Submit manuscript...
Journal of
eISSN: 2373-437X

Microbiology & Experimentation

Review Article Volume 6 Issue 3

Chagas disease: an overview of diagnosis

Gilberto Ballesteros Rodea,1 Teresa Itandehui Martinez Cuevas,2 Berenice Jimenez Ramos,2 Alberto Antonio Campos2,3

1Departamento de Patologia, Universidad Autonoma de San Luis Potosi, Mexico
2Departamento de Biomedicina Molecular, Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico Nacional, Mexico
3Departamento de Parasitologia, Escuela Nacional de Ciencias Biologicas, Instituto Politecnico Nacional, Mexico

Correspondence: Gilberto Ballesteros-Rodea, Km. 14.5 Carretera San Luis Potosi, Matehuala, Ejido Palma de la Cruz, Soledad de Graciano Sanchez, San Luis Potosi, S.L.P., CP. 78321, Tel 52 (444) 852-40-56 al 60

Received: May 08, 2018 | Published: June 7, 2018

Citation: Rodea GB, Cuevas TIM, Ramos BJ. Chagas disease: an overview of diagnosis. J Microbiol Exp. 2018;6(3):151-157. DOI: 10.15406/jmen.2018.06.00207

Download PDF


Chagas disease, caused by the flagellated protozoan Trypanosoma cruzi, afflicts millions of people, mainly affecting poor and neglected populations. The different transmission routes, the high genetic variability of the parasite, the different detection methods as well as the distinct phases of the disease, influence negatively the diagnosis accuracy of the disease. Diagnostic tests can range from simple parasitological techniques to complex molecular and serological techniques that can be used for early diagnosis in the acute phase of the disease, the determination of congenital transmission, to determine the epidemiological behavior of the disease, to detect the presence of the parasite, both in blood transfusions, as in organ transplantation, among others. This review addresses some of the most widely used tools to detect T. cruzi infection in different scenarios.

Keywords: chagas disease, Trypanosoma cruzi, diagnosis


IF, indirect immuno fluorescence; ELISA, enzyme-linked immuno-sorbent assay; IH, indirect hemagglutination; SAPA/TS, shed acute-phase antigens/trans-sialidase; TESA: trypomastigote excreted and secreted antigen; IA: immunoagglutination assay; Ab, antibodies; Ag, antigens; PCR, polymerase chain reaction


Chagas disease is caused by the flagellated protozoan Trypanosoma cruzi, which affects several species of mammals and is considered an important zoonosis.1 It’s distributed throughout the American continent, from the south of the United States to the south of Argentina; most of the cases are found in poor and rural areas of Central and South America. The endemic areas closely related to the presence of the vector, which correspond to bugs of the genus Triatoma, Rhodnius and Panstrongylus.2,3 The human is the main reservoir in the domiciliary cycle, followed by domestic animals such as the dog, the cat and some domestic rodents. Numerous species of mammals can be naturally infected in endemic areas. Therefore, animals that invade homes and peri-domiciliary areas such as raccoons and rodents can be a risk of transmitting the disease to humans.4,5 Approximately 6 to 7 million people worldwide are infected with the parasite, mainly in Latin America. In addition, and it is estimated that about 12.9% of the world population, approximately 70 million people, is at risk of contracting T. cruzi infection.6–8 Rates of higher prevalence are found in Bolivia (6.75%), Argentina (4.13%), El Salvador (3.37%), Honduras (3.05%) and Paraguay (2.54%), while in Mexico and Brazil the prevalence is low, around 1%. However, due to their large populations, approximately one third of all people infected with T. cruzi live in these two countries.9,10 Chagas disease is also transmitted through blood transfusions, organ transplants, through the placenta and through laboratory accidents.11–17


At the end of the 1980s, the documented number of Latin American immigrants in the United States from endemic countries for T. cruzi was 2.24 million, of which 1.55 million came from Mexico. More than 7 million people from the endemic countries of T. cruzi became legal residents between 1981 and 2005. The infection is most frequently associated with immigrants from Mexico, Central America and South America; it´s estimated that there are between 100,000 and 600,000 Latin American immigrants infected with Chagas disease in the United States.18 Approximately 250,000; 8,000; 200,000 immigrants from Latin America lived in Europe, Australia and Japan respectively in the late 80's.13 Immigrants in Japan are mainly Brazilians of Japanese descent whose living conditions in Brazil make it unlikely that they have been infected with T. cruzi. In Europe, Spain has become a magnet for immigrants from Latin America.19 The deaths caused by this disease are estimated between 45 thousand and 50 thousand each year, with chronic chagasic myocardiopathy being the main cause, while mortality during the acute phase occurs in approximately 5% of children under 2years of age, due to acute myocarditis or meningoencephalitis.20–23

Phases of the disease

To make a good diagnosis it’s necessary to consider the complexity of Chagas disease, the epidemiology, transmission and its distinct phases. Fever is often a suggestive sign of infection in the acute phase. The lesion at the site of entry of the parasite: chagoma (furunculoid lesion on the skin) or Romaña sign (entry through the conjunctiva) is present in 20 to 50% of acute cases. In children, hepatomegaly, splenomegaly, generalized subcutaneous edema or localized on the face and lower extremities have been observed in 30% to 50% of cases; and from 30 to 80% of patients develop persistent tachycardia. The manifestations of the acute phase are solved spontaneously in a period of between 3 and 8 weeks in approximately 90% of the individuals that have been infected.21,23,24 The Indeterminate stage is a direct progression between the acute phase and the defined phase (symptomatic). Approximately 50 to 70% of patients in the indeterminate phase never develop lesions and remain asymptomatic. The remaining 30 to 50% of patients develop cardiac or digestive dysfunction 10 to 30 years after the acute phase.21,23,25 Finally, the symptomatic stage is characterized by palpitations, dizziness, syncope and seizures, due to acute circulatory failure caused by decreased heart rate, ventricular tachycardia or cardiac arrest. In heart failure, arrhythmia, atypical precordial pain without evidence of coronary disease and dyspnea can be observed. In the presence of megaesophagus, there is regurgitation and aspiration of food, salivary gland hypertrophy, weight loss, and cachexia, signs of esophageal rupture and increased incidence of esophageal cancer. In the case of Megacolon, there is asymmetrical distention of the abdomen, bloating, intestinal occlusion and sigmoid colon volvulus.21,23,24

Clinical diagnosis

The clinical diagnosis of Chagas disease is mainly given by the patient's clinical history, in addition to cabinet and laboratory studies. The cabinet studies are mainly radiological (thoracic-abdominal), electrocardiogram, ultrasound and echocardiogram, which are most useful in the chronic phase of the disease. In the acute stage, studies are focused on the search for the blood parasite. Because the sensitivity of the used methods is variable, it’s advisable to follow a predetermined diagnostic routine.25,26 Generally the diagnosis of Chagas disease, is made by direct observation of the trypomastigotes under the microscope in a peripheral blood sample, either from a fresh sample, a smear, with the thick-film technique stained with Giemsa or after performing the Strout test.27–29 Which consists of concentrating the parasites from blood obtained without anticoagulant to favor coagulation and obtain the trypomastigotes that are suspended in the supernatant: after several cycles of centrifugation, first to eliminate the residual erythrocytes and then to concentrate the parasites, finally the sediment is analyzed under a microscope. A variant of this procedure that is very useful for the diagnosis of congenital infections is the heparinized or microhematocrit capillary tube technique; this test consists of the analysis of the parasites at the interface between the formed coagulum and the plasma of several capillaries.30–32 For the morphological identification and to be able to differentiate with T. rangeli, it is necessary to analyze the preparations stained with Giemsa: the trypomastigotes of T. cruzi are characterized by having a prominent kinetoplast, which gives the appearance of being above the body of the parasite. However, the correct morphological differentiation of these two species is very complicated, even by trained personnel; therefore, in these cases the use of molecular techniques is recommended for confirmation;33–35 that situation mainly occurs in the acute phase of infection and in areas where both species of the parasite are co-endemic.36 When parasitemia is low, which is one of the main characteristics of the chronic stage, it is essential to draw on techniques that allow amplifying some specific DNA sequence of the parasite in order to make an adequate diagnosis. The xenodiagnostic consists of using triatomines placed on the inside of the arms or legs of the patient for 30min. Afterwards, the feces of the vector are analyzed at 30,60 and 90days, in search of metacyclic trypomastigotes in movement. This technique has been modified over time, now days it can be performed artificially with the same sensitivity as a traditional xenodiagnosis, in this way; direct exposure of the patient to the triatomines is avoided. The amount of peripheral blood used is the same as that ingested by insects in the traditional way; to reach an optimal performance it is necessary that it be processed immediately or in 4 hours after it is obtained. With the optimization of artificial xenodiagnosis, it has been possible to avoid that the patient suffers from the sting of the triatomines, although the result is still obtained between 30-90days after the feeding of these insects. Some researchers have added to this technique the polymerase chain reaction (PCR) to detect the parasite and thus increase its sensitivity and reduce the time to 30 days, namely, to earlier times.37 The diagnosis of Trypanosoma cruzi infection is complex, mainly during the chronic or clinical phase of the disease, where the lack of apparent signs and symptoms, in addition to the low and intermittent parasitemia, leads to the need for more specific diagnostic methods than direct parasitologicals. Which are not so reliable due to its low sensitivity, so the development of serological and molecular tools have had a great boom thanks to their sensitivity and specificity.38

Serological diagnosis

For diagnosis in chronic phase, either asymptomatic or symptomatic phases, several immunological techniques have been developed for the detection of specific IgG antibodies against epimastigotes extracts of T. cruzi, including the Indirect Immuno fluorescence (IF), the Enzyme-Linked Immuno-Sorbent Assay (ELISA) and Indirect Hemagglutination (IH).8,39–42 The majority of immunological tests commercially available use recombinant antigens, synthetic peptides and antigens obtained from non-native strains antigenically different from local strains, which generates low specificity and sensitivity in the tests.40,43 Since no single standard reference test is available yet, diagnosis should base on the presence of IgG against various T. cruzi antigens by using at least two serological assays with different antigens. A subject is considered infected when the results of the two serological tests are positive.44 However, in some serological results, discrepancies or inconclusive results may occur. To verify these results, the use of a third technique is recommended. Several serological assays and antigens have been proposed and evaluated for this use as confirmatory or supplementary test of T. cruzi infection. Nevertheless, there is no an actual consensus establishing a reference technique, and no single test is considered the gold standard for unequivocal diagnosis of infection by this parasite.45 The use of a quantitative method such as immunoblotting (Western blot) can be very useful on inconclusive results, mainly in countries with endemic presence of other trypanosomatids such as Leishmania spp.46 Despite of this, the use of western blot has some disadvantages. For example, there are no commercial tests based on this technique at this moment46 and it is only able to detect linear epitopes (processed antigens on MHC-I), excluding the conformational structural epitopes. The use of serological diagnosis in Chagas disease is based on the use of extracts of T. cruzi epimastigotes as antigens. Although these extracts show limited specificity, they have also been reported to show high sensitivity in the chronic phase of the disease, moreover they show low sensitivity in the acute phase as well as in congenital infection.47–50 The Shed Acute-Phase Antigens/Trans-Sialidase (SAPA/TS) are used in these cases to increase sensitivity. These antigens are proteins released to the extracellular medium through the surface of the T. cruzi trypomastigotes, meanwhile in epimastigotes, SAPA is a transmembrane protein.47,51–56

A large set of extracts/antigen preparations have been used for the serological diagnosis during T. cruzi infection.57 However, Umezawa et al. quantified the sensitivity (100%) and specificity (99.4%) of IgG from patients with Chagas disease in both the acute phase and the chronic phase in 1996. For this, they used the western blot technique and Trypomastigote Excreted and Secreted Antigen (TESA) obtained from cells LLC-MK2 in culture, infected with trypomastigotes of the Y strain of T. Cruzi.45,47,50,57 In laboratory conditions, TESA blot is considered positive when is reactive with antigens of 130-200 kDa or antigens of 150-160 kDa. However, some sera also react with bands of 80-120 kDa that belong to Shed Acute-Phase Antigen (SAPA). Some chronic patients also react with SAPA bands plus a band of approximately 95 kDa.47,58 It has been reported that some members of SAPA and TESA molecules are part of T. cruzi transialidases, a superfamily of proteins implicated in the penetration and infection of host cells as result of the transference of sialic acid molecules from the host cell-surface glycoconjugates to its own surface mucin as glycoprotein. Transialidases are highly immunogenic, and both the C-terminal and the N-terminal regions stimulate strong humoral responses (B-cells); they are predominant antigens on the surfaces of bloodstream trypomastigotes, metacyclic trypomastigotes, and intracellular amastigotes.53,59–61

Despite of the fact that over time a large number of serological methods have been developed for the detection of T. cruzi in the late stages of Chagas disease, there are also classical and effective methods with acceptable sensitivity and specificity. For example, the Immunoagglutination Assay (IA), which is faster and less expensive than the above-mentioned test.62,63 The IA for the detection of antibodies (Ab) comprises mixing serum or plasma with a suspension containing antigens (Ag) bound to latex particles. This method uses small volume samples, obtaining results in short times (5min approximately), is easy to implement and it does not require sophisticated equipment. Furthermore, it’s a technique that may be used almost anywhere, when is possible detected visually the immunoagglutination reaction.62 However, when some total parasite extracts are used, this test also generates cross-reaction with homologous organisms such as T. rangeli and Leishmania spp.64,65 At the present, one of the most accepted and used tests for the detection of T. cruzi infection in samples of patients suspected of having Chagas disease, as well as in epidemiological studies, is indirect immunofluorescence (IF).44 The detection of antibodies (IgG) against T. cruzi and the parasite’s presence for prolonged periods, stimulates the prevalence of this disease.66,67 In addition to the use of IgG antibodies, is possible to use IgM antibodies. These types of immunoglobulins are the first to appear during an adaptive response and are of limited duration. Because of the latter, the use of specific IgM antibodies against T. cruzi indicates an acute phase of Chagas disease, even in urine samples (80-kilodalton Trypanosoma cruzi antigen).67–69

In addition to the use of immunofluorescence and IgM as an early marker of infection, its main application is in the detection of congenital infections. The pentameric nature of IgM prevents it from traversing the intact placenta.70 In addition, the fetus is capable of generating IgM antibodies by itself in response to foreign agents.67 Therefore, the detection of IgM antibodies against T. cruzi detected in the blood of newborns is an indicator of a transplacental infection.67,68 Nevertheless, false positives have been observed due contamination of IgM antibodies from the mother to the fetus, coming from damage to the integrity of the placenta and through maternal blood during childbirth.67,71,72 Despite the diagnostic advances and the fact that serological methods are in many cases sensitive and specific, it’s necessary to perform complementary tests (xenodiagnostic, PCR, electrocardiogram, echocardiogram, etc.) for the validation of the serological results.

Molecular diagnosis

During Chagas disease chronic phase, serologic diagnosis methods are needed to identify infected subjects. These methods involve the T. cruzi indirect detection by the searching of antibodies against the parasite.73 Indirect detection in serologic methods represent a disadvantage due to the potential anti- T. cruzi antibody transference from mother to fetus (congenital transmission), as well as a possible crossed-reaction between other tripanosomatids such as Leshmania spp. and T. rangeli which can cause false positive results.74–76 Thus, several groups have implemented the usage of PCR (polymerase chain reaction) to identification of the genetic material from the parasite, in blood and serum samples as well as tissue samples.77–80 Several types of the PCR techniques are available to detect T. cruzi DNA in serum and blood samples, among them we find: conventional PCR that amplifies a specific sequence from the parasite’s DNA, for this it’s common the use of repeated sequences or DNA from kinetoplast (kDNA).77,81,82 Hot-Start PCR which is a modification to the conventional PCR to diminish amplification of unspecific products;40nested PCR is employed to amplify DNA sequences that are found in very low quantities in the parasite genome enhancing the sensitivity in the system, it consists in the extension of a specific sequence in two successive amplification steps.83 Several tools that utilize probes to verify presence/absence of specific DNA are used too (Southern Blot or PCR and hybridization) Finally, real-time PCR usage allows to determine the parasite load by the quantification of the specific sequence amplification.40,77,85 An important advantage that the use of PCR offers as a diagnosis tool is that allows the characterization of the circulating strains in an endemic area for Chagas disease.86 Due to the wide genetic diversity in T. cruzi, the different strains of the parasite have been classified in six Discrete Typing Units (DTU). These differences are shown in the biological, pathological and immunological behavior as well as in the eco-epidemiology of the disease.87

The utilization of this technique to diagnostic purposes remains limited since it implies a higher cost than the conventional serological tools and requires trained and specialized staff. Nonetheless, it’s used in very concrete cases like mother-fetus transmission as a confirmatory test in cases where serological evidence is not concluding and for research purposes too.40,78,83,88–90 Lacking of a gold standard test as well as a biological marker that allow the identification of T. cruzi infection points toward the pursuit of several serological (like antigens) and/or molecular markers (like specie-specific genomic sequences) that enable a more reliable and accurate diagnosis. One of the major challenges is the great genetic diversity between the strains of the parasite since this suggests the existence of a distinct antigenic repertory among the different strains which is reflected, i. e., in the presence of immunological markers related to DTU.91–95

Diverse strategies have developed in order to identify antigenic proteins in the parasite that could work as potential candidates for a diagnosis tools and vaccines. A pursued characteristic is that the markers be “universal”, which means markers must be likely to be located in every strain of the parasite, no matter the genotype it belongs. One of the most employed is the usage of libraries, from complementary DNA expression libraries to peptides libraries, however here we found a weakness: the number of proteins to be identified by this method can be low.96–113 Accession of massive sequencing techniques has broadened the repertory of probable candidates that can be useful for serological diagnostic of Chagas disease.

Implementation of microarray and immunoprecipitation assays coupled to mass spectrometry, besides the availability of parasite genome project, make possible to increase the number of antigenic proteins identified.114,115 Nevertheless, this feature also signifies a difficulty due to a wide number of candidate proteins that are obtained; hence, it’s necessary to make use of bioinformatic tools that allow filtering the list of candidate peptides to be analyzed so in this way the potentially reactive epitopes can be enriched. A great advantage in this kind of assays is the utilization of biological samples derived from infected subjects with the parasite. Recent use of linear epitope mapping in the antigenic proteins, supported in peptide micromatrix platforms, sets the facility to use quick serological screening.116 However, the difficulty that this method presents is the use of in sillico tools, which although are efficient to identify the “theoretical” antigenic properties in the parasite proteins, do not assure this antigens act like that in the biological context.


The diagnosis of Chagas disease has limitations, mainly due to the great complexity of the factors that involve it, as well as to the low sensitivity of the parasitological techniques and the low specificity of the immunological tests. Molecular techniques, such as the polymerase chain reaction (PCR), which detect specific and repeated DNA sequences of the parasite, represent a suitable alternative for diagnosis in some situations, particularly in acute cases, in congenital transmission and in the evaluation and control of treatment. The PCR technique also has some limitations in terms of cost, necessary infrastructure and sensitivity in the chronic phase of the disease. To obtain better results in the diagnosis, it is necessary to combine the use of different parasitological, immunological and molecular tools according to the phase of the disease that the patient faces in each particular case.



Conflict of interest

The author declares no conflict of interest.


  1. Salazar Schettino PM, de Haro Arteaga I, Uribarren Berrueta T. Chagas disease in Mexico. Parasitol today. 1988;4(12):348–52.
  2. Martínez-Ibarra JA, Bárcenas-Ortega NM, Nogueda-Torres B, et al. Role of two Triatoma (Hemiptera: Reduviidae: Triatominae) species in the transmission of Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae) to man in the West Coast of Mexico. Mem Inst Oswaldo Cruz. 2001;96:141–144.
  3. Montfort WR, Weichsel A, Andersen JF. Nitrophorins and related antihemostatic lipocalins from Rhodnius prolixus and other blood-sucking arthropods. Biochim Biophys Acta. 2000;1482(1-2):110–118.
  4. Cruz-Reyes A, Pickering-Lopez JM. Chagas disease in Mexico: an analysis of geographical distribution during the past 76 years--a review. Mem Inst Oswaldo Cruz. 2006;101(4):345–54.
  5. Salazar Schettino PM, Bucio Torres MI, de Haro Arteaga I, et al. [Reservoirs and vectors of Trypanosoma cruzi in the state of Oaxaca]. Salud Publica Mex. 1987;29(1):26–32.
  6. Hotez PJ, Alvarado M, Basanez MG, et al. The global burden of disease study 2010: interpretation and implications for the neglected tropical diseases. PLoS Negl Trop Dis. 2014;8(7):e2865.
  8. Perez-Molina JA, Molina I. Chagas disease. Lancet. 2018;391(10115):82–94.
  9. Kirchhoff LV. Epidemiology of american trypanosomiasis (Chagas Disease). Adv Parasitol. 2011;75:1–18.
  10. Bern C, Montgomery SP. An estimate of the burden of Chagas disease in the United States. Clin Infect Dis. 2009;49(5):e52–54.
  11. Brener Z. Biology of Trypanosoma cruzi. Annu Rev Microbiol. 1973;27:347–382.
  12. Coura JR. Chagas disease: what is known and what is needed-A background article. Mem Inst Oswaldo Cruz. 2007;102(supp 1):113–122.
  13. Schmunis GA. Trypanosoma cruzi, the etiologic agent of Chagas' disease: status in the blood supply in endemic and nonendemic countries. Transfusion. 1991;31(6):547–557.
  14. Tanowitz HB, Kirchhoff LV, Simon D, et al. Chagas' disease. Clin Microbiol Rev. 1992;5(4):400–419.
  15. Vickerman K. Developmental cycles and biology of pathogenic trypanosomes. Br Med Bull. 1985;41(2):105–114.
  16. de Souza W. Cell biology of Trypanosoma cruzi. Int Rev Cytol. 1984;86:197–283.
  17. Barreto-de-Albuquerque J, Silva-dos-Santos D, Pérez AR, et al. Trypanosoma cruzi Infection through the Oral Route Promotes a Severe Infection in Mice: New Disease Form from an Old Infection? PLoS Negl Trop Dis. 2015;9(6):e0003849.
  18. Espinoza B, Manning-Cela R. An overview of mammalian cell infection by Trypanosoma cruzi: Cellular and molecular basis. In: Terrazas L, editor. Advances in the Immunobiology of Parasitic Diseases. 2007:219–311.
  19. Schmunis GA. Epidemiology of Chagas disease in non-endemic countries: the role of international migration. Mem Inst Oswaldo Cruz. 2007;102(Suppl 1):75–85.
  20. Barreto ML, Andrade ME. [Impact of Chagas' infection on some demographic characteristics: results of an ecological study]. Cadernos de saude publica. 1994;10(Suppl 2):273–280.
  21. Cubillos-Garzon LA, Casas JP, Morillo CA, et al. Congestive heart failure in Latin America: the next epidemic. Amheart J. 2004;147(3):412–417.
  22. Guzmán-Bracho C. Epidemiology of Chagas disease in Mexico: an update. Trends Parasitol. 2001;17(8):372–376.
  23. Torrico F, Alonso-Vega C, Suarez E, et al. Maternal Trypanosoma cruzi infection, pregnancy outcome, morbidity, and mortality of congenitally infected and non-infected newborns in Bolivia. Am J Trop Med Hyg. 2004;70(2):201–209.
  24. Prata A. Clinical and epidemiological aspects of Chagas disease. Lancet Infect Dis. 2001;1(2):92–100.
  25. Rassi A, Rassi A, Little WC. Chagas' heart disease. Clin Cardiol. 2000;23(12):883–889.
  26. Dumonteil E. Update on Chagas' disease in Mexico. Salud Publica Mex. 1999;41(4):322–327.
  27. Anez N, Carrasco H, Parada H, et al. Acute Chagas' disease in western Venezuela: a clinical, seroparasitologic, and epidemiologic study. Am J Trop Med Hyg. 1999;60(2):215–222.
  28. Kirchhoff LV, Votava JR, Ochs DE, et al. Comparison of PCR and microscopic methods for detecting Trypanosoma cruzi. J Clin Microbiol. 1996;34(5):1171–1175.
  29. Storino R. Consenso de enfermedad de Chagas, Topico I: Enfermedad de Chagas con parasitemia evidente. Rev Arg Cardiol. 2002;70(1):15–39.
  30. Feilij H, Muller L, Gonzalez Cappa SM. Direct micromethod for diagnosis of acute and congenital Chagas' disease. J Clin Microbiol. 1983;18(2):327–330.
  31. Sartori AM, Neto JE, Nunes EV, et al. Trypanosoma cruzi parasitemia in chronic Chagas disease: comparison between human immunodeficiency virus (HIV)-positive and HIV-negative patients. J Infect Dis. 2002;186(6):872–875.
  32. Torrico MC, Solano M, Guzman JM, et al. Estimation of the parasitemia in Trypanosoma cruzi human infection: high parasitemias are associated with severe and fatal congenital Chagas disease. Rev Soc Bras Med Trop. 2005;38(Suppl 2):58–61.
  33. Chiurillo MA, Crisante G, Rojas A, et al. Detection of Trypanosoma cruzi and Trypanosoma rangeli infection by duplex PCR assay based on telomeric sequences. Clin Diagn Lab Immunol. 2003;10(5):775–779.
  34. Moser DR, Kirchhoff LV, Donelson JE. Detection of Trypanosoma cruzi by DNA amplification using the polymerase chain reaction. J Clin Microbiol. 1989;27(7):1477–1482.
  35. Sturm NR, Degrave W, Morel C, et al. 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(3):205-14.
  36. Saldaña A, Samudio F, Miranda A, et al. Predominance of Trypanosoma rangeli infection in children from a Chagas disease endemic area in the west-shore of the Panama canal. Mem Inst Oswaldo Cruz.2005;100(7):729–731.
  37. Coronado X, Zulantay I, Reyes E, et al. Comparison of Trypanosoma cruzi detection by PCR in blood and dejections of Triatoma infestans fed on patients with chronic Chagas disease. Acta tropica. 2006;98(3):314–317.
  38. Rassi A, Rassi A, Marcondes de Rezende J. American trypanosomiasis (Chagas disease). Infect Dis Clin North Am. 2012;26(2):275–291.
  39. Ponce C, Ponce E, Vinelli E, et al. Validation of a rapid and reliable test for diagnosis of chagas' disease by detection of Trypanosoma cruzi-specific antibodies in blood of donors and patients in Central America. J Clin Microbiol. 2005;43(10):5065–5068.
  40. Brasil PE, De Castro L, Hasslocher-Moreno AM, et al. ELISA versus PCR for diagnosis of chronic Chagas disease: systematic review and meta-analysis. BMC infect dis. 2010;10:337.
  41. Nakazawa M, Rosa DS, Pereira VR, et al. Excretory-secretory antigens of Trypanosoma cruzi are potentially useful for serodiagnosis of chronic Chagas' disease. Clin Diagn Lab Immunol. 2001;8(5):1024–1027.
  42. Souza RM, Amato Neto V. Discrepancies and consequences of indirect hemagglutination, indirect immunofluorescence and ELISA tests for the diagnosis of Chagas disease. Rev Inst Med Trop Sao Paulo. 2012;54(3):141–143.
  43. Barfield CA, Barney RS, Crudder CH, et al. A highly sensitive rapid diagnostic test for Chagas disease that utilizes a recombinant Trypanosoma cruzi antigen. IEEE Trans Biomed Eng. 2011;58(3):814–817.
  45. Frade AF, Luquetti AO, Prata A, et al. Western blotting method (TESAcruzi) as a supplemental test for confirming the presence of anti-Trypanosoma cruzi antibodies in finger prick blood samples from children aged 0-5 years in Brazil. Acta tropica. 2011;117(1):10–13.
  46. Riera C, Verges M, Iniesta L, et al. Identification of a Western blot pattern for the specific diagnosis of Trypanosoma cruzi infection in human sera. Am J Trop Med Hyg. 2012;86(3):412–416.
  47. Umezawa ES, Shikanai-Yasuda MA, Gruber A, et al. Trypanosoma cruzi defined antigens in the serological evaluation of an outbreak of acute Chagas disease in Brazil (Catole do Rocha, Paraiba). Mem Inst Oswaldo Cruz. 1996;91(1):87–93.
  48. Velasquez E, Reyes L, Thors C, et al. Autoantibodies give false positive reactions in the serodiagnosis of Trypanosoma cruzi infection. Trans R Soc Trop Med Hyg. 1993;87(1):35.
  49. Chiller TM, Samudio MA, Zoulek G. IgG antibody reactivity with Trypanosoma cruzi and Leishmania antigens in sera of patients with Chagas' disease and leishmaniasis. Am J Trop Med Hyg. 1990;43(6):650–656.
  50. Umezawa ES, Shikanai-Yasuda MA, Stolf AM. Changes in isotype composition and antigen recognition of anti-Trypanosoma cruzi antibodies from acute to chronic Chagas disease. J Clin Lab Anal. 1996;10(6):407–413.
  51. Affranchino JL, Ibanez CF, Luquetti AO, et al. Identification of a Trypanosoma cruzi antigen that is shed during the acute phase of Chagas' disease. Mol Biochem Parasitol. 1989;34(3):221–228.
  52. Jazin EE, Luquetti AO, Rassi A, et al. Shift of excretory-secretory immunogens of Trypanosoma cruzi during human Chagas' disease. Infect Immun. 1991;59(6):2189–2191.
  53. Schenkman S, Eichinger D, Pereira ME, et al. Structural and functional properties of Trypanosoma trans-sialidase. Annu Rev Microbiol. 1994;48:499–523.
  54. Amaya MF, Buschiazzo A, Nguyen T, et al. The high resolution structures of free and inhibitor-bound Trypanosoma rangeli sialidase and its comparison with T. cruzi trans-sialidase. Jmolbiol. 2003;325(4):773–84.
  55. Kashif M, Moreno-Herrera A, Lara-Ramirez EE, et al. Recent developments in trans-sialidase inhibitors of Trypanosoma cruzi. J Drug Target. 2017;25(6):485–498.
  56. Agusti R, Couto AS, Campetella OE, et al. The trans-sialidase of Trypanosoma cruzi is anchored by two different lipids. Glycobiology. 1997;7(6):731–735.
  57. Berrizbeitia M, Ndao M, Bubis J, Gottschalk M, et al. Purified Excreted-Secreted Antigens from Trypanosoma cruzi Trypomastigotes as Tools for Diagnosis of Chagas' Disease. J Clin Microbiol. 2006;44(2):291–296.
  58. Araujo AB, Berne ME. Conventional serological performance in diagnosis of Chagas' disease in southern Brazil. Braz J Infect Dis. 2013;17(2):174–178.
  59. Santos MA, Garg N, Tarleton RL. The identification and molecular characterization of Trypanosoma cruzi amastigote surface protein-1, a member of the trans-sialidase gene super-family. Mol Biochem Parasitol. 1997;86(1):1–11.
  60. Cross GA, Takle GB. The surface trans-sialidase family of Trypanosoma cruzi. Annu Rev Microbiol. 1993;47:385–411.
  61. Frasch AC. Functional diversity in the trans-sialidase and mucin families in Trypanosoma cruzi. Parasitol today. 2000;16(7):282–286.
  62. Garcia VS, Gonzalez VD, Marcipar IS, et al. Immunoagglutination test to diagnose Chagas disease: comparison of different latex-antigen complexes. Trop Med Int Health. 2014;19(11):1346–1354.
  63. Gonzalez VD, Garcia VS, Vega JR, et al. Immunodiagnosis of Chagas disease: Synthesis of three latex-protein complexes containing different antigens of Trypanosoma cruzi. Colloids Surf B Biointerfaces. 2010;77(1):12–17.
  64. da Silveira JF, Umezawa ES, Luquetti AO. Chagas disease: recombinant Trypanosoma cruzi antigens for serological diagnosis. Trends Parasitol. 2001;17(6):286–291.
  65. Saez-Alquezar A, Sabino EC, Salles N, et al. Serological confirmation of Chagas' disease by a recombinant and peptide antigen line immunoassay: INNO-LIA chagas. J clin microbiol. 2000;38(2):851–854.
  66. Chandler FW, Watts JC. Immunofluorescence as an adjunct to the histopathologic diagnosis of Chagas' disease. J clin microbiol. 1988;26(3):567–569.
  67. Lorca M, Thiermann E. Valor diagnostico de la inmunofluorescencia indirecta con anti-IgM para Enfermedad de Chagas, en adultos y recién nacidos. Revista chilena de pediatría. 1982;53(6):199–204.
  68. Lorca M, Veloso C, Munoz P, et al. Diagnostic value of detecting specific IgA and IgM with recombinant Trypanosoma cruzi antigens in congenital Chagas' disease. Am J Trop Med Hyg. 1995;52(6):512–515.
  69. Corral RS, Altcheh JM, Freilij HL. Presence of IgM antibodies to Trypanosoma cruzi urinary antigen in sera from patients with acute Chagas' disease. Int J Parasitol. 1998;28(4):589–594.
  70. Reyes MB, Lorca M, Munoz P, et al. Fetal IgG specificities against Trypanosoma cruzi antigens in infected newborns. Proceedings of the National Academy of Sciences of the United States of America. 1990;87(7):2846–2850.
  71. Alford CA. Immunoglobulin determinations in the diagnosis of fetal infection. Pediatr Clin North Am. 1971;18(1):99–113.
  72. Gebrekristos HT, Buekens P. Mother-to-Child Transmission of Trypanosoma cruzi. J Pediatric Infect Dis Soc. 2014;3(Suppl 1):S36–40.
  73. WHO. Consultation on International Biological Reference - Preparations for Chagas Diagnostic Tests. Geneva, Switzerland: WHO; 2007.
  74. Gomes YM, Lorena VM, Luquetti AO. Diagnosis of Chagas disease: what has been achieved? What remains to be done with regard to diagnosis and follow up studies? Memorias do Instituto Oswaldo Cruz. 2009;104(Suppl 1):115–121.
  75. Wincker P, Bosseno MF, Britto C, 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(3):419–423.
  76. Mallimaci MC, Sosa-Estani S, Russomando G, et al. Early diagnosis of congenital Trypanosoma cruzi infection, using shed acute phase antigen, in Ushuaia, Tierra del Fuego, Argentina. Am J Trop Med Hyg. 2010;82(1):55–59.
  77. Seiringer P, Pritsch M, Flores-Chavez M, et al. Comparison of four PCR methods for efficient detection of Trypanosoma cruzi in routine diagnostics. Diagn Microbiol Infect Dis. 2017;88(3):225–232.
  78. Schijman AG, Bisio M, Orellana L, et al. International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients. PLoS Negl Trop Dis. 2011;5(1):e931.
  79. Qvarnstrom Y, Schijman AG, Veron V, et al. Sensitive and specific detection of Trypanosoma cruzi DNA in clinical specimens using a multi-target real-time PCR approach. PLoS Negl Trop Dis. 2012;6(7):e1689.
  80. Munoz-San Martin C, Apt W, Zulantay I. Real-time PCR strategy for the identification of Trypanosoma cruzi discrete typing units directly in chronically infected human blood. Infect Genet Evol. 2017;49:300–308.
  81. Avila HA, Pereira JB, Thiemann O, 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. Jclin microbiol. 1993;31(9):2421–2426.
  82. Wincker P, Britto C, Pereira JB, et al. 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(6):771–777.
  83. Marcon GEB, Andrade PD, de Albuquerque DM, 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(1):39–43.
  84. Lane JE, Ribeiro-Rodrigues R, Olivares-Villagomez D, et al. Detection of Trypanosoma cruzi DNA within murine cardiac tissue sections by in situ polymerase chain reaction. Mem Inst Oswaldo Cruz. 2003;98(3):373–376.
  85. Caldas S, Caldas IS, Diniz Lde F, et al. Real-time PCR strategy for parasite quantification in blood and tissue samples of experimental Trypanosoma cruzi infection. Acta trop. 2012;123(3):170–177.
  86. Abras A, Gallego M, Munoz C, et al. Identification of Trypanosoma cruzi Discrete Typing Units (DTUs) in Latin-American migrants in Barcelona (Spain). Parasitology international. 2017;66(2):83–88.
  87. Zingales B, Miles MA, Campbell DA, et al. The revised Trypanosoma cruzi subspecific nomenclature: rationale, epidemiological relevance and research applications. Infect Genet Evol. 2012;12(2):240–253.
  88. Gilber SR, Alban SM, Gobor L, et al. Comparison of conventional serology and PCR methods for the routine diagnosis of Trypanosoma cruzi infection. Rev Soc Bras Med Trop. 2013;46(3):310–315.
  89. Junqueira ACV, Chiari E, Whicker 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. 90(2):129–132.
  90. Ministerio da Saude. Secretaria de Vigilancia em S. Brazilian Consensus on Chagas disease. Rev Soc Bras Med Trop. 2005;38(Suppl 3):7–29.
  91. Bhattacharyya T, Brooks J, Yeo M, et al. Analysis of molecular diversity of the Trypanosoma cruzi trypomastigote small surface antigen reveals novel epitopes, evidence of positive selection and potential implications for lineage-specific serology. Int J Parasitol. 2010;40(8):921–928.
  92. Risso MG, Garbarino GB, Mocetti E, et al. Differential expression of a virulence factor, the trans-sialidase, by the main Trypanosoma cruzi phylogenetic lineages. J Infect Dis. 2004;189(12):2250–2259.
  93. Di Noia JM, Buscaglia CA, De Marchi CR, et al. A Trypanosoma cruzi small surface molecule provides the first immunological evidence that Chagas' disease is due to a single parasite lineage. J Exp Med. 2002;195(4):401–413.
  94. De Marchi CR, Di Noia JM, Frasch AC, et al. Evaluation of a recombinant Trypanosoma cruzi mucin-like antigen for serodiagnosis of Chagas' disease. Clin Vaccine Immunol. 2011;18(11):1850–1855.
  95. Mendes TAdO, Reis Cunha JL, de Almeida Lourdes R, et al. Identification of Strain-Specific B-cell Epitopes in Trypanosoma cruzi Using Genome-Scale Epitope Prediction and High-Throughput Immunoscreening with Peptide Arrays. PLoS Negl Trop Dis. 2013;7(10):e2524.
  96. Breniere SF, Bosseno MF, Noireau F, et al. Integrate study of a Bolivian population infected by Trypanosoma cruzi, the agent of Chagas disease. Mem Inst Oswaldo Cruz. 2002;97(3):289–295.
  97. Breniere SF, Yaksic N, Telleria J, et al. Immune response to Trypanosoma cruzi shed acute phase antigen in children from an endemic area for Chagas' disease in Bolivia. Mem Inst Oswaldo Cruz. 1997;92(4):503–507.
  98. Buchovsky AS, Campetella O, Russomando G, et al. Trans-sialidase inhibition assay, a highly sensitive and specific diagnostic test for Chagas' disease. Clin Diagn Lab Immunol. 2001;8(1):187–189.
  99. Burns JM, Shreffler WG, Rosman DE, et al. Identification and synthesis of a major conserved antigenic epitope of Trypanosoma cruzi. Proc Natl Acad Sci U S A. 1992;89(4):1239–1243.
  100. Cetron MS, Hoff R, Kahn S, et al. Evaluation of recombinant trypomastigote surface antigens of Trypanosoma cruzi in screening sera from a population in rural northeastern Brazil endemic for Chagas' disease. Acta trop. 1992;50(3):259–266.
  101. Cotrim PC, Paranhos-Baccala G, Santos MR, et al. Organization and expression of the gene encoding an immunodominant repetitive antigen associated to the cytoskeleton of Trypanosoma cruzi. Mol Biochem Parasitol. 1995;71(1):89–98.
  102. Frasch AC, Reyes MB. Diagnosis of Chagas disease using recombinant DNA technology. Parasitol today. 1990;6(4):137–139.
  103. Gruber A, Zingales B. Trypanosoma cruzi: characterization of two recombinant antigens with potential application in the diagnosis of Chagas' disease. Exp Parasitol. 1993;76(1):1–12.
  104. Hoft DF, Kim KS, Otsu K, et al. Trypanosoma cruzi expresses diverse repetitive protein antigens. Infect Immun. 1989;57(7):1959–1967.
  105. Houghton RL, Benson DR, Reynolds LD, et al. 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. 1999;179(5):1226–1234.
  106. Ibanez CF, Affranchino JL, Macina RA, et al. Multiple Trypanosoma cruzi antigens containing tandemly repeated amino acid sequence motifs. Mol Biochem Parasitol. 1988;30(1):27–33.
  107. Kerner N, Liegeard P, Levin MJ, Hontebeyrie-Joskowicz M. Trypanosoma cruzi: antibodies to a MAP-like protein in chronic Chagas' disease cross-react with mammalian cytoskeleton. Exp Parasitol. 1991;73(4):451–459.
  108. Lafaille JJ, Linss J, Krieger MA, et al. Structure and expression of two Trypanosoma cruzi genes encoding antigenic proteins bearing repetitive epitopes. Mol Biochem Parasitol. 1989;35(2):127–136.
  109. Levin MJ, Mesri E, Benarous R, et al. Identification of major Trypanosoma cruzi antigenic determinants in chronic Chagas' heart disease. Am J Trop Med Hyg. 1989;41(5):530–538.
  110. Peralta JM, Teixeira MG, Shreffler WG, et al. Serodiagnosis of Chagas' disease by enzyme-linked immunosorbent assay using two synthetic peptides as antigens. J Clin Microbiol. 1994;32(4):971–974.
  111. Umezawa ES, Bastos SF, Camargo ME, et al. Evaluation of recombinant antigens for serodiagnosis of Chagas' disease in South and Central America. J clin microbiol. 1999;37(5):1554–1560.
  112. Umezawa ES, Nascimento MS, Kesper N, et al. Immunoblot assay using excreted-secreted antigens of Trypanosoma cruzi in serodiagnosis of congenital, acute, and chronic Chagas' disease. J clin microbiol. 1996;34(9):2143–2147.
  113. Aznar C, Lopez-Bergami P, Brandariz S, et al. Prevalence of anti-R-13 antibodies in human Trypanosoma cruzi infection. FEMS Immunol Med Microbiol. 1995;12(3-4):231–238.
  114. Oliveira LG, Kuehn CC, dos Santos CD, et al. Protective actions of melatonin against heart damage during chronic Chagas disease. Acta tropica. 2013;128(3):652–8.
  115. Verissimo da Costa GC, Lery LM, da Silva ML, et al. The identification and characterization of epitopes in the 30-34 kDa Trypanosoma cruzi proteins recognized by antibodies in the serum samples of chagasic patients. J Proteomics. 2013;80:34–42.
  116. Carmona SJ, Nielsen M, Schafer-Nielsen C, et al. Towards High-throughput Immunomics for Infectious Diseases: Use of Next-generation Peptide Microarrays for Rapid Discovery and Mapping of Antigenic Determinants. Mol Cell Proteomics. 2015;14(7):1871–184.
Creative Commons Attribution License

©2018 Rodea, et al. This is an open access article distributed under the terms of the, which permits unrestricted use, distribution, and build upon your work non-commercially.