Resumen Introducción. Trypanosoma cruzi, agente causal de la enfermedad de Chagas, exhibe una sustancial heterogeneidad fenotípica y genotípica que puede influir en las variaciones epidemiológicas y clínicas de la enfermedad, así como en la sensibilidad a los fármacos utilizados en el tratamiento. Objetivo. Evaluar la sensibilidad in vitro al benznidazol, el nifurtimox y el posaconazol de 40 cepas clonadas de T. cruzi de Paraguay, con distintos genotipos, huéspedes y localidades de origen. Materiales y métodos. En su estado epimastigote, los parásitos se incubaron en medio de cultivo LIT (Liver Infusion Tryptose) con diferentes concentraciones de cada fármaco en ensayos por triplicado. El grado de sensibilidad se estimó a partir de las concentraciones inhibitorias del 50 y el 90% (IC50 e IC90) y se obtuvieron los valores promedio y la desviación estándar de cada cepa y fármaco. La significación estadística entre grupos se determinó mediante análisis de varianzas con el test no paramétrico de Wilcoxon/Kruskal-Wallis y valores de p<0,05. Resultados. Se observó un amplio rango de respuesta a los fármacos. Se identificaron dos grupos de parásitos (A y B) con diferencias significativas en la sensibilidad al benznidazol (p<0,0001), y tres grupos (A, B, C) en cuanto a la sensibilidad al nifurtimox y el posaconazol (p<0,0001). Conclusiones. En general, las cepas fueron más sensibles al nifurtimox que al benznidazol y el posaconazol. Estas diferencias evidencian la heterogeneidad de las poblaciones de T cruzi que circulan en Paraguay, lo que debe considerarse en el tratamiento y el seguimiento de las personas afectadas.

Abstract Introduction: Trypanosoma cruzi, the causative agent of Chagas disease, shows substantial phenotypic and genotypic heterogeneity, which can influence the epidemiological and clinical variations of the disease and the sensitivity to the drugs used in the treatment. Objective: To assess the in vitro susceptibility to benznidazole, nifurtimox, and posaconazole of 40 cloned strains of T. cruzi isolated in Paraguay belonging to different genotypes, hosts, and localities. Materials and methods: We incubated the parasites in their epimastigote stage in LIT culture medium with different concentrations of each drug in triplicate assays. The degree of susceptibility was estimated by the inhibitory concentrations of 50 and 90% (IC50 and IC90) to obtain the average values and the standard deviation for each strain and drug. We determined the statistical significance between groups by analysis of variances with the Wilcoxon/Kruskal-Wallis non-parametric test and values of p<0.05. Results: A wide range of drug response was observed. Two groups of parasites (A and B) were identified as having significant differences in susceptibility to benznidazole (p<0.0001), and three groups (A, B, C) to nifurtimox and posaconazole (p<0.0001). Conclusions: Overall, the isolates were more susceptible to nifurtimox than benznidazole and posaconazole. Such differences highlight the heterogeneity of T. cruzi populations circulating in Paraguay, an aspect to consider in the treatment and follow up of patients.

ABSTRACT Chagas disease is still endemic in the Paraguayan Chaco, damaging mainly rural or lower incomes populations, thus being the indigenous communities some of the most vulnerable populations. The objective of this work was to determine the presence of triatomine vectors of Trypanosoma cruzi in the Chaidi and Aocojadi Ayoreo Totobiegosode indigenous communities of Alto Paraguay. The characteristics of the housings and their relationship with the vectors permanence were evaluated. The search for vectors was carried out in the intradomicile and peridomicile of the houses by installing a light trap in a wild environment contiguous to one of the communities. For searching the flagellates, the faeces of the collected triatomines were examined under an optical microscope and the xenoculture technique was performed in the positive insects. Triatomines were found and identified both in the domestic and wild environments of the Chaidi and Aocojadi, Ayoreo Totobiegosode communties of Alto Paraguay during the collection period of 2016. The triatomines identified in this study, Triatoma sordida and Triatoma guasayana, are typical of wild environments, although during certain seasons of the year they act as intrusive species invading the domestic areas. The natural infection with T. cruzi-like was detected in a specimen from the Chaidi community, without ruling out the possibility that the parasite is also circulating in Aocojadi community. This study research contributes to consider the disease exposure risk of the local population because up to the time of this study there was no information on the presence or absence of T. cruzi circulation in these communities.

RESUMEN La enfermedad de Chagas es hasta hoy día endémica en el Chaco paraguayo, perjudicando principalmente a las poblaciones rurales o de menores recursos económicos, representando así las comunidades indígenas algunas de las poblaciones más vulnerables. El objetivo de este trabajo fue determinar la presencia de triatominos vectores de Trypanosoma cruzi en las comunidades indígenas Ayoreo Totobiegosode Chaidi y Aocojadi del Alto Paraguay. Se evaluaron características de las viviendas y su relación con la permanencia de vectores. La búsqueda de vectores se realizó en el intradomicilio y peridomicilio de las viviendas, y mediante la instalación de una trampa de luz en un ambiente silvestre adyacente a una de las comunidades. Para la búsqueda de flagelados fueron examinadas al microscopio óptico las heces de los triatominos colectados. Se realizó la técnica de xenocultivo para los insectos positivos. Fueron encontrados e identificados triatominos tanto en los ambientes domésticos como en el ambiente silvestre en la zona de las comunidades del Pueblo Ayoreo Totobiegosode del Alto Paraguay: Chaidi y Aocojadi, durante el periodo de colecta del 2016. Los triatominos identificados en este estudio, Triatoma sordida y Triatoma guasayana, son propios de ambientes silvestres, aunque durante ciertas épocas del año actúan como especies intrusivas invadiendo las áreas domésticas. Se detectó la infección natural con T. cruzi-like en un ejemplar proveniente de la comunidad Chaidi, sin descartar la posibilidad de que el parásito se encuentre circulando en Aocojadi. La investigación contribuye a considerar el riesgo de exposición a la enfermedad que tiene la población local, debido a que hasta el momento de este estudio no se cuenta con información sobre la presencia o ausencia de circulación de T. cruzi en estas comunidades.

Resumo Com o objetivo de verificar a infecção natural por Trypanosoma cruzi em primatas não-humanos de uma área endêmica rural da região leste do Paraguai, foi realizado o xenodiagnóstico em 35 animais pertencentes a duas espécies. Para a genotipagem foi utilizada a unidade discreta de tipagem (UDT) do T. cruzi, em uma combinação de quatro marcadores, incluindo amplificação de produtos de pequena (18S) e grande (24Sα) subunidades do gene do ácido ribonucleico ribossômico, da região intergênica de miniéxon e do gene da proteína de choque térmico 60 (HSP60/EcoRV-PCR-RFLP), pela reação em cadeia da Polimerase. Um espécime de Sapajus cay se mostrou positivo pelo UDT TcII. Este resultado constitui o primeiro relato da infecção natural pelo T. cruzi em um macaco silvestre no Paraguai, abrigando um UDT associado com a doença de Chagas grave em humanos.

Abstract To verify the occurrence of natural Trypanosoma cruzi infection in non-human primates from a rural endemic area of the east region of Paraguay, xenodiagnosis was performed in 35 animals belonging to two species. For genotyping and T. cruzi discrete typing unit (DTU) assignment, a combination of four markers was used, including amplification products of the small (18S) and large (24Sα) subunits of ribosomal ribonucleic acid gene, the intergenic region of mini-exon gene and the heat shock protein 60 Eco-RV polymerase chain reaction-restriction fragment length polymorphism (HSP60/EcoRV-PCR-RFLP). One specimen of Sapajus cay was found positive and infected by the DTU TcII. This result constitutes the first record of natural T. cruzi infection in a sylvatic monkey in Paraguay, harbouring a DTU associated with severe Chagas disease in humans.

Twenty one Trypanosoma cruzi stocks from humans, domiciliary triatomines and one sylvatic animal of different areas of Paraguay were subjected to isoenzyme analysis. Thirteen enzyme systems (15 loci in total) were studied. MN cl2 (clonets 39) and SO34 cl4 (clonets 20) were used as references. Relationships between stocks were depicted by an UPGMA dendrogram constructed using the Jaccard´s distances matrix. Among the Paraguayan stocks 14 zymodemes were identified (Par1 to Par14), Par 5 being the most frequent. Polymorphism rate and clonal diversity were 0.73 and 0.93, respectively. Average number of alleles per polymorphic locus was 2.5 (range 2-4). These measurements show a high diversity, which is confirmed by the dendrogram topology. All stocks belong to the same lineage, as MN cl2 reference strain (T. cruzi II). Moreover three distinct subgroups were identified and two of them correspond to Brazilian and Bolivian zymodemes, respectively. The third subgroup, the most common in Paraguay, is related to Tulahuen stock. The large geographical distribution of some zymodemes agrees with the hypothesis of clonality for T. cruzi populations. However sample size was not adequate to detect genetic recombination in any single locality.