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Haptoglobin genotypes in Chagas' disease
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Clinical Biochemistry 43 (2010) 314 – 316
Haptoglobin genotypes in Chagas' disease Susan E.D.C. Jorge a , Cristina Feix Abreu a , Maria Elena Guariento b , Maria de Fatima Sonati a,⁎ a
Department of Clinical Pathology, School of Medical Sciences, State University of Campinas-UNICAMP, P.O. Box 6111, Zip Code 13083-970, Campinas, state of São Paulo, Brazil b Department of Clinical Medicine, School of Medical Sciences, State University of Campinas-UNICAMP, P.O. Box 6111, Zip Code 13083-970, Campinas, state of São Paulo, Brazil Received 3 July 2009; received in revised form 20 September 2009; accepted 22 September 2009 Available online 3 October 2009
Abstract Objectives: To investigate the existence of an association between haptoglobin (Hp) genotypes and the severity of heart complications in Chagas' disease. Design and methods: Hp genotyping was performed by PCR in 107 Brazilian patients sub-classified in asymptomatic, with mild heart disease and with severe heart disease. Results: Multiple logistic regression (R2 = 24%) indicated that patients with the Hp1-1 genotype have lower probability of developing the severe heart complications. Conclusion: The Hp polymorphism may influence the clinical evolution of Chagas' disease. © 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: Cardiomyopathies; Chagas' disease; Trypanosoma cruzi; Haptoglobin; Brazil
Introduction American trypanosomiasis is a disease caused by the protozoan parasite Trypanosoma cruzi and was first described by the Brazilian physician Carlos Chagas in 1909 [1]. Also known as Chagas' disease, it is considered the most serious parasite infection in the Americas and, according to estimates, 18 endemic Latin American countries have approximately 12 to 14 million infected individuals, in addition to 60 million people who are at risk of contracting the disease. In Brazil, the mortality rate due to this disease per year, from 1981 to 1998, was about 3.75 per 100,000 inhabitants [1,2]. The disease is mainly transmitted either by blood-sucking triatomine insects (Triatoma sp.) or by blood transfusion, has variable clinical manifestations and has two phases, acute and chronic. The former is characterized by high parasitemia, which can be detected by direct blood examination. However, in the majority of patients the infection goes unrecognized in this ⁎ Corresponding author. Fax: +55 19 3521 9434. E-mail addresses: [email protected], [email protected] (M.F. Sonati).
phase because of the scarcity or absence of clinical manifestations. The chronic phase usually begins 2–4 months after the initial infection and is generally characterized by a long initial period of clinical latency in which patients are considered to have the indeterminate form of the disease and which can last for 10–30 years or even the patient's whole life. After this period, a small percentage of these patients start to manifest symptoms, which can involve cardiac manifestations (a mild or severe heart condition) and/or digestive-tract manifestations. It has been suggested that the prognosis of the disease during the chronic phase may be related to the host's immune mechanisms and/or persistence of parasites in their tissues [1]. Haptoglobin (Hp) is a plasma glycoprotein whose main known biological function is that of binding free hemoglobin to prevent iron loss and kidney damage following intravascular hemolysis. It is also an acute-phase protein with immunomodulatory activities [3]. Two co-dominant alleles, HP1 and HP2, located on chromosome 16q22, are responsible for three main genotypes/phenotypes (Hp1-1, Hp2-1 and Hp2-2), which correspond to proteins with distinct structural and functional properties. Since the Hp2-2 protein is the largest protein, it has been suggested that the Hp2-2 phenotype confers lower
0009-9120/$ - see front matter © 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2009.09.020
S.E.D.C. Jorge et al. / Clinical Biochemistry 43 (2010) 314–316
antioxidative capacity and higher immunological reactivity than the other phenotypes [3–6]. Although the Hp polymorphism has been associated with susceptibility and outcome in several important diseases as diabetes mellitus, cardiovascular and neurological disorders and malignancies [3,7], the relationship between the different Hp subtypes and the prognosis of infectious diseases is still poorly investigated. The only previous study in chagasic patients was recently carried out in Brazil and suggested that the Hp2-2 phenotype is more frequent in these patients than in healthy controls [8]. We determined the Hp genotypes of 107 patients of the same country region in order to investigate the existence of association between the Hp polymorphism and the severity of heart complications in this infectious disease. Methods The patients came from endemic areas mainly in southeastern and southern Brazil. They were attended by the Chagas' Disease Study Group (GEDoCh) at UNICAMP University Hospital, in Campinas, state of São Paulo, southeastern Brazil. The group included 49 women and 58 men aged between 18 and 76 years, 66.36% of whom were white. These patients had previously been classified as indeterminate (n = 36), moderate cardiac (n = 48) and severe cardiac (n = 23). The classification of the cardiac status was based on clinical and complementary imaging findings (chest x-ray and electrocardiogram), as standardized by the WHO [9,10]. Individuals with the digestive or conjugated form of the disease were excluded from the study, as well as those with any other concomitant diseases, such as HIV, hepatitis, diabetes or autoimmune diseases. All patients gave their voluntary written consent to take part in the study, which was approved by the local ethics committee. DNA samples were obtained from peripheral blood leukocytes (GFX Genomic Blood DNA Purification kit, GE Healthcare, Buckinghamshire, UK), and the haptoglobin genotyping was performed by allele-specific polymerase chain reaction according to Yano et al. [11]. Statistical analysis was carried out with the Statistical Analysis Systems program (version 9.1.3 for Windows, Service Pack 3- SAS Institute Inc., Cary, NC, USA). The patients were compared with 142 healthy individuals (51 men and 91 women aged between 18 and 62 years, 78% of whom were white). The Chi-square (χ2) test was used to verify the Hardy– Weinberg (HW) equilibrium hypothesis; χ2 and Fisher tests were used to compare the patient and control groups and multiple logistic regression was performed to determine whether age and Hp genotypes were associated with the prognosis of the disease. Results and discussion The frequencies of Hp genotypes for patients and controls and comparisons between them are shown in Table 1. The Hardy–Weinberg equilibrium hypothesis was rejected for the patients (p = 0.016), and this tendency was also observed for the control group (p = 0.072). Although no significant differences were found in the distribution of the genotype frequencies
315
Table 1 Hardy–Weinberg equilibrium and statistical comparison of the Hp genotypes in the patient and control groups. Hp genotypes
Patients
Controls
Hp1-1 Hp2-1 Hp2-2 N HW-χ2 P
35 (32.7%) 41 (38.3%) 31 (29%) 107 5.79 0.016
36 (25.3%) 60 (42.3%) 46 (32.4%) 142 3.23 0.072
Hp genotypes
Indeterminate
Mild heart disease
Severe heart disease
Controls
Hp1-1 Hp2-1 Hp2-2 N P (χ2)
13 (5.2%) 15 (6.0%) 8 (3.2%) 36 (14.2%)
18 (7.2%) 15 (6.0%) 15 (6.0%) 48 (19.3%) 0.411
4 11 8 23
36 (14.5%) 60 (24.1%) 46 (18.5%) 142 (57.0%)
(1.6%) (4.4%) (3.2%) (9.2%)
Hp1-1: patients with the haptoglobin 1-1 genotype; Hp2-1: patients with haptoglobin 2-1 genotype; Hp2-2: patients with haptoglobin 2-2 genotype; N: control group.
between patients and controls, multiple logistic regression showed a correlation between Hp genotypes in different age groups and clinical cardiac manifestations of the indeterminate, mild and severe forms of trypanosomiasis (Fig. 1). Accordingly, the disease had a better outcome in Hp1-1 patients than in those with the other Hp genotypes. The younger Hp1-1 patients were asymptomatic, and the probability of their developing a mild heart condition with increasing age was higher than that of developing severe heart disease (Fig. 1A). In contrast, the presence of the HP2 allele appears to confer a higher risk of developing severe heart disease, as can be seen in Figs. 1B and C. According to Fig. 1B, patients with the Hp2-1 genotype have a higher probability of developing cardiac manifestations at a younger age than Hp1-1 patients. They also have a greater chance of developing a severe cardiac form of the disease with increasing age. Fig. 1C shows that Hp2-2 patients have a higher probability of developing cardiac manifestations earlier in life than patients with the other genotypes (p = 0.0060). Our finding of an R2 value of 24% in the multiple logistic regression test indicates that, although the clinical behavior in Chagas' disease is primarily related to other genetic and environmental factors, the Hp polymorphism has also some influence on the prognosis of this infectious disease. A number of studies have shown that Hp plays an important role in modulating the balance between Th1 (pro-inflammatory) and Th2 (anti-inflammatory) lymphocytes and suppresses the synthesis of TNF-α, IL-10 and IL-12 by monocytes stimulated by lipopolysaccharides (LPS) [4]. It is also known that the HP2 allele is a weaker antioxidant than HP1 and that one of its immunomodulatory functions is to stimulate a Th1 cytokinedependent profile [6,12]. In cardiovascular diseases in diabetic and even non-diabetic patients, the HP2 allele has been found to be a risk factor [6,7]. In addition, it has recently been observed that cells in culture release more cytokines (GM-CSF, IL-1β, IL-6, IL-10 and TNF-α) after exposure to the Hp2-2-Hb
316
S.E.D.C. Jorge et al. / Clinical Biochemistry 43 (2010) 314–316
dulation are very important and may contribute with the clinical course of this kind of disease. Our findings suggest that the higher antioxidant capability and lower inflammatory reactivity of the Hp1-1 phenotype may favor a better clinical outcome in chagasic Hp1-1 patients. More patients, in different regions, should be investigated. Acknowledgments We would like to thank Mrs. Cleide Silva, of the Statistical and Research Committee of the School of Medical Sciences— UNICAMP, for helping us with the statistical analyses. Financial support from FAPESP (grant no. 08/01690-2) and CNPq/ Brazil.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.clinbiochem.2009.09.020.
References
Fig. 1. Estimated probability of developing the severe, mild or indeterminate form of Chagas' disease for patients with the (A) Hp1-1 genotype, (B) Hp2-1 genotype and (C) Hp2-2 genotype. Multiple logistic regression (R2 = 24%).
complex than after exposure to Hp-1-1-Hb complex, indicating a more powerful inflammatory response to Hp2-2 [5]. Chagas' disease is an infectious disease with inflammatory characteristics. The activities of antioxidation and immunomo-
[1] Teixeira ARL, Nitz N, Guiamaro MC, Gomes C, Santos-Buch CA. Chagas disease. Postgrad Med J 2006;82:788–98. [2] Drumond JA, Marcopito LF. Internal migration and distribution of Chagas disease mortality, Brazil, 1891–1998. Cad Saude Publica 2006;22:2131–40. [3] Langlois MR, Delanghe JR. Biological and clinical significance of haptoglobin polymorphism in humans. Clin Chem 1996;42:1589–600. [4] Arredouani MS, Kasran A, Vanoirbeek JA, Berger FG, Baumann H, Ceuppens JL. Haptoglobin dampens endotoxin-induced inflammatory effects both in vitro and in vivo. Immunology 2005;114:263–71. [5] Bora K, Natarajan K, Kutlar F, et al. In vitro exploratory studies of haptoglobin polymorphism and their effect on cytokine release from cultured mononuclear cells in sickle cell disease. ASH Annual Meeting Abstracts 2008;112:1441. [6] Quaye IK. Haptoglobin, inflammation and disease. Trans R Soc Trop Med Hyg 2008;102:735–42. [7] Shor M, Boaz M, Gavish D, Wainshtein J, Matas Z, Shargorodsky M. Relation of haptoglobin phenotype to early vascular changes in patients with diabetes mellitus. Am J Cardiol 2007;100:1767–70. [8] Calderoni DR, Andrade Tdos S, Grotto HZ. Haptoglobin phenotype appears to affect the pathogenesis of American trypanosomiasis. Ann Trop Med Parasitol 2006;100:213–21. [9] Lázzari JO, Pereira M, Antunes CM, et al. Diagnostic electrocardiography in epidemiological studies of Chagas' disease: multicenter evaluation of a standardized method. Rev Panam Salud Publica 1998;4:317–30. [10] Prata A. Clinical and epidemiological aspects of Chagas Disease. Lancet Infect Dis 2001;1:92–100. [11] Yano A, Yamamoto Y, Miyaishi S, Ishizu H. Haptoglobin genotyping by allele-specific polymerase chain reaction amplification. Acta Med Okayama 1998;52:173–81. [12] Guetta J, Strauss M, Levy NS, Fahoum L, Levy AP. Haptoglobin genotype modulates the balance of Th1/Th2 cytokines produced by macrophages exposed to free hemoglobin. Atherosclerosis 2007;191:48–53.
Clinical Biochemistry 43 (2010) 314 – 316
Haptoglobin genotypes in Chagas' disease Susan E.D.C. Jorge a , Cristina Feix Abreu a , Maria Elena Guariento b , Maria de Fatima Sonati a,⁎ a
Department of Clinical Pathology, School of Medical Sciences, State University of Campinas-UNICAMP, P.O. Box 6111, Zip Code 13083-970, Campinas, state of São Paulo, Brazil b Department of Clinical Medicine, School of Medical Sciences, State University of Campinas-UNICAMP, P.O. Box 6111, Zip Code 13083-970, Campinas, state of São Paulo, Brazil Received 3 July 2009; received in revised form 20 September 2009; accepted 22 September 2009 Available online 3 October 2009
Abstract Objectives: To investigate the existence of an association between haptoglobin (Hp) genotypes and the severity of heart complications in Chagas' disease. Design and methods: Hp genotyping was performed by PCR in 107 Brazilian patients sub-classified in asymptomatic, with mild heart disease and with severe heart disease. Results: Multiple logistic regression (R2 = 24%) indicated that patients with the Hp1-1 genotype have lower probability of developing the severe heart complications. Conclusion: The Hp polymorphism may influence the clinical evolution of Chagas' disease. © 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: Cardiomyopathies; Chagas' disease; Trypanosoma cruzi; Haptoglobin; Brazil
Introduction American trypanosomiasis is a disease caused by the protozoan parasite Trypanosoma cruzi and was first described by the Brazilian physician Carlos Chagas in 1909 [1]. Also known as Chagas' disease, it is considered the most serious parasite infection in the Americas and, according to estimates, 18 endemic Latin American countries have approximately 12 to 14 million infected individuals, in addition to 60 million people who are at risk of contracting the disease. In Brazil, the mortality rate due to this disease per year, from 1981 to 1998, was about 3.75 per 100,000 inhabitants [1,2]. The disease is mainly transmitted either by blood-sucking triatomine insects (Triatoma sp.) or by blood transfusion, has variable clinical manifestations and has two phases, acute and chronic. The former is characterized by high parasitemia, which can be detected by direct blood examination. However, in the majority of patients the infection goes unrecognized in this ⁎ Corresponding author. Fax: +55 19 3521 9434. E-mail addresses: [email protected], [email protected] (M.F. Sonati).
phase because of the scarcity or absence of clinical manifestations. The chronic phase usually begins 2–4 months after the initial infection and is generally characterized by a long initial period of clinical latency in which patients are considered to have the indeterminate form of the disease and which can last for 10–30 years or even the patient's whole life. After this period, a small percentage of these patients start to manifest symptoms, which can involve cardiac manifestations (a mild or severe heart condition) and/or digestive-tract manifestations. It has been suggested that the prognosis of the disease during the chronic phase may be related to the host's immune mechanisms and/or persistence of parasites in their tissues [1]. Haptoglobin (Hp) is a plasma glycoprotein whose main known biological function is that of binding free hemoglobin to prevent iron loss and kidney damage following intravascular hemolysis. It is also an acute-phase protein with immunomodulatory activities [3]. Two co-dominant alleles, HP1 and HP2, located on chromosome 16q22, are responsible for three main genotypes/phenotypes (Hp1-1, Hp2-1 and Hp2-2), which correspond to proteins with distinct structural and functional properties. Since the Hp2-2 protein is the largest protein, it has been suggested that the Hp2-2 phenotype confers lower
0009-9120/$ - see front matter © 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2009.09.020
S.E.D.C. Jorge et al. / Clinical Biochemistry 43 (2010) 314–316
antioxidative capacity and higher immunological reactivity than the other phenotypes [3–6]. Although the Hp polymorphism has been associated with susceptibility and outcome in several important diseases as diabetes mellitus, cardiovascular and neurological disorders and malignancies [3,7], the relationship between the different Hp subtypes and the prognosis of infectious diseases is still poorly investigated. The only previous study in chagasic patients was recently carried out in Brazil and suggested that the Hp2-2 phenotype is more frequent in these patients than in healthy controls [8]. We determined the Hp genotypes of 107 patients of the same country region in order to investigate the existence of association between the Hp polymorphism and the severity of heart complications in this infectious disease. Methods The patients came from endemic areas mainly in southeastern and southern Brazil. They were attended by the Chagas' Disease Study Group (GEDoCh) at UNICAMP University Hospital, in Campinas, state of São Paulo, southeastern Brazil. The group included 49 women and 58 men aged between 18 and 76 years, 66.36% of whom were white. These patients had previously been classified as indeterminate (n = 36), moderate cardiac (n = 48) and severe cardiac (n = 23). The classification of the cardiac status was based on clinical and complementary imaging findings (chest x-ray and electrocardiogram), as standardized by the WHO [9,10]. Individuals with the digestive or conjugated form of the disease were excluded from the study, as well as those with any other concomitant diseases, such as HIV, hepatitis, diabetes or autoimmune diseases. All patients gave their voluntary written consent to take part in the study, which was approved by the local ethics committee. DNA samples were obtained from peripheral blood leukocytes (GFX Genomic Blood DNA Purification kit, GE Healthcare, Buckinghamshire, UK), and the haptoglobin genotyping was performed by allele-specific polymerase chain reaction according to Yano et al. [11]. Statistical analysis was carried out with the Statistical Analysis Systems program (version 9.1.3 for Windows, Service Pack 3- SAS Institute Inc., Cary, NC, USA). The patients were compared with 142 healthy individuals (51 men and 91 women aged between 18 and 62 years, 78% of whom were white). The Chi-square (χ2) test was used to verify the Hardy– Weinberg (HW) equilibrium hypothesis; χ2 and Fisher tests were used to compare the patient and control groups and multiple logistic regression was performed to determine whether age and Hp genotypes were associated with the prognosis of the disease. Results and discussion The frequencies of Hp genotypes for patients and controls and comparisons between them are shown in Table 1. The Hardy–Weinberg equilibrium hypothesis was rejected for the patients (p = 0.016), and this tendency was also observed for the control group (p = 0.072). Although no significant differences were found in the distribution of the genotype frequencies
315
Table 1 Hardy–Weinberg equilibrium and statistical comparison of the Hp genotypes in the patient and control groups. Hp genotypes
Patients
Controls
Hp1-1 Hp2-1 Hp2-2 N HW-χ2 P
35 (32.7%) 41 (38.3%) 31 (29%) 107 5.79 0.016
36 (25.3%) 60 (42.3%) 46 (32.4%) 142 3.23 0.072
Hp genotypes
Indeterminate
Mild heart disease
Severe heart disease
Controls
Hp1-1 Hp2-1 Hp2-2 N P (χ2)
13 (5.2%) 15 (6.0%) 8 (3.2%) 36 (14.2%)
18 (7.2%) 15 (6.0%) 15 (6.0%) 48 (19.3%) 0.411
4 11 8 23
36 (14.5%) 60 (24.1%) 46 (18.5%) 142 (57.0%)
(1.6%) (4.4%) (3.2%) (9.2%)
Hp1-1: patients with the haptoglobin 1-1 genotype; Hp2-1: patients with haptoglobin 2-1 genotype; Hp2-2: patients with haptoglobin 2-2 genotype; N: control group.
between patients and controls, multiple logistic regression showed a correlation between Hp genotypes in different age groups and clinical cardiac manifestations of the indeterminate, mild and severe forms of trypanosomiasis (Fig. 1). Accordingly, the disease had a better outcome in Hp1-1 patients than in those with the other Hp genotypes. The younger Hp1-1 patients were asymptomatic, and the probability of their developing a mild heart condition with increasing age was higher than that of developing severe heart disease (Fig. 1A). In contrast, the presence of the HP2 allele appears to confer a higher risk of developing severe heart disease, as can be seen in Figs. 1B and C. According to Fig. 1B, patients with the Hp2-1 genotype have a higher probability of developing cardiac manifestations at a younger age than Hp1-1 patients. They also have a greater chance of developing a severe cardiac form of the disease with increasing age. Fig. 1C shows that Hp2-2 patients have a higher probability of developing cardiac manifestations earlier in life than patients with the other genotypes (p = 0.0060). Our finding of an R2 value of 24% in the multiple logistic regression test indicates that, although the clinical behavior in Chagas' disease is primarily related to other genetic and environmental factors, the Hp polymorphism has also some influence on the prognosis of this infectious disease. A number of studies have shown that Hp plays an important role in modulating the balance between Th1 (pro-inflammatory) and Th2 (anti-inflammatory) lymphocytes and suppresses the synthesis of TNF-α, IL-10 and IL-12 by monocytes stimulated by lipopolysaccharides (LPS) [4]. It is also known that the HP2 allele is a weaker antioxidant than HP1 and that one of its immunomodulatory functions is to stimulate a Th1 cytokinedependent profile [6,12]. In cardiovascular diseases in diabetic and even non-diabetic patients, the HP2 allele has been found to be a risk factor [6,7]. In addition, it has recently been observed that cells in culture release more cytokines (GM-CSF, IL-1β, IL-6, IL-10 and TNF-α) after exposure to the Hp2-2-Hb
316
S.E.D.C. Jorge et al. / Clinical Biochemistry 43 (2010) 314–316
dulation are very important and may contribute with the clinical course of this kind of disease. Our findings suggest that the higher antioxidant capability and lower inflammatory reactivity of the Hp1-1 phenotype may favor a better clinical outcome in chagasic Hp1-1 patients. More patients, in different regions, should be investigated. Acknowledgments We would like to thank Mrs. Cleide Silva, of the Statistical and Research Committee of the School of Medical Sciences— UNICAMP, for helping us with the statistical analyses. Financial support from FAPESP (grant no. 08/01690-2) and CNPq/ Brazil.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.clinbiochem.2009.09.020.
References
Fig. 1. Estimated probability of developing the severe, mild or indeterminate form of Chagas' disease for patients with the (A) Hp1-1 genotype, (B) Hp2-1 genotype and (C) Hp2-2 genotype. Multiple logistic regression (R2 = 24%).
complex than after exposure to Hp-1-1-Hb complex, indicating a more powerful inflammatory response to Hp2-2 [5]. Chagas' disease is an infectious disease with inflammatory characteristics. The activities of antioxidation and immunomo-
[1] Teixeira ARL, Nitz N, Guiamaro MC, Gomes C, Santos-Buch CA. Chagas disease. Postgrad Med J 2006;82:788–98. [2] Drumond JA, Marcopito LF. Internal migration and distribution of Chagas disease mortality, Brazil, 1891–1998. Cad Saude Publica 2006;22:2131–40. [3] Langlois MR, Delanghe JR. Biological and clinical significance of haptoglobin polymorphism in humans. Clin Chem 1996;42:1589–600. [4] Arredouani MS, Kasran A, Vanoirbeek JA, Berger FG, Baumann H, Ceuppens JL. Haptoglobin dampens endotoxin-induced inflammatory effects both in vitro and in vivo. Immunology 2005;114:263–71. [5] Bora K, Natarajan K, Kutlar F, et al. In vitro exploratory studies of haptoglobin polymorphism and their effect on cytokine release from cultured mononuclear cells in sickle cell disease. ASH Annual Meeting Abstracts 2008;112:1441. [6] Quaye IK. Haptoglobin, inflammation and disease. Trans R Soc Trop Med Hyg 2008;102:735–42. [7] Shor M, Boaz M, Gavish D, Wainshtein J, Matas Z, Shargorodsky M. Relation of haptoglobin phenotype to early vascular changes in patients with diabetes mellitus. Am J Cardiol 2007;100:1767–70. [8] Calderoni DR, Andrade Tdos S, Grotto HZ. Haptoglobin phenotype appears to affect the pathogenesis of American trypanosomiasis. Ann Trop Med Parasitol 2006;100:213–21. [9] Lázzari JO, Pereira M, Antunes CM, et al. Diagnostic electrocardiography in epidemiological studies of Chagas' disease: multicenter evaluation of a standardized method. Rev Panam Salud Publica 1998;4:317–30. [10] Prata A. Clinical and epidemiological aspects of Chagas Disease. Lancet Infect Dis 2001;1:92–100. [11] Yano A, Yamamoto Y, Miyaishi S, Ishizu H. Haptoglobin genotyping by allele-specific polymerase chain reaction amplification. Acta Med Okayama 1998;52:173–81. [12] Guetta J, Strauss M, Levy NS, Fahoum L, Levy AP. Haptoglobin genotype modulates the balance of Th1/Th2 cytokines produced by macrophages exposed to free hemoglobin. Atherosclerosis 2007;191:48–53.