Quantification of parasite burden of Trypanosoma cruzi and identification of Discrete Typing Units (DTUs) in blood samples of Latin American immigrants residing in Barcelona, Spain

Abstract

Background

Trypanosoma cruzi has a high genetic and biological diversity and has been subdivided into seven genetic lineages, named TcI-TcVI and TcBat. DTUs TcI-TcII-TcV and TcVI are agents of ChD in different regions of Latin America. Due to population movements, the disease is an emergent global public health problem. Thus, the aim of this study was to quantify the parasitic load and identify the presence of T. cruzi DTUs in 101 Latin American immigrants with chronic ChD, residing in Barcelona, Spain.

Methodology / Principal findings

5ml of peripheral blood were collected in guanidine/EDTA from each patient for DNA extraction, quantification of the parasitic load and genotyping. A great variation of the parasitic load of the patients was verified: from 0.001 to 22.2 T. cruzi DNA (fg) / Blood DNA (ng). In patients from Bolivia the parasitic load was 3.76±4.43 T. cruzi DNA (fg) / Blood DNA (ng) (mean ± SD), in patients of other countries was 0.95±1.38 T. cruzi DNA (fg) / Blood DNA (ng). No statistically significant difference was observed in the parasitic load between patients with the indeterminate and cardiac forms of ChD (p = 0,57). Parasite genotyping was performed by multilocus conventional PCR. In patients from Bolivia there was a nearly equal prevalence of DTUs TcV (27/77), TcII/TcV/TcVI (26/77), and TcII/TcVI (22/77). TcVI was detected in only 2 samples (2/77). A higher prevalence of TcII/TcVI (19/24) was verified in patients of other countries, with low prevalence of TcII/TcV/TcVI (4/24) and TcV (1/24).

Conclusions/Significance

In this study, low/medium parasitic load was found in all patients evaluated. Our data corroborate previous conclusions indicating that patients from the Bolivia, living in Spain, are predominantly infected by TcV, and TcVI DTUs. On the other hand, in Non-Bolivians patients TcII/TcVI predominated. Surprisingly, in our cohort of 101 patients no infection by TcI DTU was observed.

About the Authors:

Maykon Tavares de Oliveira

Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Resources, Validation, Visualization, Writing – original draft, Writing – review & editing

Affiliations Department of Infectious Diseases, Universitat Autònoma de Barcelona, Vall d’Hebron University Hospital. PROSICS, Barcelona. Spain, Department of Internal Medicine, Cardiology Division, Medical School of Ribeirão Preto, University of São Paulo (FMRP-USP), Ribeirão Preto, SP, Brazil

ORCID logo http://orcid.org/0000-0002-7652-3225

Elena Sulleiro

Roles Conceptualization, Data curation, Methodology, Resources, Visualization

Affiliation: Department of Microbiology, Vall d’Hebron University Hospital. Universitat Autònoma de Barcelona. PROSICS Barcelona. Spain

Aroa Silgado Gimenez

Roles Conceptualization, Methodology, Visualization

Affiliation: Department of Microbiology, Vall d’Hebron University Hospital. Universitat Autònoma de Barcelona. PROSICS Barcelona. Spain

ORCID logo http://orcid.org/0000-0001-7581-0049

Marta de Lana

Roles Investigation, Resources, Visualization, Writing – review & editing

Affiliation: School of Pharmacy and Center for Research in Biological Sciences (NUPEB), Federal University of Ouro Preto (UFOP), Ouro Preto, MG, Brazil

Bianca Zingales

Roles Investigation, Resources, Visualization, Writing – review & editing

Affiliation: Department of Biochemistry, Institute of Chemistry, University of São Paulo (USP), São Paulo, SP, Brazil

ORCID logo http://orcid.org/0000-0001-5490-007X

João Santana da Silva

Roles Investigation, Writing – review & editing

Affiliation: Department of Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo (FMRP-USP), Ribeirão Preto, SP, Brazil.

ORCID logo http://orcid.org/0000-0002-3410-3927

J. Antônio Marin-Neto

Roles Funding acquisition, Project administration, Supervision, Visualization, Writing – review & editing

¶‡ These authors are joint last authors on this work.

Affiliation: Department of Internal Medicine, Cardiology Division, Medical School of Ribeirão Preto, University of São Paulo (FMRP-USP), Ribeirão Preto, SP, Brazil

Israel Molina

Roles Conceptualization, Data curation, Project administration, Resources, Supervision, Visualization, Writing – review & editing

* E-mail: [email protected]

¶‡ These authors are joint last authors on this work.

Affiliation: Department of Infectious Diseases, Universitat Autònoma de Barcelona, Vall d’Hebron University Hospital. PROSICS, Barcelona. Spain

ORCID logo http://orcid.org/0000-0001-6642-7515

Introduction

Chagas disease (ChD) is caused by the hemoflagellate protozoan, Trypanosoma cruzi [1]. Approximately 60–70% of the chronic patients have no clinical symptoms (indeterminate form), whereas 30–40% either have or will develop cardiomyopathy, digestive megasyndromes or both [2]. According to the World Health Organization [3], 6–7 million people are chronically infected with T. cruzi worldwide, and more than 90 million individuals are at risk of infection. T. cruzi is genetically highly diverse and, at present, it has been subdivided into seven genetic lineages or discrete typing units (DTUs), named TcI to TcVI and TcBat [4,5]. T. cruzi DTUs have distinct, but not exclusive ecological and epidemiological associations [6]. With regard to ChD, DTU TcI is a major human infection agent in Amazonia, the Andean Region, Central America and Mexico, whereas DTUs TcII, TcV and TcVI are prevalent in patients in the Southern Cone region of South America [6–9].

In recent decades, the population movements from endemic to non-endemic countries have started to create notable changes in the epidemiology of ChD, as T. cruzi has spread worldwide [10,11]. The prevalence of ChD infection in Latin American immigrants living in Europe is estimated as 4.2%, with the highest prevalence among individuals from Bolivia (18.1%) and Paraguay (5.5%) [12].

Although direct vector transmission cannot occur in the European continent, infected blood transfusion, vertical transmission from mother to fetus and organ transplantation can provide parasite spreading in non-endemic countries [12]. Measures to control vertical transmission have been designed and implemented in some countries in Europe. However, these measures have not been effective [13].

Assessing the T. cruzi burden in immigrants from Latin America living in non-endemic countries has important implications for the implementation of medical care, monitoring of vertical transmission, introduction of additional controls for blood banks, training of personnel to diagnose and treat ChD, among others. In this direction, the present investigation aims at evaluating the parasitic load and the genotype of the infecting agent in immigrants from Latin America residing in Barcelona, Spain.

Materials and methods

Study population

This study included 101 ChD patients who were followed up by the clinical group of Infectious Diseases at Vall d'Hebron University Hospital, Barcelona, Spain, in the period 2015–2019. The patients had two positive serological tests for ChD, according to [3] and positive real-time PCR for T. cruzi. Patients were subjected to clinical evaluation consisting of anamnesis, ECG, resting transthoracic echocardiography, chest, esophageal and colon X-ray examination. The patients were classified into different clinical forms of chronic ChD, according to the [14]. Peripheral blood samples (5 mL) were collected and mixed with an equal volume of 6 M Guanidine Hydrochloride / 0.2 M ethylenediaminetetraacetic acid buffer (EDTA) solution, pH 8.0. The Guanidine-EDTA Blood lysates (GEB) were boiled for 15 minutes, incubated at room temperature for 24 h, and stored at 4˚C until use [15].

Ethical clearance

The study was approved by the Human Research Ethics Committee of the Vall d'Hebron University Hospital. All patients provided written informed consent.

DNA extraction

DNA was extracted from 200 μL of GEB samples and eluted with 55 μL of NucliSens easyMAG system (Biomerieux, France), according to the manufacturer's instructions.

Parasitic load quantification by qPCR

The quantitative real-time PCR (qPCR) was performed according to a methodology previously proposed [16], using the multiplex TaqMan system targeting the 166 bp region of T. cruzi satellite DNA. The qPCR reactions were carried out at 25 μL final volume containing 5 μL DNA from each sample (20 ng/μL), 400 nM of the two primers and 100 nM of the TaqMan probe. The Quantitec Multiplex PCR kit (Qiagen, Manchester, United Kingdom) was used and the CFX Real-Time PCR detection system (Bio-Rad, Hercules, CA) used for amplification. The standard curve of the qPCR results was obtained using serial dilutions of 100 ng of DNA extracted from epimastigotes of the strain SO3 cl5 (DTU TcV), with a detection limit of 0.0001 fg, as proposed by [17] and modified by [18]. Positive, negative and reagent internal controls were used in all qPCR reactions.

Genotyping of Trypanosoma cruzi

Genotyping of T. cruzi in six DTUs (TcI-TcVI) was performed based on multilocus conventional PCR in association with Nested PCR, as described by [15] and modified by [19]. The subsequent identification of genotypes was based on the analysis of the set of profiles of the amplified PCR products presented for each gene target, using the following molecular markers: (i) the intergenic region of the Spliced Leader gene (SL-IRac) using the UTCC and TCac primers; (ii) the intergenic region of Spliced Leader (SL-IR) using TCC, TC1 and TC2 primers; (iii) the variable D7 domain of the 24Sα rRNA gene, with D75, D76 and D71 primers in semi-nested PCR; (iv) the A10 nuclear fragment in semi-nested PCR, with primers Pr1, P6 and Pr3. The PCR systems, gene targets and expected sizes of the amplified products are indicated in Fig 1. In all PCR reactions, DNA control samples from reference strains belonging to the six DTUs and Tcbat were used (Colombiana—TcI; Y—TcII; X109/2—TcIII; CanIII cl1—TcIV; Bug2148 cl1- TcV; CL Brener–TcVI and Tcbat 1994—Tcbat), as well as the negative controls and reagents. All amplification reactions were prepared in a final volume of 30 μL, using 12.5 μL of Mastermix Go Taq Green 2X (Promega, Madison, USA), 5 μL T. cruzi extracted DNA, and primers. The PCR cycling conditions were as described [15], using the Thermocycler (G-Storm, model GS 0001). The PCR products were separated by agarose gel electrophoresis (2% or 3% w/v), stained with Syber (Midori Green Advanced DNA Strain, Nippon Genetics Europe Gmbh) and viewed on Biorad photo documentation platform (Molecular Imager, Gel DOC XR, Imaging System). Molecular weight markers of 100 bp (Fast Gene Genetics, MWD100) were used to estimate the product size.

[Figure omitted. See PDF.]

Fig 1. Multilocus conventional PCR for Trypanosoma cruzi genotyping in clinical samples [19].

The flowchart indicates the gene targets and the sizes of the PCR products expected for the DTUs.

https://doi.org/10.1371/journal.pntd.0008311.g001

Statistical analysis

All experiments were performed at least in two technical replicates. Categorical data were expressed as percentages, and continuous data as mean ± standard deviation (SD), or mean interval, according to the normality or nonparametric characteristic of the distribution. Student's t-test was used to analyze the significance of statistical differences. Results were deemed as statistically significant when p values were less than 0.05. Analysis was conducted using GraphPad Prism version 7.00 for Windows, GraphPad Software, La Jolla California USA, www.graphpad.com.

Results

Characteristics of the patients included in this study

This study included 101 patients with chronic ChD, not treated with Benzonidazole or Nifurtimox. All diagnosed by two positive serological tests and positive qPCR for T. cruzi, who were followed up at the Infectious Disease Clinic of the Vall d'Hebron University Hospital, Barcelona, in the period between 2015 and 2019. All patients reside in Barcelona and are immigrants from different countries of Latin America: Argentina (~ 8%), Bolivia (~ 77%), Brazil (~ 1%), Ecuador (~ 2%), Honduras (~ 1%), Paraguay (~ 4%), Uruguay (~ 5%) and Venezuela (~ 1%). Two patients were born in Spain, sons of Bolivian immigrants (Table 1). From the patients, 34 (33.7%) were male and 67 (66.3%) females (Table 1). The mean age was 48.2 years (24–80) (Table 1). The indeterminate form of ChD was diagnosed in 53 patients (52.5%) and 48 individuals (47.5%) presented the cardiac form. No patients with the digestive, nervous or mixed clinical forms of ChD were represented in our study population.

[Figure omitted. See PDF.]

Table 1. General information of patients involved in the study and criteria for genotyping of Trypanosoma cruzi.

https://doi.org/10.1371/journal.pntd.0008311.t001

Parasitic load

We observed a great variation of the parasitic load in the blood of the 101 patients: from 0.001 to 22.2 T. cruzi DNA (fg) / Blood DNA (ng). Regarding the country of origin, the mean ± SD of the parasitic load was 3.76 ± 4.43 T. cruzi DNA (fg) / Blood DNA (ng) in the Bolivian group and 0.95 ± 1.38 T. cruzi DNA (fg) / Blood DNA (ng) in the non-Bolivian group (Fig 2A). The data were statistically significant with a p value of 0.00029.

[Figure omitted. See PDF.]

Fig 2.

Parasitic load of Latin American immigrants with chronic ChD residing in Barcelona. Distribution according to (A) the country of origin; (B) the indeterminate and cardiac forms. The horizontal lines represent the mean values of the parasite load.

https://doi.org/10.1371/journal.pntd.0008311.g002

No statistically significant difference was observed in the parasitic load between patients with the indeterminate and cardiac forms of ChD (Fig 2B).

Trypanosoma cruzi genotyping

In all samples we applied the multilocus conventional PCR to perform the genotyping of the infecting DTUs. However, in samples which had a very low parasitic load we could not obtain amplified products of all the genes necessary for the molecular characterization of T. cruzi (Fig 3; Tables 1 and 2). In 27 DNA samples from Bolivian patients (27/77) the products confirmed DTU TcV infection, whereas in two Bolivian patients DTU TcVI was found. 22 Bolivian patients (22/77) had a genetic profile indicating infection by TcII/TcVI DTUs. In the remaining 26 patients from Bolivia (26/77) the amplified products suggested infection with TcII/TcV/TcVI DTUs.

[Figure omitted. See PDF.]

Fig 3. Representative gels of amplified gene products to define Trypanosoma cruzi DTUs.

Genes: (A) the SL-IRac; (B) the SLIR I and II; (C) the 24Sα rDNA; (D) the A10. (MW—Molecular Weight marker; NC—Negative control; Positive controls, amplified products of reference strains: TcI, Colombiana; TcII, Y; TcIII, X109/2; TcIV, CANIII cl1; TcV, Bug2148 cl1; TcVI, CL Brener. The numbers indicate the code of the sample (see Table 1). Patient’s sample and infecting DTU: 1, TcV; 19, TcVI; 56, TcII/TcVI; 90, TcII/TcVI; 2, TcII/TcVI; 26, TcII/TcV/TcVI; 82, TcII/TcV/TcVI.

https://doi.org/10.1371/journal.pntd.0008311.g003

[Figure omitted. See PDF.]

Table 2. Genotyping of Trypanosoma cruzi from peripheral blood of chronic chagasic immigrant patients.

https://doi.org/10.1371/journal.pntd.0008311.t002

The non-Bolivian patients (24/101) were infected by DTUs TcV (Argentina), TcII/TcVI (Argentina, Ecuador, Honduras, Paraguay, Spain and Venezuela) and TcII/TcV/TcVI (Argentina, Brazil, Uruguay and Paraguay) (Tables 1 and 2).

Discussion

T. cruzi is composed of heterogeneous subpopulations that circulate in both domestic and wild cycles [20], and this diversity can be observed at the morphological [1,21], biological [22], antigenic [23] and at a genetic level [24,25]. Moreover, the parasite species are currently subdivided into seven distinct genetic groups (DTUs TcI–TcVI), and the Tcbat [4], with the additional fact that each DTU has its own characteristics [5]. In order to better understand the disease in each geographical region, it is important to study the molecular epidemiology of this parasite, which is naturally related to the main biological characteristics that have already been mentioned.

Currently immigration from Latin American countries to Europe has increased, especially in southern European countries such as Spain and Italy [26]. Since a considerable proportion of Latin American immigrants may be infected with T. cruzi, the epidemiology of ChD, originally endemic in Latin America, has changed considerably [10,27]. As a consequence, the number of reported cases of ChD with or without cardiac involvement has increased dramatically in recent years, especially in Spain, Italy and Switzerland [28,29].

In the present study we evaluated the parasitic load in the peripheral blood of 101 individuals serologically and real-time PCR positive for ChD, residing in Barcelona, Spain. Most of the patients were from Bolivia (77%). Patients from countries of the Southern Cone (Argentina, Brazil, Paraguay and Uruguay), northern South America (Ecuador and Venezuela) and Central America (Honduras) were also included. A wide variation of the parasitic load was observed among the patients and in most of them parasitemia was low / medium. Interestingly, the mean ± SD of the parasitic load of the Bolivian group (3.76 ± 4.43 T. cruzi DNA (fg) / Blood DNA (ng)) was higher than that of the group of patients from other countries (0.95 ± 1.38 T. cruzi DNA (fg) / Blood DNA (ng)).

In support to our conclusions, several studies employing quantitative real-time PCR (qPCR) have reported low / medium parasitic load values in chronic ChD patients of different countries of Latin America [30–33].

To investigate the impact of transfusion-acquired T. cruzi infection, [34] investigated blood donors who originated from Chagas-endemic areas and resided in the Mallorca Islands (Spain). Seropositivity for ChD was found in 23 (1.9%) of 1,201 donors and T. cruzi DNA with less than 1 parasite equivalent / mL was detected in the peripheral blood of 60.86% (14 of 23). Of the 14 patients in which circulating T. cruzi DNA was detected, 10 were from Bolivia, 3 from Argentina and 1 from Venezuela.

Higher parasitic load ranging from 1.43–11.14 parasite equivalents/mL (median 2.54) was reported in 65 chronic ChD patients from different regions of Brazil [15]. In a study similar to ours, the authors characterized the infectious DTU in 28 patients. They verified the prevalence of TcVI, TcII and mixed infection TcVI + TcII. When T. cruzi genotypes were compared with the parasite load, more elevated parasite loads were observed in patients infected by TcII (median of 7.56 par. Eq./mL) in comparison to patients infected by TcVI (median of 2.35 par. Eq./mL) [15].

In the present study we observed that patients from Bolivia (~77%) showed nearly equal prevalence of infections by TcV, TcII/TcVI and TcII/TcV/TcVI genotypes. In contrast, TcII/TcVI prevailed in patients from Argentina (~8%), Paraguay (~ 4%) and Uruguay (~ 5%). Of note, TcIII and TcIV were not identified in any patient, nor was TcI.

A previous study [35] also characterized the infecting DTU in peripheral blood samples of 10 migrants from Bolivia who attended hospitals in the Barcelona area. In agreement with our observations, in five samples TcV was identified; in three samples a TcII/V/VI profile was obtained and in the remaining two samples mixed infections TcV plus TcII/VI and TcV plus TcII was reported.

The DTUs infecting Latin American migrants attending a reference Clinic in Madrid was also defined [36]. As in our cohort, patients from Bolivia predominated (~90%). Overall, the most common DTU found was TcV (55.2%), followed by TcIV (16.2%), TcII (9.5%) and TcI (3.8%).

The scenario of the distribution of T. cruzi DTUs in ChD patients in countries of North, Central and South America has been outlined [5], based on data of [9] who surveyed articles in which approximately 6,400 DTUs were classified according to their geographical origin and hosts.

Our data regarding DTUs infecting migrants from Latin American countries residing in Barcelona follows the pattern of the geographic distribution of DTUs in the countries of origin. Two aspects stand out: To the best of our knowledge, this is the first study to describe the presence of TcVI genotype in the European continent in Bolivian patients with chronic ChD. TcI DTU was not found in any sample analyzed.

TcVI is highly related to the domestic cycle of ChD in some regions of the Southern Cone [6]. It is involved in human infections in the Chaco region in Northern Argentina; in Chile [37,38], and Brazil, more specifically in an outbreak of oral transmission in Santa Catarina state [39] and in endemic disease area in Minas Gerais state [40].

TcI DTU has a wide geographical distribution. TcI isolates are prevalent in patients from North America (Mexico and the United States); countries of Central America and northern South America (Colombia and Venezuela). Human TcI are abundant in Chile and the Brazilian Amazonia [5]. The fact that we did not find TcI in our cohort most probably is due to the low representativeness of individuals from Honduras and Venezuela or to the low abundance of this DTU in the sample.

We attempted to look for a possible association between the genotype of the parasite and the clinical presentation of ChD in the chronic phase. But, as discussed previously [5] we found none. We also found no correlation between the level of the parasite load and the infecting DTU.

Thus, knowing the parasite load and genetic variability of T. cruzi in chronic immigrant patients may be crucial to understanding the public health implications of ChD in European countries. Enhancing this understanding can allow for appropriate conception and planning of more effective public health interventions to improve the health of immigrants and control vertical transmission, which is a serious problem in European today.

Conclusions

The data of this study corroborate previous reports indicating the prevalence of patients from Bolivia among the Latin American immigrants residing in Barcelona. We show differences in the infecting DTUs between Bolivian and non-Bolivian patients. This is the first study to describe the presence of TcVI genotype in European continent. Although the level of parasite burden is low / medium in the patients, it is higher in patients from Bolivia as compared with patients of other countries. The information generated in this study should impact planning of more effective public health interventions to improve the health of immigrants, control vertical transmission and treatment of ChD.

Acknowledgments

We thank Pilar Alcubilla for her technical support.

Citation: Tavares de Oliveira M, Sulleiro E, Silgado Gimenez A, de Lana M, Zingales B, Santana da Silva J, et al. (2020) Quantification of parasite burden of Trypanosoma cruzi and identification of Discrete Typing Units (DTUs) in blood samples of Latin American immigrants residing in Barcelona, Spain. PLoS Negl Trop Dis 14(6): e0008311. https://doi.org/10.1371/journal.pntd.0008311

References

1. Chagas C. Nova tripanozomiase humana. Estudos sobre a morfolojia e o ciclo evolutivo do Schizotrypanum cruzi n. gen., n. sp., ajente etilógico de nova entidade morbida do homem. Mem Inst Oswaldo Cruz. 1909; 159–218.

2. Marin-Neto JA, Rassi A Jr, Maciel BC, Simoes MV, Schmidt A 2010. Chagas heart disease. In Yusuf S, Cairns JA, Camm AJ, Fallen EL, Gersh BJ (eds.), Evidence-based cardiology, 3rd ed., BMJ Books, London, p. 823–841.

3. WHO—Chagas disease (American trypanosomiasis). Fact sheet N°340. Updated March 2018.

4. Zingales B, Andrade SG, Briones MRS, Campbell DA, Chiari E, Fernandes O, Guhl F, Lages-Silva E, Macedo AM, Machado CR, Miles MA, Romanha AJ, Sturm NR, Tibayrenc M, Schijman AG. A new consensus for Trypanosoma cruzi intraspecific nomenclature: second revision meeting recommends TcI to TcVI. Mem Inst Oswaldo Cruz. 2009; 104: 1051–1054. pmid:20027478

5. Zingales B. Trypanosoma cruzi genetic diversity: Something new for something known about Chagas disease manifestations, serodiagnosis and drug sensitivity. Acta Trop. 2018 Aug;184:38–52. Epub 2017 Sep 21. pmid:28941731

6. Zingales B, Miles AM, Campbell AD, Tibayrenc M, Macedo AM, Teixeira MMG, Schijman AG, Llewellyn SM, Lages-Silva E, Machado RC, Andrade SG, Sturm NR. The revised Trypanosoma cruzi subspecific nomenclature: Rationale, epidemiological relevance and research applications. Infect Genet Evol. 2012; 12:240–53. pmid:22226704

7. Miles MA, Llewellyn MS, Lewis MD, Yeo M, Baleela R, Fitzpatrick S, Gaunt MW, Mauricio IL 2009. The molecular epidemiology and phylogeography of Trypanosoma cruzi and parallel research on Leishmania: looking back and to the future. Parasitology 136: 1509–1528.

8. Guhl F, Ramírez JD 2011. Trypanosoma cruzi I diversity: towards the need of genetic subdivision? Acta Trop 119: 1–4.

9. Brenière S.F., Waleckx E., Barnabé C., 2016. Over six thousand Trypanosoma cruzi strains classified into discrete typing units (DTUs): attempt at an inventory. PLoS Negl. Trop. Dis. 10, e0004792.

10. Basile L, Jansa JM, Carlier Y, Salamanca DD, Angheben A (2011). Chagas disease in European countries: the challenge of a surveillance system. Euro Surveill 16.

11. Requena-Méndez A, Aldasoro E, De Lazzari E, Sicuri E, Brown M, Moore DA, Gascon J, Muñoz J. Prevalence of Chagas disease in Latin-American migrants living in Europe: a systematic review and meta-analysis. PLoS Negl Trop Dis. 2015 Feb 13;9(2): e0003540. eCollection 2015 Feb. pmid:25680190

12. Pérez-Molina JA, Molina I. Chagas disease. Lancet. 2018 Jan 6;391(10115):82–94. Epub 2017 Jun 30.

13. Munoz J, Coll O, Juncosa T, Verges M, Del Pino M, (2009) Prevalence and vertical transmission of Trypanosoma cruzi infection among pregnant Latin American women attending 2 maternity clinics in Barcelona, Spain. Clin Infect Dis 48: 1736–1740. pmid:19438393

14. Dias JC, Ramos AN Jr, Gontijo ED, Luquetti A, Shikanai-Yasuda MA, Coura JR, Torres RM, Melo JR, Almeida EA, Oliveira W Jr, Silveira AC, Rezende JM, Pinto FS, Ferreira AW, Rassi A, Fragata AA Filho, Sousa AS, Correia D Filho, Jansen AM, Andrade GM, Britto CF, Pinto AY, Rassi A Jr, Campos DE, Abad-Franch F, Santos SE, Chiari E, Hasslocher-Moreno AM, Moreira EF, Marques DS, Silva EL, Marin-Neto JA, Galvão LM, Xavier SS, Valente SA, Carvalho NB, Cardoso AV, Silva RA, Costa VM, Vivaldini SM, Oliveira SM, Valente VD, Lima MM, Alves RV. (Brazilian Consensus on Chagas Disease, 2015). Epidemiol Serv Saude. 2016 Jun;25(spe):7–86. pmid:27869914

15. Rodrigues-Dos-Santos Í, Melo MF1, de Castro L, Hasslocher-Moreno AM, do Brasil PEAA, Silvestre de Sousa A, Britto C, Moreira OC. Exploring the parasite load and molecular diversity of Trypanosoma cruzi in patients with chronic Chagas disease from different regions of Brazil. PLoS Negl Trop Dis. 2018 Nov 12;12(11):e0006939. pmid:30418976

16. Piron M, Fisa R, Casamitjana N, López-Chejade P, Puig L, Vergés M, Gascón J, Gómez i Prat J, Portús M, Sauleda S. Development of a real-time PCR assay for Trypanosoma cruzi detection in blood samples. Acta Trop. 2007 Sep;103(3):195–200. Epub 2007 Jun 23.

17. Cummings KL, Tarleton RL. Rapid quantitation of Trypanosoma cruzi in host tissue by real-time PCR. Mol Biochem Parasitol. 2003 Jun;129(1):53–9.

18. Silva MC, Davoli-Ferreira M, Medina TS, Sesti-Costa R, Silva GK, Lopes CD, Cardozo LE, Gava FN, Lyroni K, Dias FC, Frade AF, Baron M, Nakaya HI, Figueiredo F, Alves-Filho JC, Cunha FQ, Tsatsanis C, Chevillard C, Cunha-Neto E, Hirsch E, Silva JS, Cunha TM.Canonical PI3Kγ signaling in myeloid cells restricts Trypanosoma cruzi infection and dampens chagasic myocarditis. Nat Commun. 2018 Apr 17;9(1):1513. pmid:29666415

19. da Cruz Moreira Otacilio; Ramirez Juan Carlos. Genotyping of Trypanosoma cruzi from Clinical Samples by Multilocus Conventional PCR. In: Karina Andrea Gómez; Carlos Andrés Buscaglia. (Org.). Methods in Molecular Biology. 1ed.: Springer New York, 2019, v. 1955, p. 227–238.

20. Tibayrenc M, Ward P, Moya A, Ayala FJ. Natural populations of Trypanosoma cruzi, the agent of Chagas disease, have a complex multiclonal structure. Proc NatAcad. Sci. 1986; 83:115–119.

21. Brener Z, Chiari E. (Morphological variations observed in different strains of Trypanosoma cruzi). Rev Inst Med Trop Sao Paulo. 1963 Sep-Oct;5:220–4.

22. Andrade SG. Caracterização de cepas de Trypanosoma cruzi isoladas no Recôncavo Baiano. Rev Pat Trop. 1974; 65–121.

23. Maguire JH, Hoff R, Sleigh AC, Mott KE, Ramos NB, Sherlock IA.An outbreak of Chagas' disease in southwestern Bahia, Brazil. Am J Trop Med Hyg. 1986 Sep;35(5):931–6.

24. Macedo AM, Machado CR, Oliveira RP, Pena SD. Trypanosoma cruzi: genetic structure of populations and relevance of genetic variability to the pathogenesis of Chagas disease. Mem Inst Oswaldo Cruz. 2004; 99:1–12. pmid:15057339

25. Tibayrenc M, Ayala FJ. The population genetics of Trypanosoma cruzi revisited in the light of the predominant clonal evolution model. Acta Trop. 2015 Nov;151:156–65. pmid:26188332

26. Rechel B, Mladovsky P, Ingleby D, Mackenbach JP, Mckee M (2013) Migration and health in an increasingly diverse Europe. Lancet 381: 1235–1245. pmid:23541058

27. Gascon J, Bern C, Pinazo MJ. Chagas disease in Spain, the United States and other non-endemic countries. Acta Trop. 2010 Jul-Aug;115(1–2):22–7. Epub 2009 Jul 29. pmid:19646412

28. Jackson Y, Gétaz L, Wolff H, Holst M, Mauris A, Tardin A, Sztajzel J, Besse V, Loutan L, Gaspoz JM, Jannin J, Albajar Vinas P, Luquetti A, Chappuis F. Prevalence, clinical staging and risk for blood-borne transmission of Chagas disease among Latin American migrants in Geneva, Switzerland. PLoS Negl Trop Dis. 2010 Feb 2;4(2):e592. pmid:20126397

29. Coura JR, Viñas PA. Chagas disease: a new worldwide challenge. Nature. 2010 Jun 24;465(7301):S6–7. pmid:20571554

30. Duffy T, Cura CI, Ramirez JC, Abate T, Cayo NM, Parrado R, Bello ZD, Velazquez E, Muñoz-Calderon A, Juiz NA, Basile J, Garcia L, Riarte A, Nasser JR, Ocampo SB, Yadon ZE, Torrico F, de Noya BA, Ribeiro I, Schijman AG. Analytical performance of a multiplex Real-Time PCR assay using TaqMan probes for quantification of Trypanosoma cruzi satellite DNA in blood samples. PLoS Negl Trop Dis. 2013;7(1): e2000. pmid:23350002

31. Moreira OC, Ramírez JD, Velázquez E, Melo MF, Lima-Ferreira C, Guhl F, Sosa-Estani S, Marin-Neto JA, Morillo CA, Britto C. Towards the establishment of a consensus real-time qPCR to monitor Trypanosoma cruzi parasitemia in patients with chronic Chagas disease cardiomyopathy: a substudy from the BENEFIT trial. Acta Trop. 2013 Jan;125(1):23–31. pmid:22982466

32. Hernández C, Cucunubá Z, Flórez C, Olivera M, Valencia C, Zambrano P, León C, Ramírez JD. Molecular Diagnosis of Chagas Disease in Colombia: Parasitic Loads and Discrete Typing Units in Patients from Acute and Chronic Phases. PLoS Negl Trop Dis. 2016 Sep 20;10(9):e0004997. pmid:27648938

33. D'Ávila DA, Galvão LMC, Sousa GR, Britto C, Moreira OC, Chiari E. Monitoring the parasite load in chronic Chagas disease patients: comparison between blood culture and quantitative real time PCR. PLoS One. 2018 Nov 29;13(11): e0208133. pmid:30496249

34. Cancino-Faure B, Fisa R, Riera C, Bula I, Girona-Llobera E, Jimenez-Marco T. Evidence of meaningful levels of Trypanosoma cruzi in platelet concentrates from seropositive blood donors. Transfusion. 2015 Jun;55(6):1249–55. Epub 2015 Feb 13. pmid:25683267

35. Abras A, Gállego M, Muñoz C, Juiz NA, Ramírez JC, Cura CI, Tebar S, Fernández-Arévalo A, Pinazo MJ, de la Torre L, Posada E, Navarro F, Espinal P, Ballart C, Portús M, Gascón J, Schijman AG. Identification of Trypanosoma cruzi Discrete Typing Units (DTU´s) in Latin-American migrants in Barcelona (Spain). Parasitol Int. 2017 Apr;66(2):83–88. Epub 2016 Dec 7. pmid:27940065

36. Martinez-Perez A, Poveda C, Ramírez JD, Norman F, Gironés N, Guhl F, Monge-Maillo B, Fresno M, López-Vélez R. Prevalence of Trypanosoma cruzi 's Discrete Typing Units in a cohort of Latin American migrants in Spain. Acta Trop. 2016 May;157:145–50. Epub 2016 Feb 2. pmid:26851167

37. Valadares HM, Pimenta JR, de Freitas JM, Duffy T, Bartholomeu DC, Oliveira Rde P, Chiari E, Moreira MC, Filho GB, Schijman AG, Franco GR, Machado CR, Pena SD, Macedo AM. Genetic profiling of Trypanosoma cruzi directly in infected tissues using nested PCR of polymorphic microsatellites. Int J Parasitol. 2007; 38:839–50. pmid:18154957

38. Burgos JM, Diez M, Vigliano C, Bisio M, Risso M, Duffy T, Cura C, Brusses B, Favaloro L, Leguizamon MS, Lucero RH, Laguens R, Levin MJ, Favaloro R, Schijman AG. Molecular identification of Trypanosoma cruzi discrete typing units in end-stage chronic Chagas heart disease and reactivation after heart transplantation. Clin Infect Dis. 2010; 51:485–95. pmid:20645859

39. Andrade SG, Campos RF, Steindel M, Guerreiro ML, Magalhães JB, Almeida MC, Reis JN, Santos VC, Valadares HM, Reis MG, Macedo AM. Biological, biochemical and molecular features of Trypanosoma cruzi strains isolated from patients infected through oral transmission during a 2005 outbreak in the state of Santa Catarina, Brazil: its correspondence with the new T. cruzi Taxonomy Consensus (2009). Mem Inst Oswaldo Cruz. 2011 Dec;106(8):948–56.

40. Oliveira MT, de Assis GF, Oliveira e Silva JC, Machado EM, da Silva GN, Veloso VM, Macedo AM, Martins HR, de Lana M. Trypanosoma cruzi Discret Typing Units (TcII and TcVI) in samples of patients from two municipalities of the Jequitinhonha Valley, MG, Brazil, using two molecular typing strategies. Parasit Vectors. 2015 Oct 31;8:568. pmid:26520576