Outbreaks of orally transmitted Trypanosoma cruzi continue to be reported in Brazil and are associated with a high mortality rate, mainly due to myocarditis.
This study is a detailed report on the disease progression of acute Chagas disease in 13 patients who were infected during two micro-outbreaks in two northeastern Brazilian towns. Clinical outcomes as well as EKG and ECHO results are described, both before and after benznidazole treatment.
Fever and dyspnea were the most frequent symptoms observed. Other clinical findings included myalgia, periorbital edema, headache and systolic murmur. Two patients died of cardiac failure before receiving benznidazole treatment. EKG and ECHO findings frequently showed a disturbance in ventricular repolarization and pericardial effusion. Ventricular dysfunction (ejection fraction <55%) was present in 27.3% of patients. After treatment, EKG readings normalized in 91.7% of patients. Ventricular repolarization abnormalities persisted in 50% of the patients, while sinus bradycardia was observed in 18%. The systolic ejection fraction normalized in two out of three patients with initially depressed ventricular function, while pericardial effusion disappeared.
Myocarditis is frequently found and potentially severe in patients with acute Chagas disease. Benznidazole treatment may improve clinical symptoms, as well as EKG and ECHO findings.
Chagas disease is caused by a parasitic protozoan transmitted to humans by the contaminated feces of blood-feeding assassin bugs from the Triatominae subfamily. It may also be transmitted from mother to baby during pregnancy, by breastfeeding, blood transfusion or organ transplant. In rare cases, the disease can also be caused by accidental ingestion of contaminated food (sugar cane or açaí juice, drinking water, etc.). Acute Chagas disease often presents itself as a mononucleosis-like syndrome, with symptoms including fever, lymph node enlargement and muscle pain. The mortality rate of acute Chagas disease is high, mainly due to heart failure as a consequence of cardiac fiber lesions. There are few studies describing clinical outcomes and the disease progression of patients who receive therapeutic treatment, especially with regard to cardiac exam findings. In this report, the authors describe clinical findings from two micro-outbreaks occurring in impoverished towns in northeastern Brazil. Prior to receiving treatment, patient mortality rate was 28.6% in one of the outbreaks, and one pregnant woman experienced a spontaneous abortion due to the disease in the other outbreak. Most patients complained of fever, dyspnea, myalgia and periorbital edema. After receiving a two-month course of treatment, clinical symptoms improved and the number of abnormalities in cardiac exams decreased.
Citation: Bastos CJC, Aras R, Mota G, Reis F, Dias JP, et al. (2010) Clinical Outcomes of Thirteen Patients with Acute Chagas Disease Acquired through Oral Transmission from Two Urban Outbreaks in Northeastern Brazil. PLoS Negl Trop Dis 4(6): e711. doi:10.1371/journal.pntd.0000711
Editor: Photini Sinnis, New York University School of Medicine, United States of America
Received: November 25, 2009; Accepted: April 22, 2010; Published: June 15, 2010
Copyright: © 2010 Bastos et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by the Oswaldo Cruz Foundation and the Sistema Único de Saúde (SUS), Ministry of Health, Brazil. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
American trypanosomiasis, or Chagas disease, is a zoonotic protozoan disease caused by the haemoflagellate Trypanosoma cruzi. The disease is endemic throughout Central and South America where about 17 million people are estimated to be infected and 100 million are at risk of infection . In Brazil, the overall prevalence of Chagas disease is 4.2% and in the northeast region the infection rate can reach more than 5.0% –. In endemic areas, the primary infection usually occurs in children aged 15 years and under. More than 99% of acute Chagas disease cases are asymptomatic or appear as a nonspecific febrile disease. However, in untreated patients with severe symptoms of acute Chagas disease, the mortality rate rises to about 5–10% . Moreover, 30% of infected individuals can develop chronic symptoms of Chagas disease over a lifetime .
Vectorial transmission of Chagas disease has decreased over the last decade in Brazil. Other forms of transmission, such as blood transfusion, congenital, organ transplants, and laboratory accidents are reported sporadically , . The oral-accidental transmission of Trypanosoma cruzi is becoming increasingly common.
Since 1965, several outbreaks, possibly caused by oral-accidental routes, mainly due to ingestion of food, fresh water, “açaí” (Euterpe oleracea) or sugar cane juice, have occurred in many Brazilian states, including Rio Grande do Sul, Amazonas, Amapá, Santa Catarina, and Bahia –. In other Latin American countries, oral transmission has also been reported –. Oral infection with T. cruzi is associated with a high mortality rate, usually in the first two weeks after infection –. Mortality is mainly due to acute congestive heart failure, myocarditis and meningoencephalitis , . Hemorrhagic manifestations and severe gastritis have also been reported .
Myocarditis is present in 80% of patients presenting severe symptoms of acute Chagas disease , –. Electrocardiography (EKG) and echocardiography (ECHO) show alterations such as atrial fibrillation and pericardial effusion, which are associated with a poor prognosis , –. In this study, we describe the clinical outcomes of 13 patients, before and after benznidazole treatment, during two micro outbreaks of acute Chagas disease in two towns located in the State of Bahia in northeastern Brazil.
Materials and Methods
Study Area and Patients
The patients involved in this study came from two neighboring towns: Macaúbas (46,554 inhabitants) and Ibipitanga (13,109 inhabitants), both located in the south central region of the state of Bahia (approximately 700 km from the capital) in the Brazilian Northeast. In both towns, the annual per capita income is less than U$1,000 and the UN human development index is 0.62521. In May 2006, there was an outbreak of acute Chagas disease involving seven individuals from Macaúbas (cases 1–7) . All individuals were members of the same family. Acute Chagas disease was suspected by a local physician and diagnosis was laboratory-confirmed in five cases (cases 1–5). Two patients (cases 6, 7) died as a consequence of heart failure before Chagas disease was confirmed. Oral contamination with T. cruzi probably occurred via ingestion of improperly stored water, possibly contaminated by feces of infested T. sordida .
The Ibipitanga outbreak occurred a few months after that of Macaúbas and involved six cases occurring among a family of 11. On August 9, 2006, the six cases: father (case 9), three sons (cases 10, 12, 13), one daughter (case 11) and his daughter-in-law (case 8) were working on a sugarcane plantation and drank a freshly-made sugarcane juice between 8:00 and 9:00 am, which they prepared in an abandoned sugarcane mill located next to the plantation. The cases developed symptoms between 11 and 21 days (between August 20 and 30, 2006) after the day they drank the sugar cane juice. A diagnosis of acute Chagas disease was suspected by a local physician 49 days after the ingestion of sugar cane juice (September 27, 2006). On October 8, 2006, the Epidemiological Surveillance Department of the Bahia Health Secretariat investigated the outbreak. Twelve specimens of Triatoma sordida were captured at the sugarcane mill, one of which was infested with T. cruzi. The diagnosis of Chagas disease was laboratory-confirmed for all six cases based on positive serological test results from samples collected on October 8, 2006, almost 60 days after exposure (Table 1).
Table 1. Serological test results from 13 patients with acute Chagas disease in two urban outbreaks Bahia, Brazil, 2006.doi:10.1371/journal.pntd.0000711.t001
The five family members who did not develop clinical symptoms had repeated negative serological test results for Chagas disease. All five reported to have drunk the same sugar cane juice prepared on August 9, 2006 which was also drunk by the six confirmed cases. However, the five uninfected family members drank the juice more than four hours after it was prepared and two of them had boiled the juice prior to ingestion.
The diagnosis for acute Chagas disease was confirmed using results from positive T cruzi parasitological tests: thick smear or blood culture; or a positive serologic test for IgM anti-T-cruzi antibodies: conventional enzyme-linked immunosorbent assay (ELISA), ELISA with recombinant antigens , or an indirect immunofluorescence antibody test (IFAT) (Table 1).
The study was approved by the institutional review board of CPqGM-FIOCRUZ, Bahia, Brazil. All patients and/or parents signed a letter of informed consent prior to examination.
All patients were treated after the Chagas diagnosis, which occurred between seven to 14 days and 27 to 37 days after the onset of symptoms in the Macaúbas and Ibipitanga patient groups, respectively. Oral benznidazole 300mg/day for 60 days was prescribed according to the Brazilian Consensus of Chagas Disease .
Eletrocardiogram (EKG) and Two-Dimensional Doppler Echocardiography (ECHO)
The EKG and ECHO were carried out before or shortly after beginning treatment, and 180 days after the end of specific treatment for Chagas disease. The criteria to define EKG alterations were based on the AHA/ACCF/HRS Recommendations for the Standardization and Interpretation of the Electrocardiogram and on the Guidelines of the Brazilian Society of Cardiology on Analysis and Report Issuance Electrocardiographic , . The classification of the severity of the valve disease in adults was based on the American College of Cardiology/American Heart Association Practice Guidelines  and the quantification of cardiac chamber size and ventricular mass followed the criteria from American Society of Echocardiography's Guidelines and the European Association of Echocardiography .
The most frequent symptoms in the acute phase were fever (92%) and dyspnea (92%), myalgia (69.2%), periorbital edema (53.9%), headache, systolic murmur (46.2%), nausea, cough, abdominal pain, hepatomegaly (38.5%). Thoracic pain and vomiting were observed in four patients (30.8%), while palpitations were present in three patients (23.1%) (Table 2). Two patients (6, 7) from Macaúbas had heart enlargement, gallop rhythm (3rd sound), tachycardia, hypotension, and cardiac failure, resulting in death. Chest X-rays showed pleural effusion and cardiac enlargement (Figure 1A and B) in both patients. These patients were brothers and were the first in their family to develop the symptoms of this disease. In these cases, the diagnosis of Chagas disease was based on epidemiological findings alone. The mortality rate of Chagas disease, before benznidazole treatment, was 28.6% in Macaúbas and one pregnant woman from Ibipitanga experienced a spontaneous abortion prior to receiving treatment.
Figure 1. Chest radiographs showing cardiac enlargement and pulmonary congestion in acute Chagas disease.
Patient # 6 (A), 16 years old; patient # 7 (B), 09 years old.doi:10.1371/journal.pntd.0000711.g001
Table 2. Frequencies of signs and symptoms of thirteen patients with acute Chagas disease from Macaúbas and Ibipitanga, Bahia-Brazil.doi:10.1371/journal.pntd.0000711.t002
EKG and ECHO Alterations
Table 3 displays the most frequent EKG and ECHO findings. EKG data were available for 12 out of 13 patients. At initial presentation, all 12 patients had a disturbance of ventricular repolarization. Right bundle branch block was observed in three out of 12 patients (25%) (cases 2, 3, 6) and sinus bradycardia was observed in cases 2, 12 and 13. Atrial fibrillation was present only in case 4 (8.3%).
Table 3. Clinical outcome, electrocardiogram (EKG) and Two-dimensional Doppler Echocardiography (ECHO) of thirteen patients with acute Chagas disease from Macaúbas and Ibipitanga, Bahia, Brazil, after benznidazole treatment.doi:10.1371/journal.pntd.0000711.t003
ECHO results were available for 11 out of 13 patients. Seven out of 11 patients had major alterations: a mild degree of mitral regurgitation was observed in six out of 11 patients (54.6%) (cases 2, 3, 4, 10, 12, 13). Pericardial effusion was observed in cases 2, 3, 4, and 9. Tricuspide regurgitation was found in cases 4, 10 and 13 (27.3%), and dyskinetic septum was observed in cases 3 and 4 (18.2%). Ventricular dysfunction with low ejection fraction <55% was present in cases 9, 10 and 12 (27.3%). ECHO findings were normal for cases 1, 5, 8, and 11.
Effect of Benznidazole Treatment on EKG and ECHO Alterations
No adverse events during treatment with benznidazole were observed. As shown in Table 3, EKG results normalized in five out of 11 patients (91.7%), 180 days after treatment ended. Ventricular repolarization abnormalities persisted in six out of 11 patients (50%) while sinus bradycardia was observed in two patients (16.7%). The atrial fibrillation that was present in case 4 ceased after treatment. Regarding ECHO findings, mitral regurgitation persisted only in cases 3, 4 but disappeared in cases 2, 10, 12, and 13. After treatment, mitral regurgitation was present in case 9. Ventricular function normalized in cases 9, 10 and 12, and pericardial effusion was not present.
Acute Chagas disease caused by oral transmission has been increasingly reported in Brazil and other Latin American countries –, . However, few studies describe clinical outcomes after treatment with benznidazole . In this report, the post-treatment clinical evolution of acute Chagas disease in patients from two impoverished rural towns in northeastern Brazil was observed. Oral transmission was determined to be the cause of both micro-outbreaks of acute Chagas disease. In the Macaúbas outbreak, patients were purportedly infected by the ingestion of stored water contaminated by the feces of infested triatomines ; while in Ibipitanga, oral transmission was due to the ingestion of sugarcane juice prepared in an abandoned sugarcane mill, where specimens of T. sordida contaminated with T. cruzi were captured.
Fever and dyspnea were experienced by nearly all patients. Other symptoms and findings indicating myocardial involvement, such as periorbital edema, chest pain and pericardial effusion, were observed in more than one-third of patients in both outbreaks. Hematological and digestive tract symptoms, including gastrointestinal bleeding and gastritis, were not found in our series, but were observed in patients from the Santa Catarina outbreak .
Morbidity and mortality rates of severe symptomatic acute Chagas disease are notably higher in children who contract the disease –, . Moreover, orally transmitted Chagas disease causes more severe symptoms in acute phases –. Prior to receiving treatment, two children from Macaúbas died as a consequence of heart failure (28.6% mortality rate), while in Ibipitanga, one pregnant woman experienced a spontaneous abortion. In both outbreaks, almost all exposed individuals developed severe manifestations of acute Chagas disease. In the Macaúbas outbreak the attack rate was 100% . In the Ibipitanga outbreak, the six cases had drunk freshly-made sugarcane juice, while the five uninfected family members drank the juice more than four hours after it was prepared and two of them boiled the juice prior to ingestion. These five individuals remained asymptomatic and had repeated negative serological test results for Chagas disease. Insect-derived metacyclic trypomastigotes have specialized mechanisms that allow mucosal invasion. In orally-infected mice, trypomastigotes are able to invade and replicate in the gastric mucosa, causing a systemic infection –. In addition, metacyclic trypomastigotes taken from a patient who was orally infected with acute Chagas disease caused high parasitemia and a high mortality rate in orally-infected mice . The development of myocarditis in acute Trypanosoma sp. infection is associated with intense edema of the cardiac fibers and the presence of inflammatory infiltrate containing amastigote forms of T. cruzi . High levels of inflammatory cytokines are associated with myocardial damage. A recent study found higher levels of interferon-gamma, tumor necrosis factor-alpha, interleukin-10 and CCL3 in the myocardium of hamsters exhibiting acute symptoms of Chagas disease, when compared to asymptomatic animals .
EKG and ECHO abnormalities are frequently observed as a consequence of myocarditis. In the acute phase of Chagas disease, EKG findings may present alterations including low QRS voltage, prolonged PR and/or QT intervals, as well as T-wave changes , . Ventricular extra systoles, sinus tachycardia, atrial fibrillation and advanced grade right bundle branch block are all associated with a poor prognosis . In this study, during the acute phase of Chagas disease, ECHO exams were normal in only one-third of patients. Pericardial effusion was observed in 36% of patients. These findings were similar to those observed by Pinto et al during the acute phase of Chagas disease .
Six-months after the end of treatment with benznidazole, every patient showed an improvement in clinical symptoms, as well as a decrease in the number of EKG and ECHO abnormalities. Clinical signs of cardiac dysfunction, such as ejection fraction by ECHO, showed improvement in patients with more severe cardiac manifestations (cases 9, 10 and 12). In addition, 45% of patients (5 out of 11) with acute Chagas disease had a normal EKG at the end of treatment. We cannot conclude that improvements in EKG and ECHO findings were directly attributable to therapy, as opposed to the natural course of the disease. Although there is ample information on the clinical evolution of chronic Chagas disease, there are few studies that have evaluated the effect of benznidazole treatment on EKG and ECHO exams in patients with acute Chagas disease who have been followed from initial presentation to convalescence , –. In children in the early chronic phase of Chagas disease, after three to four years of follow-up, no conclusive evidence was obtained to indicate that benznidazole treatment, when compared with a placebo, could revert EKG abnormalities –. However, adults with indeterminate and chronic Chagas disease who were treated with benznidazole developed fewer electrocardiographic abnormalities when compared with untreated patients . The present study exclusively involved patients in the acute phase of Chagas disease, for whom treatment is mandatory . As such, it would be unethical to deprive these patients of treatment, making a clinical trial with a placebo control impossible. The efficacy of benznidazole in acute Chagas disease is demonstrated by a reduction in parasite load . The cure rate for parasitological acute Chagas disease ranges from 60 to 80%, depending on patient age, dosage and whether treatment was initiated at the beginning of the infection –.
We can conclude that cardiac alterations occur frequently, and are potentially severe, in the acute phase of orally transmitted Chagas disease. Furthermore, EKG and ECHO findings may have an impact on the clinical management of the disease, enabling monitoring of disease progression both during and after benznidazole treatment. Further studies are necessary to evaluate the persistence of EKG and ECHO abnormalities over the long term, as well as morbidity and mortality.
We thank Sérgio Manuel Ferreira, Verônica Mascarenhas, José Luiz Nunes Ferreira, Ana Karenina Baraúna Cunha, Eduardo M Netto and Martha Silvia Martinez-Silveira for expert technical assistance.
Conceived and designed the experiments: CJCB RA MFRG. Performed the experiments: GM FR RSdJ MSF EGdA JP. Analyzed the data: CJCB RA GM FR JPD MFRG. Contributed reagents/materials/analysis tools: RA JPD MFRG. Wrote the paper: CJCB MFRG.
- 1. World Health Organization (2002) Control of Chagas disease. World Health Organ Tech Rep Ser 1–109.
- 2. Dias JC, Machado EM, Fernandes AL, Vinhaes MC (2000) Esboço geral e perspectivas da doença de Chagas no Nordeste do Brasil. Cad Saude Publica 16: 13–34.
- 3. Aras R, Veiga M, Gomes I, Mota G, Rodrigues B, et al. (2002) Prevalence of Chagas' disease in Mulungu do Morro northeastern Brazil. Arq Bras Cardiol 78: 441–443.
- 4. Prata A (2001) Clinical and epidemiological aspects of Chagas disease. Lancet Infect Dis 1: 92–100.
- 5. Brasil , Ministério da Saúde , Secretaria de Vigilância da Saúde (2005) Doença de Chagas aguda no município de Santana - Amapá. Technical Note. Available: http://www.parasitologia.org.br/default.asp?MENU=20&EDITORIA=53. Accessed 2009 Jan 10.
- 6. Brasil , Ministério da Saúde , Secretaria de Vigilância da Saúde (2005) Doença de Chagas Aguda relacionada à ingestão de caldo de cana em Santa Catarina. Technical Note. Available: http://www.anvisa.gov.br/divulga/noticias/2005/240305_nota.pdf. Accessed 2009 Jan 3.
- 7. Brasil , Ministério da Saúde , Secretaria de Vigilância da Saúde (2007) Doença de Chagas aguda por transmissão oral. Technical Note. Available: http://portal.saude.gov.br/portal/arquivos/pdf/nota_chagas2308.pdf. Accessed 2009 Jan15.
- 8. Dias JP, Bastos C, Araujo E, Mascarenhas AV, Martins Netto E, et al. (2008) Acute Chagas disease outbreak associated with oral transmission. Rev Soc Bras Med Trop 41: 296–300.
- 9. Brasil , Ministério da Saúde , Secretaria de Vigilância em Saúde (2005) Consenso Brasileiro em Doença de Chagas. Rev Soc Bras Med Trop 38: 1–29.
- 10. Guimarães FN, da Silva NN, Clausell DT, de Mello AL, Rapone T, et al. (1968) Um surto epidêmico de doença de Chagas de provável transmissão digestiva, ocorrido em Teutônia (Estrela Rio Grande Do Sul). Hospital (Rio J) 73: 1767–1804.
- 11. Pinto AY, Harada GS, Valente V, Abud JE, Gomes F, et al. (2001) Acometimento cardíaco em pacientes com doença de Chagas aguda em microepidemia familiar, em Abaetetuba, na Amazônia Brasileira. Rev Soc Bras Med Trop 34: 413–419.
- 12. Pinto AY, Valente SA, Valente VC (2004) Emerging acute Chagas disease in Amazonian Brazil: case reports with serious cardiac involvement. Braz J Infect Dis 8: 454–460.
- 13. Shikanai-Yasuda MA, Marcondes CB, Guedes LA, Siqueira GS, Barone AA, et al. (1991) Possible oral transmission of acute Chagas' disease in Brazil. Rev Inst Med Trop Sao Paulo 33: 351–357.
- 14. Valente SA, da Costa Valente V, das Neves Pinto AY, de Jesus Barbosa Cesar M, dos Santos MP, et al. (2009) Analysis of an acute Chagas disease outbreak in the Brazilian Amazon: human cases, triatomines, reservoir mammals and parasites. Trans R Soc Trop Med Hyg 103: 291–297.
- 15. Anez N, Carrasco H, Parada H, Crisante G, Rojas A, et al. (1999) Acute Chagas' disease in Western Venezuela: a clinical, seroparasitologic, and epidemiologic study. Am J Trop Med Hyg 60: 215–222.
- 16. Carme B, Aune I, Nguyen G, Aznar C, Beaudet B (2001) Four cases of acute chagasic myocarditis in French Guiana. Am J Trop Med Hyg 64: 162–163.
- 17. Punukollu G, Gowda RM, Khan IA, Navarro VS, Vasavada BC (2007) Clinical aspects of the Chagas' heart disease. Int J Cardiol 115: 279–283.
- 18. Acquatella H (2007) Echocardiography in Chagas heart disease. Circulation 115: 1124–1131.
- 19. Benchimol-Barbosa PR (2009) Predictors of mortality in Chagas' disease: the impact of atrial fibrillation and oral transmission on infected population. Int J Cardiol 133: 275–277.
- 20. Houghton RL, Benson DR, Reynolds LD, McNeill PD, Sleath PR, et al. (1999) A multi-epitope synthetic peptide and recombinant protein for the detection of antibodies to Trypanosoma cruzi in radioimmunoprecipitation-confirmed and consensus-positive sera. J Infect Dis 179: 1226–1234.
- 21. Surawicz B, Childers R, Deal BJ, Gettes LS (2009) AHA/ACCF/HRS recommendations for the standardization and interpretation of the electrocardiogram: part III: intraventricular conduction disturbances: a scientific statement from the American Heart Association Electrocardiography and Arrhythmias Committee, Council on Clinical Cardiology; the American College of Cardiology Foundation; and the Heart Rhythm Society. Endorsed by the International Society for Computerized Electrocardiology. J Am Coll Cardiol 53: 976–981.
- 22. Pastore CA, Pinho C, Germiniani H, Samesima N, Mano R, et al. (2009) Diretrizes da Sociedade Brasileira de Cardiologia sobre Análise e Emissão de Laudos Eletrocardiográficos. Arq Bras Cardiol 93: 1–19.
- 23. Bonow RO, Carabello BA, Chatterjee K, de Leon AC Jr, Faxon DP, et al. (2006) ACC/AHA 2006 guidelines for the management of patients with valvular heart disease: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines. J Am Coll Cardiol 48: e1–e148.
- 24. Lang RM, Bierig M, Devereux RB, Flachskampf FA, Foster E, et al. (2005) Recommendations for chamber quantification: a report from the American Society of Echocardiography's Guidelines and Standards Committee and the Chamber Quantification Writing Group, developed in conjunction with the European Association of Echocardiography, a branch of the European Society of Cardiology. J Am Soc Echocardiogr 18: 1440–1463.
- 25. Coura JR (2007) Chagas disease: what is known and what is needed–a background article. Mem Inst Oswaldo Cruz 102: 113–122.
- 26. Pinto AYN, Ferreira AG, Valente VC, Harada GS, Valente SAS (2009) Urban outbreak of acute Chagas disease in Amazon region of Brazil: four-year follow-up after treatment with benznidazole. Rev Panam Salud Publica 25: 77–83.
- 27. Parada H, Carrasco HA, Anez N, Fuenmayor C, Inglessis I (1997) Cardiac involvement is a constant finding in acute Chagas' disease: a clinical, parasitological and histopathological study. Int J Cardiol 60: 49–54.
- 28. Hoft DF (1996) Differential mucosal infectivity of different life stages of Trypanosoma cruzi. Am J Trop Med Hyg 55: 360–364.
- 29. Hoft DF, Farrar PL, Kratz-Owens K, Shaffer D (1996) Gastric invasion by Trypanosoma cruzi and induction of protective mucosal immune responses. Infect Immun 64: 3800–3810.
- 30. Yoshida N (2008) Trypanosoma cruzi infection by oral route: how the interplay between parasite and host components modulates infectivity. Parasitol Int 57: 105–109.
- 31. Bilate AM, Teixeira PC, Ribeiro SP, Brito T, Silva AM, et al. (2008) Distinct outcomes of Trypanosoma cruzi infection in hamsters are related to myocardial parasitism, cytokine/chemokine gene expression, and protein expression profile. J Infect Dis 198: 614–623.
- 32. de Andrade AL, Zicker F, de Oliveira RM, Almeida Silva S, Luquetti A, et al. (1996) Randomised trial of efficacy of benznidazole in treatment of early Trypanosoma cruzi infection. Lancet 348: 1407–1413.
- 33. Sosa Estani S, Segura EL, Ruiz AM, Velazquez E, Porcel BM, et al. (1998) Efficacy of chemotherapy with benznidazole in children in the indeterminate phase of Chagas' disease. Am J Trop Med Hyg 5: 526–529.
- 34. Viotti R, Vigliano C, Lococo B, Bertocchi G, Petti M, et al. (2006) Long-term cardiac outcomes of treating chronic Chagas disease with benznidazole versus no treatment: a nonrandomized trial. Ann Intern Med 144: 724–734.
- 35. Rodrigues Coura J, de Castro SL (2002) A critical review on Chagas disease chemotherapy. Mem Inst Oswaldo Cruz 97: 3–24.
- 36. Caldas IS, Talvani A, Caldas S, Carneiro CM, de Lana M, et al. (2008) Benznidazole therapy during acute phase of Chagas disease reduces parasite load but does not prevent chronic cardiac lesions. Parasitol Res 103: 413–421.
- 37. Cancado JR (2002) Long term evaluation of etiological treatment of Chagas disease with benznidazole. Rev Inst Med Trop Sao Paulo 44: 29–37.