Comparison of a PCR test based on the histone H2A/SIRE genes with classical serological tests for the diagnosis of chronic Chagas disease in Colombian patients
Keywords:
polymerase chain reaction, histones, Chagas myocardiopathy, Trypanosoma cruzi
Abstract
Introduction. Diagnosis of Chagas disease in its latent and chronic phase is difficult because of the low parasitemia levels. Therefore, serological and molecular techniques are necessary to achieve an appropriate diagnosis.Objective. The polymerase chain reaction based on the amplification of the SIRE element inserted into H2A encoding genes was compared with classical serological tests for the diagnosis of Chagas disease in Colombian patients.
Materials and methods. An agreement study was carried out by comparing the PCR with ELISA (enzyme linked immuno sorbent assay) and IFAT (indirect immunofluorescence) tests. In addition, the PCR sensitivity and specificity were determined. A sample of 156 individuals was tested with the H2A PCR primers after a Chagas disease classification based on clinical, epidemiological and serological data associated with each patient. In addition, 97 out of 156 samples were also compared with the S35/S36 PCR primers.
Results. Eighty-nine of 156 samples (57%) were positive by both IFAT and ELISA and 84 (53.8%) presented the expected 234 bp amplification fragment. The sensitivity of the TcH2AF/R PCR was 88% (95% C.I.: 75%--95%) and its specificity 92.5% (95% C.I.: 87.7%--97.2%). The kappa index for concordance between serological tests and TcH2AF/R PCR was 0.8 (95% C.I.:73%--86%), and between the TcH2AF/R and S35/S36 PCR primers was 0.9 (95% C.I.: 84%--96%). These indices indicated a "good" and "almost perfect" agreement, respectively.
Conclusions. The TcH2AF/R PCR is a promising diagnostic tool for the detection of T. cruzi and is suggested as a tool complementary to the classical serological tests.
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References
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6. Machado CR, Augusto-Pinto L, McCulloch R, Teixeira SM. DNA metabolism and genetic diversity in Trypanosomes. Mutat Res 2006;612:40-57.
7. Saravia NG, Holguin 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.
8. Rodríguez P, Montilla M, Nicholls RS, Zarante I, Puerta, C. Isoenzymatic characterization of Colombian strains of Trypanosoma cruzi. Mem Inst Oswaldo Cruz 1998;93:739-40.
9. Marquez E, Arcos-Burgos M, Triana O, Moreno J, Jaramillo N. Clonal population structure of Colombian sylvatic Trypanosoma cruzi. J Parasitol 1998;84:1143-9.
10. Triana O, Jaramillo N, Moreno J. Genetic variability of Colombian populations of Trypanosoma cruzi and Trypanosoma rangeli. Biol Res 1999;32:1-10.
11. 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.
12. Rodríguez P, Escalante M, Díez H, Cuervo C, Montilla M, Nicholls RS, et al. Estudio de la variabilidad de seis cepas colombianas de Trypanosoma cruzi mediante polimorfismos de longitud de fragmentos de restricción (RFLP) y amplificación aleatoria de ADN polimórfico (RAPD). Biomédica 2002;22:263-71.
13. Mejia AM, Triana O. Análisis por LSSP-PCR de la variabilidad genética de Trypanosoma cruzi en sangre y órganos de ratones infectados. Biomédica 2005;25:76-86.
14. D'Alessandro A, Saravia N. Trypanosoma rangeli. En: Gilles HM, editor. Protozoal diseases. London: Arnold Press;1999. p.398-412.
15. Guhl F, Vallejo GA. Trypanosoma (Herpetosoma) rangeli Tejera, 1920: an updated review. Mem Inst Oswaldo Cruz 2003;98:435-42.
16. 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.
17. Guhl F, Jaramillo C, Carranza JC, Vallejo GA. Molecular characterization and diagnosis of Trypanosoma cruzi and Trypanosoma rangeli. Arch Med Res 2002;33:362-70.
18. Vallejo GA, Gulh F, Chiari E, Macedo AM. Species specific detection of Trypanosoma cruzi and Trypanosoma rangeli in vector and mammalian hosts by polymerase chain reaction amplification of kinetoplast minicircle DNA. Acta Trop 1999;72:203-12.
19. Vargas N, Souto RP, Carranza JC, Vallejo GA, Zingales B. Amplification of a specific repetitive DNA sequence for Trypanosoma rangeli identification and its potential application in epidemiological investigations. Exp Parasitol 2000;96:147-59.
20. Nitz N, Gomes C, de Cassia 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.
21. Pavía P, Cuervo C, Montilla M, Nicholls S, Puerta C. Diseño y estandarización de una prueba de PCR para la detección específica de Trypanosoma cruzi. Infectio 2003;7:129-36.
22. Beltrán M, Duque S, Guhl F, Herrera CP, López MC, Moreno AL, et al. Prueba de ELISA y prueba de inmunofluorescencia indirecta (IFI). En: Guhl F, Nicholls RS, editores. Manual de procedimientos para el diagnóstico de la enfermedad de Chagas. Bogotá: Ministerio de Salud; 2001. p.32-48.
23. Sambrook JF, Russell DW. Gel electrophoresis of DNA. En: Molecular cloning A laboratory manual. Tercera edición. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 2001.p.5.1-17.
24. Puerta C, Martin J, Alonso C, Lopez MC. Isolation and characterization of the gene encoding histone H2A from Trypanosoma cruzi. Mol Biochem Parasitol 1994;64:1-10.
25. Sackett D, Haynes R, Guyatt G, Tugwell P. Selección de pruebas diagnosticas. En: Epidemiologia Clínica. Segunda Edición. México, D.F.: Editorial Médica Panamericana; 1998. p.64-7.
26. Wincker P, Britto C, Pereira JB, Cardoso MA, Oelemann W, Morel CM. Use of a simplified polymerase chain reaction procedure to detect Trypanosoma cruzi in blood samples from chronic chagasic patients in a rural endemic area. Am J Trop Med Hyg 1994;51:771-7.
27. Thomas MC, Olivares M, Escalante M, Maranon 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.
28. Vázquez MP, Schijman AG, Levin MJ. A short interspersed repetitive element provides a new 3' acceptor site for trans-splicing in certain ribosomal P2 beta protein genes of Trypanosoma cruzi. Mol Biochem Parasitol 1994;64:327-36.
29. Brisse S, Verhoef J, Tibayrenc M. Characterisation of large and small subunit rRNA and miniexon genes further support the distinction of six Trypanosoma cruzi lineages. Int J Parasitol 2001;31:1218-26.
30. Urrea DA, Carranza JC, Cuba CA, Gurgel-Goncalves R, Guhl F, Schofield CJ, et al. Molecular characterisation of Trypanosoma rangeli strains isolated from Rhodnius ecuadoriensis in Peru, R. colombiensis in Colombia and R. pallescens in Panama, supports a co-evolutionary association between parasites and vectors. Infect Genet Evol 2005;5:123-9.
31. Gutierrez R, Angulo VM, Tarazona Z, Britto C, Fernandes O. Comparison of four serological tests for the diagnosis of Chagas disease in a Colombian endemic area. Parasitology 2004;129:439-44.
32. Gomes ML, Galvao LM, Macedo AM, Pena SD, Chiari E. Chagas' disease diagnosis: comparative analysis of parasitologic, molecular, and serologic methods. Am J Trop Med Hyg 1999;60:205-10.
33. Britto C, Cardoso MA, Vanni CM, Hasslocher-Moreno A, Xavier SS, Oelemann W, et al. Polymerase chain reaction detection of Trypanosoma cruzi in human blood samples as a tool for diagnosis and treatment evaluation. Parasitology 1995;110:241-7.
34. Wincker P, Bosseno MF, Britto C, Yaksic N, Cardoso MA, Morel CM, et al. High correlation between Chagas' disease serology and PCR-based detection of Trypanosoma cruzi kinetoplast DNA in Bolivian children living in an endemic area. FEMS Microbiol Lett 1994;124:419-23.
35. Avila HA, Pereira JB, Thiemann O, De Paiva E, DeGrave W, Morel CM, et al. Detection of Trypanosoma cruzi in blood specimens of chronic chagasic patients by polymerase chain reaction amplification of kinetoplast minicircle DNA: comparison with serology and xenodiagnosis. J Clin Microbiol 1993;31:2421-6.
36. Junqueira AC, Chiari E, Wincker P. Comparison of the polymerase chain reaction with two classical parasitological methods for the diagnosis of Chagas disease in an endemic region of north-eastern Brazil. Trans R Soc Trop Med Hyg 1996;90:129-32.
37. 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.
38. Marcon GE, Andrade PD, de Albuquerque DM, Wanderley Jda S, de Almeida EA, Guariento ME, et al. Use of a nested polymerase chain reaction (N-PCR) to detect Trypanosoma cruzi in blood samples from
chronic chagasic patients and patients with doubtful serologies. Diagn Microbiol Infect Dis 2002;43:39-43.
39. Luquetti AO. Megaesofago e anticorpos anti-Trypanosoma cruzi. Rev Goiana Med 1987;33:1-16.
40. Salomone OA, Basquiera AL, Sembaj A, Aguerri AM, Reyes ME, Omelianuk M, et al. Trypanosoma cruzi in persons without serologic evidence of disease, Argentina. Emerg Infect Dis 2003;9:1558-62.
41. Vacca M, Mercado M. Determinación de las características operativas de las pruebas serológicas con cepas colombianas de Trypanosoma cruzi utilizadas para el diagnóstico de la enfermedad de
Chagas. (Tesis de Maestría en Epidemiología Clínica). Bogotá D.C.: Unidad de Epidemiología Clínica, Hospital Universitario San Ignacio, Pontificia Universidad Javeriana; 2006.
2. Moncayo A. Chagas disease: current epidemiological trends after the interruption of vectorial and transfusional transmission in the Southern Cone countries. Mem Inst Oswaldo Cruz 2003;98:577-91.
3. Macedo AM, Machado CR, Oliveira RP, Pena SD. Trypanosoma cruzi: genetic structure of populations and relevance of genetic variability to the pathogenesis of Chagas disease. Mem Inst Oswaldo Cruz 2004;99:1-12.
4. Huete-Perez JA, Flores-Obando RE, Ghedin E, Caffrey CR. Genomic and proteomic approaches for Chagas' disease: critical analysis of diagnostic methods. Expert Rev Mol Diagn 2005;5:521-30.
5. D'Avila DA, Gontijo ED, Lages-Silva E, Meira WS, Chiari E, Galvao LM. Random amplified polymorphic DNA profiles of Trypanosoma cruzi isolates from chagasic patients with different clinical forms. Parasitol Res 2006;98:455-61.
6. Machado CR, Augusto-Pinto L, McCulloch R, Teixeira SM. DNA metabolism and genetic diversity in Trypanosomes. Mutat Res 2006;612:40-57.
7. Saravia NG, Holguin 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.
8. Rodríguez P, Montilla M, Nicholls RS, Zarante I, Puerta, C. Isoenzymatic characterization of Colombian strains of Trypanosoma cruzi. Mem Inst Oswaldo Cruz 1998;93:739-40.
9. Marquez E, Arcos-Burgos M, Triana O, Moreno J, Jaramillo N. Clonal population structure of Colombian sylvatic Trypanosoma cruzi. J Parasitol 1998;84:1143-9.
10. Triana O, Jaramillo N, Moreno J. Genetic variability of Colombian populations of Trypanosoma cruzi and Trypanosoma rangeli. Biol Res 1999;32:1-10.
11. 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.
12. Rodríguez P, Escalante M, Díez H, Cuervo C, Montilla M, Nicholls RS, et al. Estudio de la variabilidad de seis cepas colombianas de Trypanosoma cruzi mediante polimorfismos de longitud de fragmentos de restricción (RFLP) y amplificación aleatoria de ADN polimórfico (RAPD). Biomédica 2002;22:263-71.
13. Mejia AM, Triana O. Análisis por LSSP-PCR de la variabilidad genética de Trypanosoma cruzi en sangre y órganos de ratones infectados. Biomédica 2005;25:76-86.
14. D'Alessandro A, Saravia N. Trypanosoma rangeli. En: Gilles HM, editor. Protozoal diseases. London: Arnold Press;1999. p.398-412.
15. Guhl F, Vallejo GA. Trypanosoma (Herpetosoma) rangeli Tejera, 1920: an updated review. Mem Inst Oswaldo Cruz 2003;98:435-42.
16. 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.
17. Guhl F, Jaramillo C, Carranza JC, Vallejo GA. Molecular characterization and diagnosis of Trypanosoma cruzi and Trypanosoma rangeli. Arch Med Res 2002;33:362-70.
18. Vallejo GA, Gulh F, Chiari E, Macedo AM. Species specific detection of Trypanosoma cruzi and Trypanosoma rangeli in vector and mammalian hosts by polymerase chain reaction amplification of kinetoplast minicircle DNA. Acta Trop 1999;72:203-12.
19. Vargas N, Souto RP, Carranza JC, Vallejo GA, Zingales B. Amplification of a specific repetitive DNA sequence for Trypanosoma rangeli identification and its potential application in epidemiological investigations. Exp Parasitol 2000;96:147-59.
20. Nitz N, Gomes C, de Cassia 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.
21. Pavía P, Cuervo C, Montilla M, Nicholls S, Puerta C. Diseño y estandarización de una prueba de PCR para la detección específica de Trypanosoma cruzi. Infectio 2003;7:129-36.
22. Beltrán M, Duque S, Guhl F, Herrera CP, López MC, Moreno AL, et al. Prueba de ELISA y prueba de inmunofluorescencia indirecta (IFI). En: Guhl F, Nicholls RS, editores. Manual de procedimientos para el diagnóstico de la enfermedad de Chagas. Bogotá: Ministerio de Salud; 2001. p.32-48.
23. Sambrook JF, Russell DW. Gel electrophoresis of DNA. En: Molecular cloning A laboratory manual. Tercera edición. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 2001.p.5.1-17.
24. Puerta C, Martin J, Alonso C, Lopez MC. Isolation and characterization of the gene encoding histone H2A from Trypanosoma cruzi. Mol Biochem Parasitol 1994;64:1-10.
25. Sackett D, Haynes R, Guyatt G, Tugwell P. Selección de pruebas diagnosticas. En: Epidemiologia Clínica. Segunda Edición. México, D.F.: Editorial Médica Panamericana; 1998. p.64-7.
26. Wincker P, Britto C, Pereira JB, Cardoso MA, Oelemann W, Morel CM. Use of a simplified polymerase chain reaction procedure to detect Trypanosoma cruzi in blood samples from chronic chagasic patients in a rural endemic area. Am J Trop Med Hyg 1994;51:771-7.
27. Thomas MC, Olivares M, Escalante M, Maranon 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.
28. Vázquez MP, Schijman AG, Levin MJ. A short interspersed repetitive element provides a new 3' acceptor site for trans-splicing in certain ribosomal P2 beta protein genes of Trypanosoma cruzi. Mol Biochem Parasitol 1994;64:327-36.
29. Brisse S, Verhoef J, Tibayrenc M. Characterisation of large and small subunit rRNA and miniexon genes further support the distinction of six Trypanosoma cruzi lineages. Int J Parasitol 2001;31:1218-26.
30. Urrea DA, Carranza JC, Cuba CA, Gurgel-Goncalves R, Guhl F, Schofield CJ, et al. Molecular characterisation of Trypanosoma rangeli strains isolated from Rhodnius ecuadoriensis in Peru, R. colombiensis in Colombia and R. pallescens in Panama, supports a co-evolutionary association between parasites and vectors. Infect Genet Evol 2005;5:123-9.
31. Gutierrez R, Angulo VM, Tarazona Z, Britto C, Fernandes O. Comparison of four serological tests for the diagnosis of Chagas disease in a Colombian endemic area. Parasitology 2004;129:439-44.
32. Gomes ML, Galvao LM, Macedo AM, Pena SD, Chiari E. Chagas' disease diagnosis: comparative analysis of parasitologic, molecular, and serologic methods. Am J Trop Med Hyg 1999;60:205-10.
33. Britto C, Cardoso MA, Vanni CM, Hasslocher-Moreno A, Xavier SS, Oelemann W, et al. Polymerase chain reaction detection of Trypanosoma cruzi in human blood samples as a tool for diagnosis and treatment evaluation. Parasitology 1995;110:241-7.
34. Wincker P, Bosseno MF, Britto C, Yaksic N, Cardoso MA, Morel CM, et al. High correlation between Chagas' disease serology and PCR-based detection of Trypanosoma cruzi kinetoplast DNA in Bolivian children living in an endemic area. FEMS Microbiol Lett 1994;124:419-23.
35. Avila HA, Pereira JB, Thiemann O, De Paiva E, DeGrave W, Morel CM, et al. Detection of Trypanosoma cruzi in blood specimens of chronic chagasic patients by polymerase chain reaction amplification of kinetoplast minicircle DNA: comparison with serology and xenodiagnosis. J Clin Microbiol 1993;31:2421-6.
36. Junqueira AC, Chiari E, Wincker P. Comparison of the polymerase chain reaction with two classical parasitological methods for the diagnosis of Chagas disease in an endemic region of north-eastern Brazil. Trans R Soc Trop Med Hyg 1996;90:129-32.
37. 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.
38. Marcon GE, Andrade PD, de Albuquerque DM, Wanderley Jda S, de Almeida EA, Guariento ME, et al. Use of a nested polymerase chain reaction (N-PCR) to detect Trypanosoma cruzi in blood samples from
chronic chagasic patients and patients with doubtful serologies. Diagn Microbiol Infect Dis 2002;43:39-43.
39. Luquetti AO. Megaesofago e anticorpos anti-Trypanosoma cruzi. Rev Goiana Med 1987;33:1-16.
40. Salomone OA, Basquiera AL, Sembaj A, Aguerri AM, Reyes ME, Omelianuk M, et al. Trypanosoma cruzi in persons without serologic evidence of disease, Argentina. Emerg Infect Dis 2003;9:1558-62.
41. Vacca M, Mercado M. Determinación de las características operativas de las pruebas serológicas con cepas colombianas de Trypanosoma cruzi utilizadas para el diagnóstico de la enfermedad de
Chagas. (Tesis de Maestría en Epidemiología Clínica). Bogotá D.C.: Unidad de Epidemiología Clínica, Hospital Universitario San Ignacio, Pontificia Universidad Javeriana; 2006.
How to Cite
1.
Gil J, Pavía P, Montilla M, Florez AC, Quintero C, Mercado M, et al. Comparison of a PCR test based on the histone H2A/SIRE genes with classical serological tests for the diagnosis of chronic Chagas disease in Colombian patients. biomedica [Internet]. 2007 Jan. 1 [cited 2024 May 11];27(1esp):83-91. Available from: https://revistabiomedica.org/index.php/biomedica/article/view/251
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