ABSTRACT Background: Leishmaniasis is a vector-borne disease caused by a parasite protozoon from the genus Leishmania. Among the molecular techniques applied for detecting these parasites, real-time PCR with High Resolution Melting (PCR-HRM) proved advantageous since it simultaneously determines both the presence and species of the pathogen in one step, through amplification and later analysis of curves generated by melting temperature. Methods: Based on this molecular technique, the goal of this study was to estimate the PCR-HRM sensitivity for Leishmania spp. detection in different canine tissues by evaluating biological samples obtained from popliteal, submandibular, and pre-scapular lymph nodes, from bone marrow and ear pinnae of 28 stray dogs captured in the metropolitan area of Asunción (Paraguay). Results: The rk39 immunochromatographic test showed that 25/28 tested dogs (89%) presented antibodies against L. infantum. In 20/25 dogs that tested positive for rk39 (80%), it was possible to detect Leishmania spp. by PCR-HRM and determine that the species corresponded entirely to L. infantum. Regarding the analysis of different tissues, the parasite was detected in all popliteal lymph node samples, followed by high detection in submandibular (at 95%) and pre-scapular lymph nodes (at 90%), bone marrow (at 85%), and ear pinnae (at 85%). Conclusions: This study demonstrated that the use of real-time PCR-HRM using the molecular marker hsp70 was a highly sensitive method for simultaneously detecting and identifying Leishmania species in different tissues taken from infected dogs. In addition, the usefulness of ear pinnae as easily accessible tissue for molecular diagnosis was emphasized.

ABSTRACT Leishmaniasis and Chagas disease affect millions of people worldwide, particularly in Latin America where poor and vulnerable rural populations are mostly affected. Although joint efforts have been made to fight these neglected diseases, the combination of different factors such as inefficient treatment, lack of interest in investing in the search for new therapeutic sources, and the emergence of resistant strains of parasites end up complicating prospects. This situation makes it vitally important to develop new strategies aimed at identifying molecules with potential application in therapy or that may exert a synergistic effect when supplemented with current chemotherapy. One of the approaches proposed for this purpose is the search for these agents based on pharmacological targets present in the parasites causing Leishmaniasis and Chagas. Among these molecular targets there are enzymes involved in essential pathways of the parasite, such as Trypanothione Reductase (TR), involved in combating oxidative stress generated by reactive oxygen species produced by host macrophages during infection. An important tool in the search for inhibitors for this enzyme can be found in the application of in silico tools, which is a relatively inexpensive and fast method of selecting potential molecules from a variety of sources. Assays based on virtual screening allow the selection of molecules in a rational way, which through complementary tests can lead to the future emergence of new drugs.

RESUMEN La Leishmaniasis y la Enfermedad de Chagas afectan a millones de personas en América Latina y en otras regiones del mundo, siendo las poblaciones rurales pobres y vulnerables las más afectadas. Aunque se han realizado esfuerzos conjuntos para el combate de estas enfermedades desatendidas, la combinación de diferentes factores como el tratamiento ineficiente, la falta de interés para invertir en la búsqueda de nuevas fuentes terapéuticas y el surgimiento de cepas de parásitos resistentes acaban complicando las perspectivas. Esta situación torna de vital importancia el desarrollo de nuevas estrategias que apunten a la identificación de moléculas con potencial aplicación en la terapia o que puedan ejercer un efecto sinérgico cuando se complementan con la quimioterapia actual. Uno de los enfoques propuestos para este fin consiste en la búsqueda de estos agentes basados en blancos farmacológicos presentes en los parásitos causantes de las citadas enfermedades. Entre estos blancos moleculares se encuentran las enzimas implicadas en vías esenciales del parásito, como la Tripanotiona Reductasa (TR), involucrada en el combate al estrés oxidativo generado por las especies reactivas de oxígeno producidas por macrófagos del huésped durante la infección. Una herramienta importante en la búsqueda de inhibidores para esta enzima puede ser encontrada a través de la aplicación de herramientas in silico, que resulta un método relativamente rápido y de bajo costo, el cual permite seleccionar moléculas con potencial a partir de una gran variedad de fuentes. Los ensayos basados en el tamizaje (“screening”) virtual permiten la selección de moléculas de forma racional, que mediante ensayos complementarios pueden dar lugar al surgimiento futuro de nuevos fármacos.

Abstract INTRODUCTION Leishmaniasis, Chagas disease, and malaria cause morbidity globally. The drugs currently used for treatment have limitations. Activity of cinnamic acid analogs against Leishmania spp., Trypanosoma cruzi, and Plasmodium falciparum was evaluated in the interest of identifying new antiprotozoal compounds. METHODS In vitro effects of analogs against L. braziliensis, L. infantum chagasi, T. cruzi, and P. falciparum, and hemolytic and cytotoxic activities on NCTC 929 were determined. RESULTS Three analogs showed leishmanicidal and tripanocidal activity. No antiplasmodial, hemolytic, or cytotoxic activity was observed. CONCLUSIONS Antiprotozoal activity of analogs against L. infantum braziliensis, L. infantum chagasi, and T. cruzi was demonstrated.