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T cell-mediated immune response plays a crucial role in controlling Trypanosoma cruzi infection and parasite burden, but it is also involved in the clinical onset and progression of chronic Chagas´ disease. Therefore, the study of T cells is central to the understanding of the immune response against the parasite and its implications for the infected organism. The complexity of the parasite-host interactions hampers the identification and characterization of T cell-activating epitopes. We approached this issue by combining in silico and in vitro methods to interrogate patients´ T cells specificity. Fifty T. cruzi peptides predicted to bind a broad range of class I and II HLA molecules were selected for in vitro screening against PBMC samples from a cohort of chronic Chagas´ disease patients, using IFN-γ secretion as a readout. Seven of these peptides were shown to activate this type of T cell response, and four out of these contain class I and II epitopes that, to our knowledge, are first described in this study. The remaining three contain sequences that had been previously demonstrated to induce CD8+ T cell response in Chagas´ disease patients, or bind HLA-A*02:01, but are, in this study, demonstrated to engage CD4+ T cells. We also assessed the degree of differentiation of activated T cells and looked into the HLA variants that might restrict the recognition of these peptides in the context of human T. cruzi infection. Fil: Acevedo, Gonzalo Raúl. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Juiz, Natalia Anahí. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Ziblat, Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Perez Perri, Lucas. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Girard, Magalí Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Ossowski, Micaela Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Fernandez, Marisa Liliana. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina Fil: Hernández, Yolanda. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina Fil: Chadi, Raúl. Gobierno de la Ciudad Autónoma de Buenos Aires. Hospital General de Agudos Dr. Ignacio Pirovano; Argentina Fil: Wittig, Michael. Christian Albrechts Universität zu Kiel. Institut für Klinische Molekularbiologie; Alemania Fil: Franke, Andre. Christian-Albrechts-Universität zu Kiel. Institut für Klinische Molekularbiologie; Alemania Fil: Nielsen, Morten. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina. Technical University of Denmark. Department of Health Technology; Dinamarca Fil: Gomez, Karina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina

This study aimed to evaluate well-documented diagnostic antigens, named B13, 1F8 and JL7 recombinant proteins, as potential markers of seroconversion in treated chagasic patients. Prospective study, involving 203 patients treated with benznidazole, was conducted from endemic areas of northern Argentina. Follow-up was possible in 107 out of them and blood samples were taken for serology and PCR assays before and 2, 3, 6, 12, 24 and 36 months after treatment initiation. Reactivity against Trypanosoma cruzi lysate and recombinant antigens was measured by ELISA. The rate of decrease of antibody titers showed nonlinear kinetics with an abrupt drop within the first three months after initiation of treatment for all studied antigens, followed by a plateau displaying a low decay until the end of follow-up. At this point, anti-B13, anti-1F8 and anti-JL7 titers were relatively close to the cut-off line, while anti-T. cruzi antibodies still remained positive. At baseline, 60.8% (45/74) of analysed patients tested positive for parasite DNA by PCR and during the follow-up period in 34 out of 45 positive samples (75.5%) could not be detected T. cruzi DNA. Our results suggest that these antigens might be useful as early markers for monitoring antiparasitic treatment in chronic Chagas disease. Fil: Niborski, Leticia Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Grippo, Vanina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Lafon, Sonia Olga. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Levitus, Gabriela Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: García Bournissen, Facundo. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Ramirez Gomez, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Burgos, Juan Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Bisio, Margarita María Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Juiz, Natalia Anahí. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Ayala, Vilma. Centro Asistencial Cáritas Diocesana; Argentina Fil: Coppede, María. Centro Asistencial Cáritas Diocesana; Argentina Fil: Herrera, Verónica. Centro Asistencial Cáritas Diocesana; Argentina Fil: López, Crescencia. Centro Asistencial Cáritas Diocesana; Argentina Fil: Contreras, Ana. Centro Asistencial Cáritas Diocesana; Argentina Fil: Gomez, Karina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Elean, Juan C.. Centro Asistencial Cáritas Diocesana; Argentina Fil: Mujica, Hugo D.. Centro Asistencial Cáritas Diocesana; Argentina. Hospital Zonal de Añatuya; Argentina Fil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Levin, Mariano Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Longhi, Silvia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina

Several studies have proposed different genetic markers of susceptibility to develop chronic Chagas cardiomyopathy (CCC). Many genes may be involved, each one making a small contribution. For this reason, an appropriate approach for this problematic is to study a large number of single nucleotide polymorphisms (SNPs) in individuals sharing a genetic background. Our aim was to analyze two CCR2 and seven CCR5 SNPs and their association to CCC in Argentina. A case-control study was carried out in 480 T. cruzi seropositive adults from Argentinean Gran Chaco endemic region (Wichi and Creole) and patients from Buenos Aires health centres. They were classified according to the Consensus on Chagas-Mazza Disease as non-demonstrated (non-DC group) or demonstrated (DC group) cardiomyopathy, i.e. asymptomatic or with CCC patients, respectively. Since, after allelic analysis, 2 out of 9 studied SNPs did not fit Hardy–Weinberg equilibrium in the unaffected non-DC group from Wichi patients, we analyzed them as a separate population. Only rs1800024T and rs41469351T in CCR5 gene showed significant differences within non-Wichi population (Creole + patients from Buenos Aires centres), being the former associated to protection, and the latter to risk of CCC. No evidence of association was observed between any of the analyzed CCR2-CCR5 gene polymorphisms and the development of CCC; however, the HHE haplotype was associated with protection in Wichi population. Our findings support the hypothesis that CCR2-CCR5 genes and their haplotypes are associated with CCC; however, depending on the population studied, different associations can be found. Therefore, the evolutionary context, in which the genes or haplotypes are associated with diseases, acquires special relevance., Author summary Chagas disease caused by the infection with the protozoan Trypanosoma cruzi is endemic in Latin America. In Argentina, it is estimated 1.5 million patients have Chagas disease and 2.2 million people in risk of T. cruzi infection. The endemic area covers the north of the country where the conditions, such as high levels of poverty and social exclusion and low population density, mostly rural, favor T. cruzi infection. Most affected people remains asymptomatic after infection for the rest of their lives, but around one third of infected people may develop clinical symptoms of visceral damage. Chronic Chagas Cardiomyopathy (CCC), the most frequent and severe consequence of the chronic infection by T. cruzi, is manifested predominately as an arrhythmogenic cardiomyopathy. The pathogenesis of CCC is not completely understood, but it is believed that the human genetic variation may be a determinant factor of disease progression. We studied in Wichi and in admixed populations from Argentina the CCR2-CCR5 genes, two CC chemokine receptors involved in the trafficking of several immune cells and in the pathogenesis of cardiovascular diseases. Our results showed that CCR2-CCR5 genes are associated with CCC and highlight the relevance of the evolutionary context in which disease-associated genes are found.

This work evaluated a serial blood sampling procedure to enhance the sensitivity of duplex real-time quantitative PCR (qPCR) for baseline detection and quantification of parasitic loads and posttreatment identification of failure in the context of clinical trials for treatment of chronic Chagas disease, namely, DNDi-CH-E1224-001 (ClinicalTrials.gov registration no. NCT01489228) and the MSF-DNDi PCR Sampling Optimization Study (NCT01678599)., This work evaluated a serial blood sampling procedure to enhance the sensitivity of duplex real-time quantitative PCR (qPCR) for baseline detection and quantification of parasitic loads and posttreatment identification of failure in the context of clinical trials for treatment of chronic Chagas disease, namely, DNDi-CH-E1224-001 (ClinicalTrials.gov registration no. NCT01489228) and the MSF-DNDi PCR Sampling Optimization Study (NCT01678599). Patients from Cochabamba (n = 294), Tarija (n = 257), and Aiquile (n = 220) were enrolled. Three serial blood samples were collected at each time point, and qPCR triplicates were tested for each sample. The first two samples were collected during the same day and the third one 7 days later. A patient was considered PCR positive if at least one qPCR replicate was detectable. Cumulative results of multiple samples and qPCR replicates enhanced the proportion of pretreatment sample positivity from 54.8% to 76.2%, 59.5% to 77.8%, and 73.5% to 90.2% in Cochabamba, Tarija, and Aiquile cohorts, respectively. This strategy increased the detection of treatment failure from 72.9% to 91.7%, 77.8% to 88.9%, and 42.9% to 69.1% for E1224 low-, short-, and high-dosage regimens, respectively, and from 4.6% to 15.9% and 9.5% to 32.1% for the benznidazole arm in the DNDi-CH-E1224-001 and MSF-DNDi studies, respectively. The addition of the third blood sample and third qPCR replicate in patients with nondetectable PCR results in the first two samples gave a small, non-statistically significant improvement in qPCR positivity. No change in clinical sensitivity was seen with a blood volume increase from 5 to 10 ml. The monitoring of patients treated with placebo in the DNDi-CH-E1224-001 trial revealed fluctuations in parasitic loads and occasionally nondetectable results. In conclusion, a serial sampling strategy enhanced PCR sensitivity to detecting treatment failure during follow-up and has the potential for improving recruitment capacity in Chagas disease trials, which require an initial positive qPCR result for patient admission.

Background: Chagas disease is caused by Trypanosoma cruzi, a parasite endemic to Latin America. Most infections occur in children by vector or congenital transmission. Trypanosoma cruzi establishes a complexity of specific molecular parasite-host cell interactions to invade the host. However, most studies have been mainly focused on the interaction between the parasite and different cell types, but not on the infection and invasion on a tissue level. During congenital transmission, T. cruzi must cross the placental barrier, composed of epithelial and connective tissues, in order to infect the developing fetus. Here we aimed to study the global changes of transcriptome in the placental tissue after a T. cruzi challenge. Results: Strong changes in gene expression profiling were found in the different experimental conditions, involving the reprogramming of gene expression in genes involved in the innate immune response. Conclusions: Trypanosoma cruzi induces strong changes in genes involved in a wide range of pathways, especially those involved in immune response against infections. Fil: Castillo, Christian. Universidad de Chile; Chile Fil: Carrillo, Ileana. Universidad de Chile; Chile Fil: Libisch, Gabriela. Universidad de la República; Uruguay Fil: Juiz, Natalia Anahí. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Robello, Carlos. Universidad de la República; Uruguay Fil: Kemmerling, Ulrike. Universidad de Chile; Chile

Trypanosoma cruzi infection in women of reproductive age is associated with congenital transmission and adverse pregnancy outcomes. The placenta is a key barrier to infection. Gene expression profiles of term placental environment from T. cruzi–seropositive (SP) and -seronegative (SN) mothers were characterized by RNA-Seq. Nine pools of placental RNA paired samples were used: three from SN and six from SP tissues. Each pool consisted of female/male newborns and vaginal/cesarean delivery binomials. No newborn was congenitally infected. T. cruzi satellite DNA quantitative PCR in placental tissues and maternal and neonatal blood, and parasite 18S quantitative RT-PCR from placental RNA were negative, except in three SP women's bloodstream. To identify pathways associated with maternal T. cruzi infection, a gene-set association analysis was implemented: SP placental samples showed overexpression of inflammatory response and lymphocytic activation, whereas numerous biosynthetic processes were down-regulated. About 42 genes showed a significant fold-change between SP and SN groups. KISS1 and CGB5 were down-regulated, whereas KIF12, HLA-G, PRG2, TAC3, FN1, and ATXN3L were up-regulated. Several expressed genes in SP placentas encode proteins associated with preeclampsia and miscarriage. This first transcriptomics study in human term placental environment shows a placental response that may affect the fetus while protecting it from parasite infection; this host response could be responsible for the low rate of congenital transmission in chronic Chagas disease. Fil: Juiz, Natalia Anahí. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Torrejón, Irma del Rosario. Universidad Nacional de Jujuy; Argentina Fil: Burgos, Marianela. Hospital Nacional Professor Dr. Alejandro Posadas; Argentina Fil: Torres, Ana María Fernanda. Universidad Nacional de Jujuy; Argentina Fil: Duffy, Tomás. University Of Southern California; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Cayo, Nelly Melina. Universidad Nacional de Jujuy; Argentina Fil: Tabasco, Anahí. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Salvo, Miriam. Hospital Nacional Professor Dr. Alejandro Posadas; Argentina Fil: Longhi, Silvia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina

Congenital infection of Trypanosoma cruzi allows transmission of this parasite through generations. Despite the problematic that this entails, little is known about the placenta environment genetic response produced against infection. We performed functional genomics by microarray analysis in C57Bl/6J mice comparing placentas from uninfected animals and from animals infected with two different T. cruzi strains: K98, a clone of the non-lethal myotropic CA-I strain (TcI), and VD (TcVI), isolated from a human case of congenital infection. Analysis of networks by GeneMANIA of differentially expressed genes showed that “Secretory Granule” was a pathway down-regulated in both infected groups, whereas “Innate Immune Response” and “Response to Interferon-gamma” were pathways up-regulated in VD infection but not in K98. Applying another approach, the GSEA algorithm that detects small changes in predetermined gene sets, we found that metabolic processes, transcription and macromolecular transport were down-regulated in infected placentas environment and some pathways related to cascade signaling had opposite regulation: over-represented in VD and down-regulated in K98 group. We also have found a stronger tropism to the placental organ by VD strain, by detection of parasite DNA and RNA, suggesting living parasites. Our study is the first one to describe in a murine model the genetic response of placental environment to T. cruzi infection and suggests the development of a strong immune response, parasite genotype-dependent, to the detriment of cellular metabolism, which may contribute to control infection preventing the risk of congenital transmission., Author summary Congenital transmission of Trypanosoma cruzi, the causative agent of Chagas disease, remains a problem of global public health impact in endemic areas where vectorial and transfusional transmission have been controlled and in non-endemic countries due to migration movements. Little is known about how the parasite´s presence and genetic variability affect placental capacity to protect the fetus. This study explores, for the first time, the effects of placental environment infection by analyzing parasite persistence and gene expression using functional genomics and biological network analyses in mice infected by two strains of T. cruzi, with differential capacity of congenital transmission. The infection with the strain with a stronger placental tropism was associated to a higher degree of up-regulation in genes related to innate immunity and response to interferon-gamma. Our findings suggest that the placental environment exerts a strong immune response in detriment of cellular metabolism modulated by the parasite strain. These findings constitute a significant contribution to better understand the mechanisms causing congenital infection of T. cruzi.

Trypanosoma cruzi, the causative agent of Chagas disease, is divided into six Discrete Typing Units (DTUs): TcI–TcVI. We aimed to identify T. cruzi DTUs in Latin-American migrants in the Barcelona area (Spain) and to assess different molecular typing approaches for the characterization of T. cruzi genotypes. Seventy-five peripheral blood samples were analyzed by two real-time PCR methods (qPCR) based on satellite DNA (SatDNA) and kinetoplastid DNA (kDNA). The 20 samples testing positive in both methods, all belonging to Bolivian individuals, were submitted to DTU characterization using two PCR-based flowcharts: multiplex qPCR using TaqMan probes (MTq-PCR), and conventional PCR. These samples were also studied by sequencing the SatDNA and classified as type I (TcI/III), type II (TcII/IV) and type I/II hybrid (TcV/VI). Ten out of the 20 samples gave positive results in the flowcharts: TcV (5 samples), TcII/V/VI (3) and mixed infections by TcV plus TcII (1) and TcV plus TcII/VI (1). By SatDNA sequencing, we classified the 20 samples, 19 as type I/II and one as type I. The most frequent DTU identified by both flowcharts, and suggested by SatDNA sequencing in the remaining samples with low parasitic loads, TcV, is common in Bolivia and predominant in peripheral blood. The mixed infection by TcV–TcII was detected for the first time simultaneously in Bolivian migrants. PCR-based flowcharts are very useful to characterize DTUs during acute infection. SatDNA sequence analysis cannot discriminate T. cruzi populations at the level of a single DTU but it enabled us to increase the number of characterized cases in chronically infected patients. Fil: Abras, Alba. Universidad de Barcelona; España. Universidad de Girona; España. Barcelona Centre for International Health Research; España Fil: Gállego, Montserrat. Universidad de Barcelona; España. Barcelona Centre for International Health Research; España Fil: Muñoz, Cármen. Hospital de la Santa Creu i Sant Pau; España. Universitat Autònoma de Barcelona; España Fil: Juiz, Natalia Anahí. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Ramirez Gomez, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Cura, Carolina Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular “Dr. Héctor N. Torres” ; Argentina Fil: Tebar, Silvia. Universidad de Barcelona; España. Barcelona Centre for International Health Research; España Fil: Fernández Arévalo, Ana. Universidad de Barcelona; España. Barcelona Centre for International Health Research; España. Hospital de la Santa Creu i Sant Pau; España Fil: Pinazo, María Jesús. Barcelona Centre for International Health Research; España Fil: de la Torre, Leonardo. Barcelona Centre for International Health Research; España Fil: Posada, Elizabeth. Barcelona Centre for International Health Research; España Fil: Navarro, Ferran. Universitat Autònoma de Barcelona; España. Hospital de la Santa Creu i Sant Pau; España Fil: Espinal, Paula. Hospital de la Santa Creu i Sant Pau; España Fil: Ballart, Cristina. Universidad de Barcelona; España. Barcelona Centre for International Health Research; España Fil: Portús, Montserrat. Universidad de Barcelona; España Fil: Gascón, Joaquim. Barcelona Centre for International Health Research; España Fil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina

Most indigenous ethnias from Northern Argentina live in rural areas of "the Gran Chaco" region, where Trypanosoma cruzi is endemic. Serological and parasitological features have been poorly characterized in Aboriginal populations and scarce information exist regarding relevant T. cruzi discrete typing units (DTU) and parasitic loads. This study was focused to characterize T. cruzi infection in Qom, Mocoit, Pit'laxá and Wichi ethnias (N = 604) and Creole communities (N = 257) inhabiting rural villages from two highly endemic provinces of the Argentinean Gran Chaco.DNA extracted using Hexadecyltrimethyl Ammonium Bromide reagent from peripheral blood samples was used for conventional PCR targeted to parasite kinetoplastid DNA (kDNA) and identification of DTUs using nuclear genomic markers. In kDNA-PCR positive samples from three rural Aboriginal communities of "Monte Impenetrable Chaqueño", minicircle signatures were characterized by Low stringency single primer-PCR and parasitic loads calculated using Real-Time PCR.Seroprevalence was higher in Aboriginal (47.98%) than in Creole (27.23%) rural communities (Chi square, p = 4.e-8). A low seroprevalence (4.3%) was detected in a Qom settlement at the suburbs of Resistencia city (Fisher Exact test, p = 2.e-21).The kDNA-PCR positivity was 42.15% in Aboriginal communities and 65.71% in Creole populations (Chi square, p = 5.e-4). Among Aboriginal communities kDNA-PCR positivity was heterogeneous (Chi square, p = 1.e-4). Highest kDNA-PCR positivity (79%) was detected in the Qom community of Colonia Aborigen and the lowest PCR positivity in two different surveys at the Wichi community of Misión Nueva Pompeya (33.3% in 2010 and 20.8% in 2014).TcV (or TcII/V/VI) was predominant in both Aboriginal and Creole communities, in agreement with DTU distribution reported for the region. Besides, two subjects were infected with TcVI, one with TcI and four presented mixed infections of TcV plus TcII/VI. Most minicircle signatures clustered according to their original localities, but in a few cases, signatures from one locality clustered with signatures from other village, suggesting circulation of the same strains in the area. Parasitic loads ranged from undetectable to around 50 parasite equivalents/mL, showing higher values than those generally observed in chronic Chagas disease patients living in urban centers of Argentina. Our findings reveal the persistence of high levels of infection in these neglected populations. Fil: Lucero, R. H.. Universidad Nacional del Nordeste; Argentina Fil: Brusés, B. L.. Universidad Nacional del Nordeste; Argentina Fil: Cura, Carolina Inés. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Formichelli, L. B.. Universidad Nacional del Nordeste; Argentina Fil: Juiz, Natalia Anahí. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Fernández, G. J.. Universidad Nacional del Nordeste; Argentina Fil: Bisio, Margarita María Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires; Argentina Fil: Deluca, Gerardo Daniel. Universidad Nacional del Nordeste; Argentina Fil: Besuschio, Susana Alicia. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Hernández, D. O.. Universidad Nacional del Nordeste; Argentina Fil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires; Argentina

Background. It is currently unclear why only a proportion of children born to Trypanosoma cruzi?infected mothers acquire the infection. We have examined the association of 11 single-nucleotide polymorphisms (SNPs) located in genes coding for placental expression enzymes as genetic markers of susceptibility to congenital T. cruzi infection (hereafter, ?congenital infection?): rs2014683and rs1048988 in ALPP; rs11244787 and rs1871054 in ADAM12; rs243866, rs243865, rs17859821, rs243864, and rs2285053 in MMP2; and rs3918242 and rs2234681 in MMP9.Methods. Two groups of children born to mothers seropositive for T. cruzi were compared: 101 had congenital infection, and 116 were uninfected. Novel high-resolution melting and capillary electrophoresis genotyping techniques were designed and used.Results. Logistic regression analysis showed that mutations in rs11244787 and rs1871054 (in ADAM12) and rs243866, rs17859821, and rs2285053 (in MMP2) were associated with susceptibility to congenital infection. Multifactor dimensionality reduction revealed that genotyping results for rs11244787, rs1871054, rs243866, rs17859821 and rs243864 sites would be a good predictorof congenital infection.Conclusions. Our results suggest an important role of human polymorphisms in proteins involved in extracellular matrix remodeling and the immune response during congenital infection. To our knowledge, this is the first study demonstrating the association between mutations in placentally expressed genes and susceptibility to congenital infection. Fil: Juiz, Natalia Anahí. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular ; Argentina Fil: Cayo, Nelly M.. Universidad Nacional de Jujuy. Instituto de Biología de la Altura; Argentina Fil: Burgos, Marianela. Hospital Nacional Prof. Alejandro Posadas; Argentina Fil: Salvo, Miriam E.. Hospital Nacional Prof. Alejandro Posadas; Argentina Fil: Nasser, Julio Rubén. Universidad Nacional de Salta; Argentina Fil: Bua, Jacqueline Elena. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología ; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Longhi, Silvia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular ; Argentina Fil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular ; Argentina

An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease.