeiro-Rodrigues R, Olivares-Villagomez D, Vnencak-Jones CL, McCurley TL, Carter CE. Detection of Trypanosoma cruzi DNA within murine cardiac tissue sections by in situ polymerase chain reaction. Mem Inst Oswaldo Cruz. 2003;98:373-6.
31. Elias FE, Vigliano CA, Laguens RP, Levin MJ, Berek C. Analysis of the presence of Trypanosoma cruzi in the heart tissue of three patients with chronic Chagas’ heart disease. Am J Trop Med Hyg. 2003;68:242-7.
32. Thomas MC, Olivares M, Escalante M, Marañón C, Montilla M, Nicholls S, et al. Plasticity of the histone H2A genes in a Brazilian and six Colombian strains of Trypanosoma cruzi. Acta Trop. 2000;75:203-10.
33. Saravia NG, Holguín AF, Cibulskis RE, D’ Alessandro A. Divergent isoenzyme profiles of sylvatic and domiciliary Trypanosoma cruzi in the eastern plains, piedmont, and highlands of Colombia. Am J Trop Med Hyg. 1987;36:59-69.
34. Triana O, Jaramillo N, Moreno J. Genetic variability of Colombian populations of Trypanosoma cruzi and Trypanosoma rangeli. Biol Res. 1999; 32:1-10.
35. Nitz N, Gomes C, de Cássia Rosa A, D’souza-Ault MR, Moreno F, Lauria-Pires L, et al. Heritable integration of kDNA minicircle sequences from Trypanosoma cruzi into the avian genome: insights into human Chagas disease. Cell. 2004;118:175-86.
36. Diez M, Favaloro L, Bertolotti A, Burgos JM, Vigliano C, Lastra MP, et al. Usefulness of PCR strategies for early diagnosis of Chagas’ disease reactivation and treatment follow-up in heart transplantation. Am J Transplant. 2007;7:1633-40.
37. Maldonado C, Albano S, Vettorazzi L, Salomone O, Zlocowski JC, Abiega C, et al. Using polymerase chain reaction in early diagnosis of re-activated Trypanosoma cruzi infection after heart transplantation. J Heart Lung Transplant. 2004;23:1345-8.
38. Benvenuti LA, Roggério A, Sambiase NV, Fiorelli A, Higuchi M. Polymerase chain reaction in endomyocardial biopsies for monitoring reactivation of Chagas’ disease in heart transplantation: a case report and review of the literature. Cardiovasc Pathol. 2005;14:265-8.
How to Cite
1.
Puerta CJ, Guevara JM, Pavía PX, Montilla M, Nicholls RS, Parra E, et al. Evaluation of TcH2AF-R and S35-S36 primers in PCR tests for the detection of Trypanosoma cruzi in mouse cardiac tissue. biomedica [Internet]. 2008 Dec. 1 [cited 2024 May 10];28(4):616-2. Available from: https://revistabiomedica.org/index.php/biomedica/article/view/68
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