ORIGINAL RESEARCH published: 20 May 2021 doi: 10.3389/fcvm.2021.665624 Intra-Discrete Typing Unit TcV Genetic Variability of Trypanosoma cruzi in Chronic Chagas’ Disease Bolivian Immigrant Patients in Barcelona, Spain Maykon Tavares de Oliveira 1,2*, Elena Sulleiro 3 , Maria Cláudia da Silva 4 , Aroa Silgado 3 , Marta de Lana 5 , João Santana da Silva 6 , Israel Molina 1† and J. Antônio Marin-Neto 2† Edited by: 1 Department of Infectious Diseases, Vall d’Hebron University Hospital, Universitat Autònoma de Barcelona, PROSICS Walderez Ornelas Dutra, Barcelona, Barcelona, Spain, 2 Cardiology Division, Department of Internal Medicine, Medical School of Ribeirão Preto, Federal University of Minas University of São Paulo, São Paulo, Brazil, 3 Department of Microbiology, Vall d’Hebron University Hospital, Universitat Gerais, Brazil Autònoma de Barcelona, PROSICS Barcelona, Barcelona, Spain, 4 Department of Biochemistry and Immunology, Ribeirão Reviewed by: Preto Medical School, University of São Paulo, São Paulo, Brazil, 5 Pharmacy School and Center of Research in Biological Luisa Magalhaes, Sciences, Federal University of Ouro Preto, Ouro Preto, Brazil, 6 Fiocruz-Bi-Institutional Translational Medicine Plataform, Universidade Federal de Minas Ribeirão Preto, Brazil Gerais, Brazil Lúcia Maria Da Cunha Galvão, Federal University of Rio Grande do Background: Trypanosoma cruzi has a high rate of biological and genetic variability, Norte, Brazil and its population structure is divided into seven distinct genetic groups (TcI-TcVI and *Correspondence: Tcbat). Due to immigration, Chagas disease (ChD), caused by T. cruzi, has become a Maykon Tavares de Oliveira serious global health problem including in Europe. Therefore, the aim of this study was to maykontavares@yahoo.com.br evaluate the existence of genetic variability within discrete typing unit (DTU) TcV of T. cruzi † These authors share last authorship in Bolivian patients with chronic ChD residing in Barcelona, Spain. Specialty section: Methods: The DNA was extracted from the peripheral blood of 27 patients infected with This article was submitted to T. cruzi DTU TcV and the fragments of the genetic material were amplificated through the General Cardiovascular Medicine, a section of the journal low stringency single primer-polymerase chain reaction (LSSP-PCR). The data generated Frontiers in Cardiovascular Medicine after amplification were submitted to bioinformatics analysis. Received: 08 February 2021 Results: Of the 27 patients evaluated in the study, 8/27 (29.6%) were male and Accepted: 20 April 2021 Published: 20 May 2021 19/27 (70.4%) female, 17/27 (62.9%) were previously classified with the indeterminate Citation: clinical form of Chagas disease and 10/27 (37.1%) with Chagas cardiomyopathy. Oliveira MT, Sulleiro E, Silva MCd, The LSSP-PCR detected 432 band fragments from 80 to 1,500 bp. The unweighted Silgado A, de Lana M, da Silva JS, Molina I and Marin-Neto JA (2021) pair-group method analysis and principal coordinated analysis data demonstrated the Intra-Discrete Typing Unit TcV Genetic existence of three distinct genetic groups with moderate-high rates of intraspecific Variability of Trypanosoma cruzi in genetic variability/diversity that had shared parasite’s alleles in patients with the Chronic Chagas’ Disease Bolivian Immigrant Patients in Barcelona, indeterminate and cardiomyopathy forms of ChD. Spain. Front. Cardiovasc. Med. 8:665624. Conclusions: This study demonstrated the existence of a moderate to high rate of doi: 10.3389/fcvm.2021.665624 intra-DTU TcV variability in T. cruzi. Certain alleles of the parasite were associated with Frontiers in Cardiovascular Medicine | www.frontiersin.org 1 May 2021 | Volume 8 | Article 665624 Oliveira et al. Intra-DTU TcV Genetic Variability the absence of clinical manifestations in patients harboring the indeterminate form of ChD. These results support the need to search for increasingly specific targets in the genome of T. cruzi to be correlated with its main biological properties and clinical features in patients with chronic ChD. Keywords: Chagas disease, Trypanosoma cruzi, DTU TcV, genetic variability, cardiac form INTRODUCTION was confirmed for all 27 patients via two positive serological tests and real-time PCR, according to the WHO recommendations. Chagas disease (ChD) is caused by the hemoflagellate protozoan All patients were clinically evaluated at Vall d’Hebron University Trypanosoma cruzi and chronic Chagas cardiomyopathy is the Hospital, Barcelona, Spain, between 2015 and 2019. From all most severe manifestation (1). According to recent data from the patients, 5 mL of peripheral blood were collected and mixed with World Health Organization (WHO), ∼6 to 7 million people are an equal volume of guanidine 6 M/EDTA 0.2 M pH 8 (13). The chronically infected with T. cruzi worldwide, and more than 75 Guanidine-EDTA Blood lysates (GEB) were boiled for 15 min, million individuals are at risk of infection (2). incubated at room temperature for 24 h, and stored at 4◦ C until The genetic structure of T. cruzi is currently divided into use (14). seven distinct genetic groups, also known as discrete typing units (DTUs), TcI–TcVI, and TcBat (3, 4). T. cruzi presents Extraction of DNA From Blood/Guanidine a high rate of biological and genetic variability (5) and these and EDTA Samples differences may be linked to the main biological parameters of DNA was extracted from 200 µL of guanidine/EDTA blood the different strains of T. cruzi, such as geographical distribution (GEB) samples and eluted in 55 µL using the NucliSens and human clinical manifestations of ChD. However, no previous easyMAG R system (Biomerieux, France), according to the studies have presented sufficient data to confirm effectively such manufacturer’s instructions. potential correlations (6). The area of spread of ChD is wide across the American Clinical Evaluation of Patients continent. One of the most notable changes in the epidemiology The 27 patients with positive serology and real-time PCR for of parasitic diseases in recent decades is the emergence of ChD, were clinically evaluated at the Infectious Diseases Division, ChD in European countries, and the associated risk of T. cruzi Vall d’Hebron University Hospital, Barcelona, Spain, through transmission outside endemic areas (7). Europe is currently anamnesis, 12-lead electrocardiogram, chest, esophageal, and hosting large immigrant populations, with recent data estimating colon X-rays, and rest transthoracic echocardiography. The that immigrant populations represented 8.7% of the total patients were classified as having different clinical forms of European population in 2010 (8). The prevalence of ChD chronic ChD (15, 16). infection in Latin American immigrants living in Europe is estimated in 4.2%, with the highest prevalence among migrants Intra-DTU TcV Genetic Variability from Bolivia (18.1%) and Paraguay (5.5%) (9). Population To assess the intraspecific genetic variability of the previously movement over recent years has led to an increased prevalence of genotyped T. cruzi DTU TcV (11) present in the peripheral ChD in these countries, primarily due to high numbers of Latin blood of patients with chronic ChD, low stringency single primer American immigrants chronically infected with T. cruzi (10). (LSSP-PCR) methodology (17) was performed. To obtain the Although direct vector transmission cannot occur in genetic signature of T. cruzi kDNA, the following steps were Europe, ChD can be transmitted in non-endemic countries via performed: (A) amplification of the 330 bp fragment specific blood transfusion and organs transplantation, or even vertical to T. cruzi kDNA (18). The amplified products were run on a transmission (9). Measures to control vertical transmission have 1.5% agarose gel (Sigma R ), stained with Syber (Midori Green been designed and implemented in some countries to avoid Advanced DNA Strain, Nippon Genetics Europe Gmbh) and spreading ChD in Europe, although these measures have not viewed on the Biorad photo documentation platform (Molecular been shown to be entirely viable (11). Imager, Gel DOC XR, Imaging System). The 330 bp fragments Assessing the real burden and implications for public health were removed from the agarose gel, heated to 100◦ C, and diluted of ChD in European countries is crucial. Therefore, this study in ultrapure water at a 1:10 dilution. (B) DNA of the diluted 330 aimed to evaluate the intraspecific genetic variability of DTU TcV bp band fragments was subjected to a new amplification cycle from T. cruzi in immigrant patients with chronic ChD residing using the LSSP-PCR technique with the S35G∗ primer (5′ -AAA in Barcelona, Spain, and to correlate the genetic differences TAA TGT ACG GGG GAG AT-3′ ). A volume of 1 µL of diluted intra-DTU with the correspondent clinical forms of ChD. DNA was added to the 10 µL of the reaction mixture containing 6.38 µL of sterile milli-Q water, 2.0 µL of sample buffer, 0.2 µL MATERIALS AND METHODS of each deoxynucleotide (dATP, dCTP, dGTP, and dTTP–Sigma, St. Louis, MO, USA), 0.1 µL of the S35G ∗ primer (450 µM), Patients and Blood Samples and 0.32 µL of Taq DNA polymerase (Go Taq-Promega) (18). Were evaluated twenty-seven patients with ChD infected by DTU Amplification occurred under the following conditions: an initial TcV previously genotyped by our group (12). Diagnosis of ChD DNA denaturation stage at 95◦ C for 5 min, annealing at 30◦ C Frontiers in Cardiovascular Medicine | www.frontiersin.org 2 May 2021 | Volume 8 | Article 665624 Oliveira et al. Intra-DTU TcV Genetic Variability for 1 min, and extension at 72◦ C for 1 min, followed by 40 TABLE 1 | General information of patients with Chagas disease involved in amplification cycles consisting of a denaturation step at 94◦ C for the study. 1 min, one cycle of annealing at 30◦ C for 1 min, followed by a final Sample code Age Gender Clinical form DTU extension step at 72◦ C for 10 min. The LSSP-PCR products were (years) separated in 6% polyacrylamide gel electrophoresis and revealed with NaOH and formaldehyde after silver staining (19). After 1 32 F Cardiac TcV photo documentation the differences in the patterns of band 2 27 F Indeterminate TcV profiles were analyzed via bioinformatics tests. 3 43 M Cardiac TcV 4 65 F Cardiac TcV Bioinformatics Analysis 5 68 F Cardiac TcV The LSSP-PCR profiles were used to build a presence/absence 6 39 M Indeterminate TcV matrix of each visualized band, allowing a similarity analysis to be 7 60 F Indeterminate TcV built using NTSYSpc software. The relationships between T. cruzi 8 41 M Indeterminate TcV strains were estimated using a dendrogram representative of 9 46 M Indeterminate TcV the LSSP-PCR data. These were constructed based on the 10 38 F Cardiac TcV coefficient of association (20) and the analysis of unweighted 11 56 F Indeterminate TcV peer groups [unweighted pair-group method analysis (UPGMA)] 12 67 F Indeterminate TcV using Mega 6.04 Beta software. To estimate Shannon’s diversity, 13 35 M Indeterminate TcV the cophenetic correlation coefficient, heterozygosity by locus 14 44 F Indeterminate TcV (He), and principal coordinated analysis (PCoA) were performed 15 56 F Indeterminate TcV using the GenAlEx 6.5 software. In this case, a genetic distance 16 28 M Indeterminate TcV matrix was built. This calculation of genetic distances took 17 27 F Indeterminate TcV place in pairs for binaries. Data followed the methods of Huff 18 39 F Indeterminate TcV and collaborators (21), wherein any comparison with the same 19 37 F Indeterminate TcV state generates a value of 0 (for both 0 vs. 0 comparisons and 20 56 F Indeterminate TcV comparisons 1 vs. 1), while any comparison of different states (0 21 60 F Cardiac TcV vs. 1 or 1 vs. 0) generates a value of 1. 22 53 F Cardiac TcV Ethical Approval 23 56 F Indeterminate TcV 24 44 M Cardiac TcV This study was approved by the Human Research Ethics Committee of the Vall d’Hebron University Hospital. All patients 25 62 M Cardiac TcV who agreed to participate in the study signed an informed 26 43 F Cardiac TcV consent form. 27 50 F Indeterminate TcV F, Female; M, Male; TcV, T. cruzi DTU; DTU, Discrete Typing Unit. RESULTS Patient Characteristics This study included 27 patients with chronic ChD, identified via positive serology (IgG anti-T. cruzi) and real-time PCR for ChD, who were clinically managed at the Infectious Disease Clinic of the Vall d’Hebron University Hospital, Barcelona, Spain between 2015 and 2019. All patients reside in Barcelona, Spain, and are immigrants from Bolivia, 8/27 (29.6%) being male and 19/27 (70.4%) female (Table 1). The mean age of the patients was 47.1 ± 12.44 years (Table 1). Taking into account clinical features, echocardiography (ECG) and radiological exams, 17/27 FIGURE 1 | Gel representative of the amplified products of the LSSP-PCR. (62.9%) of the patients, presented the indeterminate form of ChD MW–Molecular Weight: 1 kb; 1, 5, 2, 24, 20, 27, 13, and 14 patient samples. and 10/27 (37.1%) the cardiomyopathy form. All patients with Chagas cardiomyopathy’ were categorized in stage B1 of clinical evolution according to the European Society of Cardiology (13). No patient had the digestive, nervous, or mixed clinical forms samples from patients infected with TcV DTU. After the second of ChD. amplification cycle, a total of 432 fragments were detected, and the products amplified were evaluated. The sizes of the bands Intra DTU TcV Genetic Variability of varied between 80 and 1,500 bp (Figure 1). Sixteen profiles Trypanosoma cruzi of the amplified products of the LSSP-PCR were obtained The LSSP-PCR analysis showed that the reaction amplified and only one (6.25%) was shared among all samples. The the 330 pb fragment of the T. cruzi kDNA in all 27 DNA percentage of polymorphic loci was 93.84%, demonstrating the Frontiers in Cardiovascular Medicine | www.frontiersin.org 3 May 2021 | Volume 8 | Article 665624 Oliveira et al. Intra-DTU TcV Genetic Variability FIGURE 2 | Phenogram containing UPGMA (unweighted pair-group method analysis) of the 27 samples of T. cruzi belonging to DTU TcV. The numbers on the horizontal scale were obtained from the similarity coefficient of data. Different colors represent the clinical forms (indeterminate and cardiac) and gender of each patient (male or female). existence of a high rate of genetic variability among the samples. range 0 to ± 1 (Figure 3). When comparing DNA samples from The cophenetic correlation coefficient, which verifies that the patients with the Indeterminate clinical form of Chagas disease, dendrogram preserved the distances in pairs between the original it was observed that the color intensity in Figure 3 tends to unmodeled data points, obtained a value of 0.851, confirming the become darker, approaching the number 1 staining score. This high representation of the similarity matrices in the dendrogram. may indicate a high degree of similarity between these samples. The UPGMA dendrogram distinguished T. cruzi DTU TcV The analysis of the principal coordinated analysis (PCoA) samples into three distinct groups (Figure 2). Patients with the in the two axes demonstrated 80.30% (35.1 + 45.2%) of indeterminate form of ChD were clearly grouped in two groups the variability between the components (Figure 4) and, in (group 1 and 2) of the dendrogram, what showed that the total, three distinct groups were formed, as shown in the genomes of these protozoa share certain alleles that may indicate UPGMA dendrogram. The average expected He was 0.296, common genetic characteristics and less intraspecific variability. indicative of moderate-high genetic diversity among the 27 For patients with the cardiac form of ChD, no sharing of distinct samples. In addition, Shannon Weaver’s diversity index (H′ alleles was observed, indicating a higher rate of intraspecific = 3,159), a quantitative measure that defines the genetic genetic variability and complexity (Figure 2). diversity of species taking into account their variability, was The DICE similarity coefficient corroborated the data also considered moderate to high. This assessment was used contained in the dendrogram and UPGMA. This analysis was to describe the richness of the intra-DTU TcV variability used to assess the variability of alleles in each sample in the of T. cruzi. Frontiers in Cardiovascular Medicine | www.frontiersin.org 4 May 2021 | Volume 8 | Article 665624 Oliveira et al. Intra-DTU TcV Genetic Variability FIGURE 3 | DICE similarity coefficient represented in the range of 0 to ± 1 for the 27 samples of T. cruzi (DTU TcV), (p, patient). DISCUSSION ChD is endemic in Latin America; however, the epidemiology of this infection has changed, mainly due to recent population Trypanosoma Cruzi specie includes heterogeneous migration for countries of distinct continents. At present, ChD subpopulations that circulate in both domestic and wild cycles is an important public health problem in other non-endemic (22), and this diversity can be observed at the morphological regions, such as Europe (8). Immigration to Europe from (23), biological, antigenic (24), and genetic (5, 25) levels. Latin American countries has increased steadily over the last Moreover, T. cruzi is currently subdivided into seven distinct 2 years, especially to southern European countries, such as genetic groups (DTUs TcI-TcVI and Tcbat) (4), and each DTU Spain and Italy. More recently, there is evidence for Latin has its own characteristics (3). In order to better understand the American immigration to northern countries in Europe as disease in each geographical region it is necessary to study the well (10, 26). These population movements have increased the molecular epidemiology of this parasite, inherently related to the occurrence of ChD in these countries (9), since a considerable main biological characteristics, which consequently have clinical proportion of Latin American immigrants are chronically implications on Chagas disease clinical features and evolution. infected with T. cruzi, added to the occurrence of autochthone Frontiers in Cardiovascular Medicine | www.frontiersin.org 5 May 2021 | Volume 8 | Article 665624 Oliveira et al. Intra-DTU TcV Genetic Variability FIGURE 4 | Principal coordinated analysis (PCoA) for the 27 samples of T. cruzi belonging to the TcV discreet typing unit (DTU). cases transmitted by mechanisms independent to triatomine study, a pioneering moderate-high rate of genetic variability vectors. Consequently, the number of reported cases of ChD within DTU TcV of T. cruzi in a specific population of Bolivian with or without cardiac involvement has increased dramatically immigrants in Spain was assessed by LSSP-PCR. Furthermore, an in recent years, especially in European countries, such as Spain, association was detected between the indeterminate form of ChD Italy, and Switzerland, where most Latino immigrants have and sharing certain alleles present in the parasite’s genome. settled (27, 28). Thus, studies aiming to evaluate the possible It is relevant to emphasize that the LSSP-PCR methodology association between the intraspecific genetic variability of T. cruzi had been described and widely used to identify T. cruzi with the specific clinical forms of ChD are necessary. underlines before the first genotyping criteria emerged in It is known that DTU TcV is closely related to the domestic 2009 (31–33). Burgos and collaborators used this methodology cycle of ChD associated with human disease in countries such to evaluate the decrease of certain subclasses of the parasite as Argentina, Bolivia, Chile and Paraguay (6). This genotype kDNA minicircles in the peripheral blood and brain tissue is related to the presentation of clinical cardiac and digestive of a patient infected with human immunodeficiency virus symptoms in patients from countries in the southern cone and and with T. cruzi (TcII) during treatment for Chagas disease little is known about their natural mammalian reservoirs (3). (34). Similarly, Costales and collaborators employed LSSP- Previous studies have demonstrating the existence of genetic PCR to investigate the presence of polyclonal infection of the variability within TcI (29) and TcII (30) isolates. In the present parasite belonging to the TcI genotype in a cardiomyopathic Frontiers in Cardiovascular Medicine | www.frontiersin.org 6 May 2021 | Volume 8 | Article 665624 Oliveira et al. Intra-DTU TcV Genetic Variability patient with reactivation of the infection after heart Barcelona, Spain, is crucial to understanding the public health transplantation (35). implications of ChD in European countries. Improving this Lages-Silva et al. (30) also tried to establish the association understanding could contribute for the adequate design and of intra-DTU genetic variability with the different clinical planning of more effective public health interventions to improve manifestations of patients infected with T. cruzi. It is worth the health of the immigrants and control the vertical transmission mentioning that the authors used the same molecular technique of ChD, which is a serious problem in Europe nowadays. (LSSP-PCR) employing different genes, and as in the present study, they were unable to detect an effective correlation, what CONCLUSIONS demonstrated the need to search for genetic targets that are increasingly intrinsic in the parasite’s genome. In the work This study demonstrated the existence of a moderate to high involving DTU TcI (29), the study group sought to assess the rate of intra-DTU TcV variability in T. cruzi, in a specific association between the TcI genotype and the home and wild population of Bolivian immigrants in Spain. Being demonstrated distribution of the genetic lineage in its hosts and biological association of sharing of certain alleles of the parasite with aspects of the strains. It is worth mentioning that the TcV DTU is the absence of clinical manifestations in patients harboring the a hybrid strain, originating from several hybridization processes indeterminate clinical form of ChD, a trend to be assessed in a between TcI and TcII, with loss of heterozygosity between the larger population. The information provided in this study could progeny to produce TcIII and TcIV, followed by a second more affect the planning of more effective public health interventions recent hybridization event between TcII and TcIII to produce to improve the health of immigrants, vertical transmission both, TcV and TcVI (36). control, and improvement of ChD treatment in countries with Lima and collaborators evaluated a family with two predominance of infection by TcV genotype. The results of this generations of patients with chronic ChD infected with study support the need to search for increasingly intrinsic and DTU TcII, and attempted to establish the DTU/clinical forms specific targets in the genome of T. cruzi to be correlated with its correlation. However, no satisfactory results were obtained, main biological properties and clinical features in patients with possibly due to the low number of clinical samples. Despite of chronic ChD. similarly to the present study, in addition, they reported low genetic variability/diversity in samples of the parasite belonging to the TcII genotype (37). DATA AVAILABILITY STATEMENT In our study the UPGMA analysis of the samples The original contributions presented in the study are included demonstrated the existence of three large groups within the in the article/supplementary material, further inquiries can be T. cruzi TcV genotype, including a high rate of intraspecific directed to the corresponding author/s. genetic variability/diversity of this DTU. Similar data were obtained by some of us using T. cruzi DTU TcII and TcVI genotypes (38). We demonstrated, through UPGMA and PCoA ETHICS STATEMENT of random amplification of polymorphic DNA, the existence of a high rate of genetic variability within DTU TcII and TcVI of T. The studies involving human participants were reviewed cruzi in samples from patients with chronic ChD residing in an and approved by Human Research Ethics Committee of the important endemic region of Minas Gerais state, Brazil. Vall d’Hebron University Hospital. The patients/participants Additionally, in accordance with the UPGMA results of provided their written informed consent to participate in our study, Macchiaverna and collaborators showed that the this study. sequencing data of the T. cruzi TcMK (mevalonate kinase) gene in clinical samples from chronic patients in Argentina, revealed a AUTHOR CONTRIBUTIONS low rate of genetic variability within the DTU TcV, and showing apparently two robust subgroups of isolates (39). MO, ES, AS, and IM: conceptualization. MO, ES, and IM: Few studies have undertaken a more detailed intra-DTU data curation. MO: formal analysis, validation, and writing— approach in an attempt to detect the appropriate correlations original draft. MO and JM-N: funding acquisition. MO, ML, with the clinical forms of ChD presented by the patients. A and JS: investigation. MO, ES, and AS: methodology. JM-N and more common approach has been to search correlation between IM: project administration and supervision. MO, ES, ML, and T. cruzi DTUs and the clinical forms of ChD. However, it IM: resources. MO, ES, AS, ML, JM-N, and IM: visualization. is reasonable to speculate that small genetic modifications or MO, MS, ML, JS, JM-N, and IM: writing—review and editing. alterations in parasite genomes at intra-DTU level may be more All authors contributed to the article and approved the related to the induction of distinct clinical manifestations of submitted version. this disease, and even responses to a specific treatment against the T. cruzi, favoring a better understanding of the clinical and FUNDING epidemiological aspects of this disease in endemic and non- endemic regions. This work was supported by São Paulo Research Foundation Therefore, knowing the genetic variability of T. cruzi intra- (FAPESP)- fapesp.br/en (Grant No. 2018/22093-4), São Paulo DTU TcV in immigrant patients with chronic ChD residing in Research Foundation (FAPESP)- fapesp.br/en (Grant No. Frontiers in Cardiovascular Medicine | www.frontiersin.org 7 May 2021 | Volume 8 | Article 665624 Oliveira et al. Intra-DTU TcV Genetic Variability 2019/11943-0), São Paulo Research Foundation (FAPESP)- ACKNOWLEDGMENTS fapesp.br/en (Grant No. 2016/25403-9), and Universitat Autònoma de Barcelona, Vall d’Hebron University Hospital, We thank Pilar Alcubilla for her technical support and Mercedes PROSICS Barcelona, Spain. Guerrero Murillo for bioinformatics analysis. REFERENCES 17. Vago AR, Macedo AM, Oliveira RP, Andrade LO, Chiari E, Galvão LMC, et al. Kinetoplast DNA signatures of Trypanosoma cruzi strains obtained directly 1. Rassi A, Rassi A, Marcondes de Rezende J. American trypanosomiasis from infected tissues. Am J Pathol. (1996) 149:2153–59. (Chagas disease). Infect Dis Clin North Am. (2012) 26:275–91. 18. Gomes ML, Macedo AM, Vago AR, Pena SDJ, Galvão LMC, Chiari E. doi: 10.1016/j.idc.2012.03.002 Trypanosoma cruzi: optimization of polymerase chain reaction for detection 2. WHO. Chagas Disease (American trypanosomiasis). Fact sheet No. 340. in human blood. Exp Parasitol. (1998) 88:28–33. doi: 10.1006/expr.1998.4191 WHO (2018). 19. Santos FR, Pena SDJ, Epplen JT. Genetic and population study of a Y- 3. Zingales B. Trypanosoma cruzi genetic diversity: something linked tetranucleotide repeat DNA polymorphism with a simple non-isotopic new for something known about Chagas disease manifestations, technique. Hum Genet. (1993) 90:655–6. doi: 10.1007/BF00202486 serodiagnosis and drug sensitivity. Acta Trop. (2018) 184:38–52. 20. Dice LR. Measures of the amount of ecologic association between species. doi: 10.1016/j.actatropica.2017.09.017 Ecology. (1945) 26:297–302. doi: 10.2307/1932409 4. Zingales B, Andrade SG, Briones MRS, Campbell DA, Chiari E, Fernandes 21. Huff DR, Peakall R, Smouse PE. RAPD variation within and among O, et al. A new consensus for Trypanosoma cruzi intraspecific nomenclature: natural populations of outcrossing buffalograss [Buchloë dactyloides (Nutt.) second revision meeting recommends TcI to TcVI. Mem Inst Oswaldo Cruz. Engelm.]. Theor Appl Genet. (1993) 86:927–34. doi: 10.1007/BF00211043 (2009) 104:1051–4. doi: 10.1590/S0074-02762009000700021 22. Tibayrenc M, Ward P, Moya A, Ayala FJ. Natural populations of Trypanosoma 5. Tibayrenc M. Genetic subdivisions within Trypanosoma cruzi (discrete typing cruzi, the agent of Chagas disease, have a complex multiclonal structure. Proc units) and their relevance for molecular epidemiology and experimental Natl Acad Sci USA. (1986) 83:115–9. doi: 10.1073/pnas.83.1.115 evolution. Kinetoplastid Biol Dis. (2003) 2:12. doi: 10.1186/1475-9292-2-12 23. Brener Z, Chiari E. Varia C Oes morfol’ogicas observadas em diferentes 6. Zingales B, Miles MA, Campbell DA, Tibayrenc M, Macedo AM, Teixeira amostras DE. Rev Inst Med Trop São Paulo. (1963) 19:220–224. MMG, et al. The revised Trypanosoma cruzi subspecific nomenclature: 24. Maguire JH, Hoff R, Sleigh AC, Mott KE, Ramos NB, Sherlock IA. An rationale, epidemiological relevance and research applications. Infect Genet outbreak of Chagas’ disease in southwestern Bahia, Brazil. Am J Trop Med Evol. (2012) 12:240–53. doi: 10.1016/j.meegid.2011.12.009 Hyg. (1986) 35:931–6. doi: 10.4269/ajtmh.1986.35.931 7. Requena-Méndez A, Aldasoro E, de Lazzari E, Sicuri E, Brown M, Moore 25. Tibayrenc M, Ayala FJ. The population genetics of Trypanosoma cruzi DAJ, et al. Prevalence of chagas disease in Latin-American migrants living in revisited in the light of the predominant clonal evolution model. Acta Trop. Europe: a systematic review and meta-analysis. PLoS Negl Trop Dis. (2015) (2015) 151:156–65. doi: 10.1016/j.actatropica.2015.05.006 9:e0003540. doi: 10.1371/journal.pntd.0003540 26. Gascon J, Bern C, Pinazo MJ. Chagas disease in Spain, the United States 8. Rechel B, Mladovsky P, Ingleby D, Mackenbach JP, McKee M. Migration and other non-endemic countries. Acta Trop. (2010) 115:22–7. and health in an increasingly diverse Europe. Lancet. (2013) 381:1235–45. doi: 10.1016/j.actatropica.2009.07.019 doi: 10.1016/S0140-6736(12)62086-8 27. Jackson Y, Gétaz L, Wolff H, Holst M, Mauris A, Tardin A, et al. Prevalence, 9. Pérez-Molina JA, Molina I. Chagas disease. Lancet. (2018) 391:82–94. clinical staging and risk for blood-borne transmission of chagas disease among doi: 10.1016/S0140-6736(17)31612-4 Latin American migrants in Geneva, Switzerland. PLoS Negl Trop Dis. (2010) 10. Basile L, Jansà JM, Carlier Y, Salamanca DD, Angheben A, Bartoloni A, et al. 4:e592. doi: 10.1371/journal.pntd.0000592 Chagas disease in european countries: the challenge of a surveillance system. 28. Coura JR, Vias PA. Chagas disease: a new worldwide challenge. Nature. (2010) Eurosurveillance. (2011) 16:3. doi: 10.2807/ese.16.37.19968-en 465:S6–7. doi: 10.1038/nature09221 11. Muñoz J, Coll O, Juncosa T, Vergés M, Pino M Del, Fumado V, et al. 29. León CM, Hernández C, Montilla M, Ramírez JD. Retrospective distribution Prevalence and vertical transmission of Trypanosoma cruzi infection among of Trypanosoma cruzi I genotypes in Colombia. Mem Inst Oswaldo Cruz. pregnant latin american women attending 2 maternity clinics in barcelona, (2015) 110:387–93. doi: 10.1590/0074-02760140402 spain. Clin Infect Dis. (2009) 48:1736–40. doi: 10.1086/599223 30. Lages-Silva E, Ramírez LE, Pedrosa AL, Crema E, Da Cunha Galvão LM, Pena 12. de Oliveira MT, Sulleiro E, Gimenez AS, de Lana M, Zingales B, da Silva SDJ, et al. Variability of kinetoplast DNA gene signatures of Trypanosoma JS, et al. Quantification of parasite burden of Trypanosoma cruzi and cruzi II strains from patients with different clinical forms of Chagas’ disease identification of discrete typing units (Dtus) in blood samples of latin in Brazil. J Clin Microbiol. (2006) 44:2167–71. doi: 10.1128/JCM.02124-05 american immigrants residing in Barcelona, Spain. PLoS Negl Trop Dis. (2020) 31. Vago AR, Andrade LO, Leite AA, d’Ávila Reis D, Macedo AM, Adad SJ, et al., 14:e0008311. doi: 10.1371/journal.pntd.0008311 Filho GB, Pena SDJ. Genetic characterization of Trypanosoma cruzi directly 13. Avila HA, Sigman DS, Cohen LM, Millikan RC, Simpson L. Polymerase chain from tissues of patients with chronic chagas disease: differential distribution reaction amplification of Trypanosoma cruzi kinetoplast minicircle DNA of genetic types into diverse organs. Am J Pathol. (2000) 156:1805–9. isolated from whole blood lysates: diagnosis of chronic Chagas’ disease. Mol doi: 10.1016/S0002-9440(10)65052-3 Biochem Parasitol. (1991) 48:11–21. doi: 10.1016/0166-6851(91)90116-n 32. Franco DJ, Vago AR, Chiari E, Meira FCA, Galvão LMC, Machado 14. Britto C, Cardoso MA, Wincker P, Morel CM. A simple protocol for the CRS. Trypanosoma cruzi: mixture of two populations can modify physical cleavage of Trypanosoma cruzi kinetoplast DNA present in blood virulence and tissue tropism in rat. Exp Parasitol. (2003) 104:54–61. samples and its use in polymerase chain reaction (PCR)-based diagnosis doi: 10.1016/S0014-4894(03)00119-X of chronic Chagas disease. Mem Inst Oswaldo Cruz. (1993) 88:171–2. 33. Segatto M, Rodrigues CM, Machado CR, Franco GR, Pena SDJ, Macedo AM. doi: 10.1590/s0074-02761993000100030 LSSP-PCR of Trypanosoma cruzi: how the single primer sequence affects the 15. Dias JCP, Ramos AN, Gontijo ED, Luquetti A, Shikanai-Yasuda MA, Coura kDNA signature. BMC Res Notes. (2013) 6:174. doi: 10.1186/1756-0500-6-174 JR, et al. Aspectos Gerais da epidemiologia da doença de chagas com especial 34. Burgos JM, Begher SB, Freitas JM, Bisio M, Duffy T, Altcheh J, et al. Molecular atenção ao Brasil. Epidemiol Serv Saude Rev Sist Unico Saude Bras. (2016) diagnosis and typing of Trypanosoma cruzi populations and lineages in 25:7–86. doi: 10.5123/S1679-49742016000500002 cerebral chagas disease in a patient with AIDS. Am J Trop Med Hyg. (2005) 16. Mora G. Chagas cardiomyopathy. E J Cardiol Pract? (2016) 14:31–28. https:// 73:1016–8. doi: 10.4269/ajtmh.2005.73.1016 www.escardio.org/Journals/E-Journal-of-Cardiology-Practice/Volume-14/ 35. Costales JA, Kotton CN, Zurita-Leal AC, Garcia-Perez J, Llewellyn MS, Chagas-cardiomyopathy Messenger LA, et al. Chagas disease reactivation in a heart transplant patient Frontiers in Cardiovascular Medicine | www.frontiersin.org 8 May 2021 | Volume 8 | Article 665624 Oliveira et al. Intra-DTU TcV Genetic Variability infected by domestic Trypanosoma cruzi discrete typing unit i (TcIDOM). 39. Macchiaverna NP, Enriquez GF, Buscaglia CA, Balouz V, Gürtler RE, Cardinal Parasit Vect. (2015) 8:435. doi: 10.1186/s13071-015-1039-3 MV. New human isolates of Trypanosoma cruzi confirm the predominance 36. Freitas JM, Augusto-Pinto L, Pimenta JR, Bastos-Rodrigues L, of hybrid lineages in domestic transmission cycle of the Argentinean Chaco. Gonçalves VF, Teixeira SMR, et al. Ancestral genomes, sex, and the Infect Genet Evol. (2018) 66:229–35. doi: 10.1016/j.meegid.2018.10.001 population structure of Trypanosoma cruzi. Plos Pathog. (2006) 2:e24. doi: 10.1371/journal.ppat.0020024 Conflict of Interest: The authors declare that the research was conducted in the 37. Lima APB, de Oliveira MT, Silva RR, Torres RM, Veloso VM, de Lana absence of any commercial or financial relationships that could be construed as a M, et al. Evaluation of parasite and host genetics in two generations potential conflict of interest. of a family with Chagas disease. Parasitol Res. (2018) 117:3009–13. doi: 10.1007/s00436-018-5969-5 Copyright © 2021 Oliveira, Sulleiro, Silva, Silgado, de Lana, da Silva, Molina and 38. De Oliveira MT, MacHado De Assis GF, Oliveira E Silva JCV, MacHado Marin-Neto. This is an open-access article distributed under the terms of the Creative EMM, Da Silva GN, Veloso VM, et al. Trypanosoma cruzi Discret Typing Commons Attribution License (CC BY). The use, distribution or reproduction in Units (TcII and TcVI) in samples of patients from two municipalities other forums is permitted, provided the original author(s) and the copyright owner(s) of the Jequitinhonha Valley, MG, Brazil, using two molecular typing are credited and that the original publication in this journal is cited, in accordance strategies. Parasites and Vectors. (2015) 8: doi: 10.1186/s13071-015-1 with accepted academic practice. No use, distribution or reproduction is permitted 161-2 which does not comply with these terms. Frontiers in Cardiovascular Medicine | www.frontiersin.org 9 May 2021 | Volume 8 | Article 665624