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The relationship among genetic diversity of Trypanosoma cruzi and clinical forms of Chagas disease remain elusive. In order to assess the possible association between different T. cruzi Discrete Typing Units (DTUs) and the clinical pictures of the disease, 205 chronic patients from Salta province, Argentina, were analysed. One hundred and twenty-two of these patients were clinically categorized as: cardiac 38.5% (47/122), digestive 15% (18/122), cardio-digestive 16% (20/122) and asymptomatic 30% (37/122). From each patient, blood samples were taken for both, Polymerase Chain Reaction (PCR) targeting kDNA and blood culture analyses. The presence of T. cruzi kDNA was detected in 43% (88/205) of the patients. T. cruzi DTUs were identified in 74% (65/88) of the kDNA positive patients by PCR-hybridization using specific probes. We detected the presence of DTUs TcI, TcII, TcV and TcVI. Single infections (i.e. presence of only one DTU in the sample) were detected in 38.64% of the samples (34/88), while mixed infections were 35.23% (31/88). TcV was the most prevalent DTU (60.3%- 53/88). The association analyses showed, for the first time to the best of our knowledge, that TcV and TcVI were associated with the digestive form of Chagas Disease (Fisher p = .0001). Fil: Monje Rumi, Maria Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Floridia Yapur, Noelia Aldana del Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Zago, María Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Ragone, Paula Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Pérez Brandán, Jimena María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Nuñez, Sonia. Gobierno de la Provincia de Salta. Hospital San Bernardo.; Argentina Fil: Barrientos, Natalia. Gobierno de la Provincia de Salta. Hospital San Bernardo.; Argentina Fil: Tomasini, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Diosque, Patricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina

In regions where Chagas disease is endemic, canine Trypanosoma cruzi infection is highly correlated with the risk of transmission of the parasite to humans. Herein we evaluated the novel TcTASV protein family (subfamilies A, B, C), differentially expressed in bloodstream trypomastigotes, for the detection of naturally infected dogs. A gene of each TcTASV subfamily was cloned and expressed. Indirect enzyme-linked immunosorbent assays (ELISA) were developed using recombinant antigens individually or mixed together. Our results showed that dogs with active T. cruzi infection differentially reacted against the TcTASV-C subfamily. The use of both TcTASV-C plus TcTASV-A proteins (Mix A+C-ELISA) enhanced the reactivity of sera from dogs with active infection, detecting 94% of the evaluated samples. These findings agree with our previous observations, where the infected animals exhibited a quick anti-TcTASV-C antibody response, coincident with the beginning of parasitaemia, in a murine model of the disease. Results obtained in the present work prove that the Mix A+C-ELISA is a specific, simple and cheap technique to be applied in endemic areas in screening studies. The Mix A+C-ELISA could help to differentially detect canine hosts with active infection and therefore with high impact in the risk of transmission to humans. Fil: Floridia Yapur, Noelia Aldana del Rosario. Universidad Nacional de Salta. Facultad de Cs.naturales. Escuela de Biología. Cátedra de Química Biologica; Argentina. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Monje Rumi, Maria Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Ragone, Paula Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Lauthier, Juan José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Tomasini, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Alberti D'amato, Anahí Maitén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Diosque, Patricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Cimino, R.. Universidad Nacional de Salta. Sede Regional Oran. Instituto de Investigación de Enfermedades Tropicales; Argentina Fil: Marco, Jorge Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Barroso, Paola Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Sanchez, Daniel Oscar. 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 Fil: Nasser, Julio Rubén. Universidad Nacional de Salta. Sede Regional Oran. Instituto de Investigación de Enfermedades Tropicales; Argentina Fil: Tekiel, Valeria Sonia. 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

Highlights • This is the first meta-analysis of individual data in chronic Trypanosoma cruzi infection after treatment. • The probability of seroreversion is variable along the course of follow-up. • An interaction was found between age at treatment and country setting. • The course of parasitological/molecular tests after treatment needs to be assessed., Objective To determine the course of serological tests in subjects with chronic Trypanosoma cruzi infection treated with anti-trypanosomal drugs. Methods A systematic review and meta-analysis was conducted using individual participant data. Survival analysis and the Cox proportional hazards regression model with random effects to adjust for covariates were applied. The protocol was registered in the PROSPERO database (http://www.crd.york.ac.uk/PROSPERO; CRD42012002162). Results A total of 27 studies (1296 subjects) conducted in eight countries were included. The risk of bias was low for all domains in 17 studies (63.0%). Nine hundred and thirteen subjects were assessed (149 seroreversion events, 83.7% censored data) for enzyme-linked immunosorbent assay (ELISA), 670 subjects (134 events, 80.0% censored) for indirect immunofluorescence assay (IIF), and 548 subjects (99 events, 82.0% censored) for indirect hemagglutination assay (IHA). A higher probability of seroreversion was observed within a shorter time span in subjects aged 1–19 years compared to adults. The chance of seroreversion also varied according to the country where the infection might have been acquired. For instance, the pooled adjusted hazard ratio between children/adolescents and adults for the IIF test was 1.54 (95% confidence interval 0.64–3.71) for certain countries of South America (Argentina, Bolivia, Chile, and Paraguay) and 9.37 (95% confidence interval 3.44–25.50) for Brazil. Conclusions The disappearance of anti-T. cruzi antibodies was demonstrated along the course of follow-up. An interaction between age at treatment and country setting was found.

The transmission cycles of Trypanosoma cruzi in the Gran Chaco are complex networks involving domestic and wild components, whose interrelationships are not well understood. Knowing the circuit of transmission of the different Discrete Typing Units (DTUs) of T. cruzi in the complex environment of the Chaco region is relevant to understanding how the different components (reservoirs, vectors, ecotopes) interact. In the present study we identified the DTUs infecting humans and dogs in two rural areas of the Gran Chaco in Argentina, using molecular methods which avoid parasite culture. Blood samples of humans and dogs were typified by PCR-DNA blotting and hybridization assays with five specific DNA probes (TcI, TcII, TcIII, TcV and TcVI). PCR analyses were performed on seropositive human and dog samples and showed the presence of T. cruzi DNA in 41.7% (98/235) and 53% (35/66) samples, respectively. The identification of infective DTUs was determined in 83.6% (82/98) and 91.4% (32/35) in human and dog samples, respectively. Single infections (36.7% – 36/98) and a previously not detected high proportion of mixed infections (47.9% – 47/98) were found. In a 15.3% (15/98) of samples the infecting DTU was not identified. Among the single infections TcV was the most prevalent DTU (30.6% – 30/98) in human samples; while TcVI (42.8% – 15/35) showed the highest prevalence in dog samples. TcV/TcVI was the most prevalent mixed infection in humans (32.6% – 32/98); and TcI/TcVI (14.3% – 5/35) in dogs. Significant associations between TcV with humans and TcVI with dogs were detected. For the first time, the presence of TcIII was detected in humans from this region. The occurrence of one human infected whit TcIII (a principally wild DTU) could be suggested the emergence of this, in domestic cycles in the Gran Chaco. Fil: Monje Rumi, Maria Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; Argentina Fil: Perez Brandan, Cecilia Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; Argentina Fil: Ragone, Paula Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; Argentina Fil: Tomasini, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; Argentina Fil: Lauthier, Juan José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; Argentina Fil: Alberti D'Amato, Anahi M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; Argentina Fil: Cimino, Ruben O.. Universidad Nacional de Salta. Facultad de Cs.naturales. Escuela de Biologia. Catedra de Quimica Biologica; Argentina. Universidad Nacional de Salta. Sede Regional Oran. Instituto de Investigación de Enfermedades Tropicales; Argentina Fil: Orellana, Viviana. Universidad Nacional de Salta. Facultad de Ciencias de la Salud; Argentina Fil: Basombrio, Miguel Angel Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina Fil: Diosque, Patricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; Argentina

SUMMARYThe rTSSA-II (recombinant Trypomastigote Small Surface II) antigen was evaluated by ELISA to detect anti-Trypanosoma cruziantibodies in sera from naturally infected dogs and humans. For this evaluation ELISA-rTSSA-II was standardized and groups were classified according to the results obtained through xenodiagnosis, ELISA and PCR. Sensitivity (Se), Specificity (Sp), Kappa index (KI) and area under curve (AUC) were determined. The Se was determined by using 14 sera from dogs infected withT. cruziVI (TcVI) whereas Sp was determined by using 95 non-chagasic sera by xenodiagnosis, ELISA-Homogenate and PCR. The performance of ELISA-rTSSA-II in dog sera was high (AUC=0·93 and KI=0·91). The Se was 92·85% (1 false negative) and Sp was 100%. Two sera from dogs infected with TcI and 1 with TcIII were negative. For patients infected withT. cruzi, reactivity was 87·8% (36/41), there was only 1 indeterminate, and Sp was 100%. Fifty-four sera from non-chagasic and 68 sera from patients with cutaneous leishmaniasis did not react with rTSS-II. ELISA-rTSSA-II showed a high performance when studying sera from naturally infected dogs and it also presented 100% Sp. This assay could be an important tool to carry out sero-epidemiological surveys on the prevalence ofT. cruzicirculating lineages in the region.

Dogs are considered the main mammal reservoir of Trypanosoma cruzi in domiciliary environments. Consequently, accurate detection of T. cruzi infection in canine populations is epidemiologically relevant. Here we analyzed the utility of the T. cruzi recombinant antigens FRA, SAPA, CP1, Ag1 and a SAPA/TSSA VI mixture, in an ELISA format. We used a positive control group of sera obtained from 38 dogs from the Chaco region in Argentina with positive Homogenate-ELISA reaction, all of them also positive by xenodiagnosis and/or PCR. The negative group included 19 dogs from a non-endemic area. Sensitivity, specificity, Area Under the Curve (AUC) of the Receiver-Operating Charactheristic (ROC) curve and Kappa index were obtained in order to compare the diagnostic efficiency of the tests. The SAPA/TSSA VI had the highest performance, with a sensitivity of 94.7% and an AUC ROC of 0.99 that indicates high accuracy. Among individual antigens, SAPA-ELISA yielded the highest sensitivity (86.8%) and AUC ROC (0.96), whereas FRA-ELISA was the least efficient test (sensitivity=36.8%; AUC ROC=0.53). Our results showed that the use of SAPA/TSSA VI in ELISA assays could be a useful tool to study dogs naturally infected with T. cruzi in endemic areas. Fil: Floridia Yapur, Noelia Aldana del Rosario. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; Argentina. Universidad Nacional de Salta. Facultad de Ciencias Naturales. Escuela de Biología. Cátedra de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta; Argentina Fil: Vega Benedetti, Ana Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Monje Rumi, Maria Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Ragone, Paula Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Lauthier, Juan José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Tomasini, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Alberti D'amato, Anahí Maitén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: López Quiroga, Inés Raquel. Universidad Nacional de Salta. Facultad de Ciencias Naturales. Escuela de Biología. Cátedra de Química Biológica; Argentina. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; Argentina Fil: Diosque, Patricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Marcipar, Iván Sergio. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Tecnología Inmunológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta; Argentina Fil: Nasser, Julio Rubén. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; Argentina. Universidad Nacional de Salta. Facultad de Ciencias Naturales. Escuela de Biología. Cátedra de Química Biológica; Argentina Fil: Cimino, Rubén Oscar. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta; Argentina. Universidad Nacional de Salta. Facultad de Ciencias Naturales. Escuela de Biología. Cátedra de Química Biológica; Argentina

A total of 221 children from two rural settlements in Northeast Argentina were examined for T. cruzi infection. Blood samples were taken for serology tests and PCR assays. In addition, T. cruzi Discrete Typing Units (DTUs) were determined by hybridization with specific DNA probes of the minicircle hypervariable regions (mHVR). Serological results indicated that 26% (57/215) were reactive against T. cruzi antigens. PCR analyses were performed on seropositive samples showing presence of parasite DNA in 31 out of 53 samples (58.5%). All seropositive children underwent specific chemotherapy with Benznidazole (5mg/kg/day) for a period of two months and were monitored two and five years after treatment. Overall the treatment was well tolerated and low side effects were observed. Serological conversion was observed at two years post -treatment in one child form Pampa Avila and at five years in two children from Tres Estacas. However, at the end of the follow-up period, T. cruzi DNA could not be detected by PCR in samples from treated children, except in two cases. In addition, the results of hybridizations with specific DNA probes showed that DTU TcV was detected in 68% (21/31), TcVI in 7% (2/31) and TcV/VI in 3% (1/31) of the samples. Altogether, results of the follow-up of treated children showed a low rate of seroconversion; however trend toward seroconversion was evident at five years post-treatment. On the other hand, detection of T. cruzi DNA by PCR significantly decreased after Benznidazole treatment. The existence of data regarding serological and molecular follow-ups from controlled studies in the Chaco Region will be important for future treatment efforts against T. cruzi infection in this region. The results obtained in the present study represent a contribution in this regard.