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An efficacious topical treatment for cutaneous leishmaniasis (CL) is highly desirable but still an ongoing challenge. Systemic risedronate (Ris) has been reported to have anti-leishmanial properties and Eudragit EPO (EuE) has shown in vitro activity against L. (L.) amazonensis. The aim of this work was to investigate the in vivo efficacy of topical Ris and EuE-Ris complexes on CL. Surface charge and Ris release kinetics from the different dispersions were analyzed. BALB/c mice were infected intradermally with promastigotes of L. (L.) amazonensis. Ulcers were treated with Ris or EuE-Ris hydrogels. All the lesions that received topical Ris or EuE-Ris showed an improvement with respect to control: reduction of ulcer average size, cicatrization, flattened edges and no signs of necrosis. In addition, a marked parasitic inhibition of 69.5 and 73.7% was observed in the groups treated with Ris and EuE-Ris, respectively, with the IgG2a levels indicating a tendency towards cure. The results are promising and the system should now be enhanced to achieve total parasite elimination., Cutaneous leishmaniasis; Risedronate; Eudragit; Topical treatment; Leishmania amazonensis.

Cutaneous leishmaniasis (CL) is a neglected tropical disease endemic in ~ 90 countries, with an increasing incidence. Presently available pharmacotherapy implies the systemic administration of moderately/very toxic drugs. Miltefosine (Milt) is the only FDA-approved drug to treat CL via the oral route (Impavido®). It produces side effects; in particular, teratogenic effects are of concern. A topical treatment would have the great advantage of minimising the systemic circulation of the drug, preventing side effects. We prepared dispersions containing Milt and liposomes of different compositions to enhance/modulate trans-epidermal penetration and evaluated in vitro and in vivo efficacy and toxicity, in vitro release rate of the drug and particles size stability with time. Treatments were topically administered to BALB/c mice infected with Leishmania (Leishmania) amazonensis. The dispersions containing 0.5% Milt eliminated 99% of the parasites and cured the lesions with a complete re-epithelisation, no visible scar and re-growth of hair. Fluid liposomes decreased the time to heal the lesion and the time needed to eliminate viable amastigotes from the lesion site. Relapse of the infection was not found 1 month after treatment in any case. Ultraflexible liposomes on the other hand had no significant in vitro effect but decreased in vivo efficacy. A topical Milt formulation including fluid liposomes seems a promising treatment against CL.

Cutaneous leishmaniasis (CL) is a neglected tropical disease endemic in ~90 countries, with an increasing incidence. Presently available pharmacotherapy implies the systemic administration of moderately/very toxic drugs. Miltefosine (Milt) is the only FDA approved drug to treat CL via the oral route (Impavido®). It produces side effects; in particular, teratogenic effects are of concern. A topical treatment would have the great advantage of minimising the systemic circulation of the drug, preventing side effects. We prepared dispersions containing Milt and liposomes to modulate trans-epidermal penetration and evaluated in vivo efficacy. Treatments were topically administered to BALB/c mice infected with Leishmania (Leishmania) amazonensis. The dispersions containing 0.5 % Milt eliminated 99 % of the parasites and cured the lesions with a complete re-epithelisation, no visible scar and re-growth of hair. Fluid liposomes decreased the time to heal the lesion and the time needed to eliminate viable amastigotes from the lesion site. Relapse of the infection was not found one month after treatment in any case. A topical Milt formulation including fluid liposomes seems a promising treatment against CL. Fil: Peralta, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigación y Tecnología Química. Universidad Tecnológica Nacional. Facultad Regional Córdoba. Centro de Investigación y Tecnología Química; Argentina Fil: Bracamonte, María Estefanía. 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: Guzman, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica; Argentina Fil: Olivera, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica; 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: Carrer, Dolores Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina

The diagnosis of American tegumentary leishmaniasis (ATL) still requires the design of more effective tools. Leishmania (Viannia) braziliensis is the causal agent of the 90% of Argentinean ATL cases. Considering the current knowledge, an ELISA based crude antigen (CA) for the diagnosis was designed. Ninety-nine subjects diagnosed as ATL, 27 as no-ATL, and 84 donors from non-ATL-endemic areas were included in this study. The current ATL diagnosis was based four techniques, dermal smear microscopic examination (parasitological test), PCR, Leishmanin skin test, and clinical records. We obtained CA extracts from promastigotes and amastigotes from macrophage cultures of different zymodemes of endemic Leishmania species circulating in the study area. Crude antigens from the ‘local’ main zymodeme of L. (V.) braziliensis showed the highest reactivity against anti-Leishmania antibodies compared to the other included species. The CA of amastigotes of this zymodeme was 3.4 fold more reactive than promastigotes one. Moreover, amastigote-membrane CA (MCA) were 3.6 fold more reactive than the soluble antigens. The MCA-ELISA reached a sensitivity and specificity of 98% (CI = 94.7%-100%) and 63.6% (53.9–73.1), respectively. When anti-Trypanosoma cruzi reactive sera were excluded, the specificity reached 98.4% (94.4–100), while the sensitivity was similar, with a positive predictive value (PV) of 98.6% (94.6–100) and negative PV of 96.3% (91.6–100). The performance of the MCA-ELISA results strongly contribute to the final diagnostic decision, since a non-reactive serological result almost discards the suspected ATL, because of its high negative PV. The developed MCA-ELISA showed a high diagnostic performance, which makes it a good candidate for ATL diagnosis, for seroprevalence studies, or for monitoring treatments efficacy. Fil: Bracamonte, María Estefanía. 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: Moya Alvarez, Agustin. 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: Sosa, Andrea Mabel. 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: Hoyos, Carlos Lorenzo. 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. Kochi University. Kochi Medical School; Japón. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Cajal, Silvana Pamela. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; Argentina Fil: Juarez, Marisa del Valle. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; Argentina Fil: Uncos, Renato Exequiel. 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: Sánchez Valdéz, Fernando Javier. 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: Acuña, Leonardo. 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: Basombrío, Miguel Ángel Manuel. 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: Nasser, Julio Rubén. Universidad Nacional de Salta; Argentina Fil: Hashiguchi, Yoshihisa. Kochi University. Kochi Medical School; Japón Fil: Korenaga, Masataka. Kochi University. Kochi Medical School; Japón 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: 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

This paper describes a rare case of leishmaniasis with simultaneous presentation of cutaneous and mucosal lesions in endemic areas for leishmaniasis. Usually, such a case does not occur simultaneously in cutaneous (CL) or mucocutaneous leishmaniasis. Besides, we performed a molecular characterization of genetic variants of the causal agent, proposed a hypothesis for the interpretation of the finding, and highlighted the relevance for searching of the signs of mucosal lesions in suspected CL cases. Fil: Hoyos, Carlos Lorenzo. 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. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; Argentina Fil: Quipildor, Marcelo Omar. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; Argentina Fil: Bracamonte, María Estefanía. 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. Kochi University. Kochi Medical School. Dept.of Parasitology; Japón Fil: Cajal, Silvana Pamela. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; Argentina Fil: Uncos, Delfor Alejandro. 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: Korenaga, Masataka. Kochi University. Kochi Medical School. Dept.of Parasitology; Japón Fil: Hashiguchi, Yoshihisa. Kochi University. Kochi Medical School. Dept.of Parasitology; Japón. Universidad Católica Santiago de Guayaquil; Ecuador 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: 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

American visceral leishmaniasis (AVL) has two main scenarios of transmission as follows: scattered cases in rural areas andurban outbreaks. Urban AVL is in active dispersion from the northeastern border of Argentina-Paraguay-Brazil to the South.The presence of Lutzomyia longipalpis was initially reported in urban environments in the northwestern border of the country.The presence of Lu. longipalpis, environmental variables associated with its distribution, and its genetic diversity were assessedin Salvador Mazza, Argentina, on the border with Bolivia. The genetic analysis showed high haplotype diversity, low nucleotidediversity, and low nucleotide polymorphism index. We discuss the hypothesis of an expanding urban population with introgressivehybridisation of older haplogroups found in their path in natural forest or rural environments, acquiring a new adaptability tourban environments, and the possibility of changes in vector capacity. Fil: Quintana, María Gabriela. Ministerio de Salud. Instituto Nacional de Medicina Tropical; Argentina. Red de Investigación de la Leishmaniasis en la Argentina; Argentina Fil: Pech May, Angélica del Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina. Red de Investigación de la Leishmaniasis en la Argentina; Argentina. Ministerio de Salud. Instituto Nacional de Medicina Tropical; Argentina Fil: Fuenzalida, Ana Denise. Ministerio de Salud. Instituto Nacional de Medicina Tropical; Argentina. Red de Investigación de la Leishmaniasis en la Argentina; Argentina. Universidad Nacional de Tucumán. Facultad de Ciencias Naturales e Instituto Miguel Lillo. Instituto Superior de Entomología; Argentina Fil: Direni Mancini, José Manuel. Red de Investigación de la Leishmaniasis en la Argentina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Tucumán. Facultad de Ciencias Naturales e Instituto Miguel Lillo. Instituto Superior de Entomología; Argentina Fil: Barroso, Paola Andrea. Ministerio de Salud. Instituto Nacional de Medicina Tropical; Argentina. 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: Yadon, Zaida Estela. Ministerio de Salud. Instituto Nacional de Medicina Tropical; Argentina Fil: Zaideneberg, Mario. Ministerio de Salud y Acción Social; Argentina Fil: Salomón, Oscar Daniel. Ministerio de Salud. Instituto Nacional de Medicina Tropical; Argentina. Red de Investigación de la Leishmaniasis en la Argentina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; 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

The treatment of leishmaniasis includes pentavalent antimony drugs but, because of the side effects, toxicity and cases of treatment failure or resistance, the search of new antileishmanial compounds are necessary. The aims of this study were to evaluate and compare the in vitro antileishmanial activity of four green tea catechins, and to assess the efficacy of topical (&minus, )-epigallocatechin gallate in a cutaneous leishmaniasis model. The antileishmanial activity of green tea catechins was evaluated against intracellular amastigotes, and cytotoxicity was performed with human monocytic cell line. BALB/c mice were infected in the ear dermis with Leishmania (Leishmania) amazonensis and treated with topical 15% (&minus, )-epigallocatechin gallate, intraperitoneal Glucantime, and control group. The efficacy of treatments was evaluated by quantifying the parasite burden and by measuring the lesions size. (&minus, )-Epigallocatechin gallate and (&minus, )-epigallocatechin were the most active compounds with IC50 values &lt, 59.6 &micro, g/mL and with a selectivity index &gt, 1. Topical treatment with (&minus, )-epigallocatechin gallate decreased significantly both lesion size and parasite burden (80.4% inhibition) compared to control group (p &lt, 0.05), and moreover (&minus, )-epigallocatechin gallate showed a similar efficacy to Glucantime (85.1% inhibition), the reference drug for leishmaniasis treatment.

Since the description of the Leishmania genus, its identification and organization have been a challenge. A high number of molecular markers have been developed to resolve phylogenetic differences at the species level and for addressing key epidemiological and population genetics questions. Based on Multilocus enzyme electrophoresis (MLEE), Multilocus sequence typing (MLST) schemes have been developed using different gene candidates. From 38 original gene targets proposed by other authors, 27 of them were chosen. In silico selection was made by analyzing free access genomic sequence data of 33 Leishmania species, one Paraleishmania representative, and one outgroup, in order to select the best 15 loci. De novo amplifications and primers redesign of these 15 genes were analyzed over a panel of 20 reference strains and isolates. Phylogenetic analysis was made at every step. Two MLST schemes were selected. The first one was based on the analysis of three-gene fragments, and it is suitable for species assignment as well as basic phylogenetic studies. By the addition of seven-genes, an approach based on the analysis of ten-gene fragments was also proposed. This is the first work that two optimized MLST schemes have been suggested, validated against a phylogenetically diverse panel of Leishmania isolates. MLST is potentially a powerful phylogenetic approach, and most probably the new gold standard for Leishmania spp. characterization.

Cutaneous leishmaniasis is endemic in Salta province, which belongs to the northwest of Argentina. Leishmania spp. DNA from Giemsa-stained slides of up to 12 years in storage of patients from Salta was characterized through polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP). One hundred smears positive for microscopy, classified in a semiquantitative scale for amastigote density, were analyzed. Also, Leishmanin skin test (LST) results were included. DNA extraction was carried out applying lysis buffer with proteinase K, and then DNA was amplified with ribosomal internal transcribed spacer1primers. PCR products were digested with HaeIII enzyme. All PCR-positive smears (74/100) belonged to Viannia subgenus. A statistically significant, directly proportional relationship between semiquantitative microscopy and PCR results was detected. All patients had LST-positive results (induration ≥ 5 mm), and the smears of those with smaller induration (LST < 19 mm) gave a higher proportion of positive PCR results. This study determined that smear age did not affect PCR positivity, which allows retrospective analyzes and suggests smears might be useful for molecular complementary diagnosis. Because Leishmania (Viannia) braziliensis is the main circulating species in the study area, determining Viannia subgenus in all analyzed samples confirms previous findings. PCR positivity showed statistically significant differences according to semiquantitative microscopy, highlighting the importance of parasite burden in the diagnostic sensitivity of the method. Considering that smears of patients with smaller LST induration were more positive in PCR, a negative smear from patients with positive LST response, but

Cutaneous leishmaniasis is endemic in Salta province, which belongs to the northwest of Argentina. Leishmania spp. DNA from Giemsa-stained slides of up to 12 years in storage of patients from Salta was characterized through polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP). One hundred smears positive for microscopy, classified in a semiquantitative scale for amastigote density, were analyzed. Also, Leishmanin skin test (LST) results were included. DNA extraction was carried out applying lysis buffer with proteinase K, and then DNA was amplified with ribosomal internal transcribed spacer 1 primers. PCR products were digested with HaeIII enzyme. All PCR-positive smears (74/100) belonged to Viannia subgenus. A statistically significant, directly proportional relationship between semiquantitative microscopy and PCR results was detected. All patients had LST-positive results (induration ≥ 5 mm), and the smears of those with smaller induration (LST < 19 mm) gave a higher proportion of positive PCR results. This study determined that smear age did not affect PCR positivity, which allows retrospective analyzes and suggests smears might be useful for molecular complementary diagnosis. Because Leishmania (Viannia) braziliensis is the main circulating species in the study area, determining Viannia subgenus in all analyzed samples confirms previous findings. PCR positivity showed statistically significant differences according to semiquantitative microscopy, highlighting the importance of parasite burden in the diagnostic sensitivity of the method. Considering that smears of patients with smaller LST induration were more positive in PCR, a negative smear from patients with positive LST response, but < 19 mm, could actually represent a false-negative result.

Background: Some sand flies are of medical importance because they are vectors of Leishmania parasites that are responsible for leishmaniasis. The aim of this study was to make a retrospective epidemiological analysis of tegumentary leishmaniasis (TL), to identify Leishmania spp. from patient isolates and to describe the diversity of sand flies from a border area between Bolivia and Argentina. Methods: TL cases included in the study were diagnosed in an endemic area of the north of Argentina from 1985 to 2017. The parasites isolated were characterized by the cytochrome B method. Sand flies were captured with Centers for Disease Control traps in Aguas Blancas and Media Luna-Algarrobito localities. Results: A total of 118 cases of TL were analysed. Eight isolates were characterized as Leishmania (Viannia) braziliensis. A total of 1291 sand flies were captured, including Nyssomyia neivai, Cortelezzii complex, Evandromyia sallesi, Migonemyia migonei and Micropygomyia quinquefer. Within the area, sand flies were found in the backyards of houses. Conclusions: In this region there exists the possibility of peridomestic transmission of TL in the neighbourhoods peripheral to the urban area and in rural environments as well as the risk of transmission to travellers that pass through the customs offices. Fil: Copa, Griselda Noemi. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta; Argentina. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; Argentina. Universidad Nacional de Salta. Facultad de Ciencias Naturales; Argentina Fil: Almazán, María Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta; Argentina. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; Argentina. Universidad Nacional de Salta. Facultad de Ciencias Naturales; Argentina Fil: Aramayo, Lorena Vanesa. Universidad Nacional de Salta; Argentina Fil: Krolewiecki, Alejandro Javier. Universidad Nacional de Salta; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta; Argentina Fil: Cajal, Silvana Pamela. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; Argentina Fil: Juarez, Marisa. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; Argentina Fil: Lauthier, Juan José. Kochi University. Kochi Medical School; Japón Fil: Korenaga, Masataka. Kochi University. Kochi Medical School; Japón 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: Nasser, Julio Rubén. Universidad Nacional de Salta. Sede Regional Orán. 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: Gil, José Fernando. 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. Instituto de Investigaciones en Energía no Convencional. Universidad Nacional de Salta. Facultad de Ciencias Exactas. Departamento de Física. Instituto de Investigaciones en Energía no Convencional; Argentina

Leishmaniases comprise zoonotic diseases caused by protozoan flagellates of the Leishmania genus. They are endemic to South America, and the visceral form has been recently reported in Argentina. Dogs can play different roles in the Leishmania transmission cycles, depending mainly on the species of parasite involved. Here we focused on the clinical characterization of canine leishmaniasis (CanL) in Northeast Argentina and on the molecular typing of its etiological agent. The nested polymerase chain reaction and sequence analysis of the Leishmania cytochrome b (cyt b) gene was performed on DNA templates purified from lymph nodes, bone marrow or spleen aspirates obtained from 48 dogs previously diagnosed by the observation of Leishmania amastigotes on smears from these aspirates. Their clinical and epidemiological data were also recorded. Systemic abnormalities were observed in 46 subjects (95.8%), most frequently lymphadenopathy, and emaciation (89.6 and 75%). Furthermore, 87% also presented tegumentary abnormalities, such as alopecia (54.2%) or secondary skin lesions (47.9%), among others. Twenty three dogs were positive for cyt b amplification. The sequence analysis showed the presence of two genotypes, LiA1 and LiA2, assigned to Leishmania (Leishmania) infantum, with 99.9 and 100% homology with the reference strain MHOM/TN/80/IPT1 respectively. LiA1 was identified in 18 cases (78.3%) and LiA2 in five (21.7%). Two cyt b variants of L. (L.) infantum were incriminated as the causative agents of CanL cases from three cities: Posadas, Garupá, and Ituzaingó. All three cities are located in the northeastern area of the country, where these parasites seem to be spreading in urban areas.

OBJECTIVES To determine the efficacy and the immunomodulatory function of Z-100 alone or combined with meglumine antimoniate on Leishmania amazonensis infection. METHODS The effect of the compounds was evaluated by microscopic counting of intracellular amastigotes in macrophages stained with Giemsa, or axenic promastigotes, and IC(50) was determined by linear regression. The antileishmanial effect of the compounds was assessed in infected BALB/c mice by a limiting dilution analysis and the production of gamma interferon (IFN-gamma), interleukin 10 (IL-10), IL-4, IgG1 and IgG2a was measured by ELISA. RESULTS In vitro, Z-100 showed antileishmanial activity against intracellular amastigotes of L. amazonensis with an IC(50) of 13 mg/L. Moreover, infected macrophages treated with Z-100 (12 mg/L) showed smaller parasitophorous vacuoles with fewer parasites than the control. In addition, the efficacy of Z-100 plus meglumine antimoniate [14 mg/L pentavalent antimony (Sb(v))] was higher (46% inhibition) than either Z-100 or meglumine antimoniate alone. Nevertheless, no effect of Z-100 on axenic promastigotes was observed. Infected BALB/c mice treated with Z-100 (100 microg/kg) alone did not show any antileishmanial effects in comparison with the control group, and IFN-gamma, as well as IL-10 and IL-4, was up-regulated by the treatment. In addition, both IgG1 and IgG2a were also increased by the Z-100 treatment. Although Z-100 plus meglumine antimoniate (14 or 28 mg/kg Sb(v)) controlled both the parasite load and the footpad swelling in comparison with control mice, no significant differences were found with meglumine antimoniate alone. CONCLUSIONS In vitro, Z-100 alone or combined with meglumine antimoniate showed an antileishmanial effect on L. amazonensis. However, no effect was observed in infected BALB/c mice treated with Z-100, suggesting that the up-regulation of IL-10 and IL-4 production by the treatment could be interfering with the development of a protective Th1-type response. For further understanding of the effects of Z-100 in vivo, another strain of mice such as C57BL/6 should be tested in future.

The area around Río Blanco, in the Orán department in the north of the Argentinian province of Salta, is endemic for American tegumentary leishmaniasis. In an attempt to facilitate the identification of the Lutzomyia species in this area, sequences of the gene coding for the 18S ribosomal RNA (rRNA) of sandflies caught in a Shannon trap were explored, by a combination of PCR and analysis of restriction-fragment-length polymorphism (RFLP). The products from the PCR, which employed two primers developed specifically for this study (Lu.18S 1S and Lu.18S AR), were cloned into a commercial vector (pGEM-T Easy) so that their nucleotide sequences could be investigated. In the RFLP analysis, the products of single and double digestion with the AfaI and HapII restriction enzymes were separated by electrophoresis in 3% or 4% agarose. Taken together with the results of a morphological investigation of the flies, the resultant DNA fragment patterns were sufficient to identify most of the sandflies caught as Lu. neivai. Although two other species, Lu. cortelezzii and Lu. sallesi, were collected, they were relatively rare and only identified morphologically. A single digestion of the 18S-rRNA gene sequences with AfaI or HapII appeared sufficient and useful for the identification of Lu. neivai from the north of Salta province, and for several other Lutzomyia species. Fil: Barroso, Paola Andrea. Kochi University; Japón. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Marco, Jorge Diego. Kochi University; Japón. 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: Kato, H.. Yamaguchi University; Japón Fil: Tarama, R.. Yamaguchi University; Japón Fil: Rueda, P.. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; Argentina Fil: Cajal, S. P.. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; Argentina Fil: Basombrío, Miguel Ángel Manuel. 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: Korenaga, M.. Kochi University; Japón Fil: Taranto, Nestor Juan. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; Argentina Fil: Hashiguchi, Y.. Kochi University; Japón

Seventeen Leishmania stocks isolated from cutaneous lesions of Pakistani patients were studied by multilocus enzyme electrophoresis and by polymerase chain reaction amplification and sequencing of the cytochrome b (Cyt b) gene. Eleven stocks that expressed nine zymodemes were assigned to L. (Leishmania) major. All of them were isolated from patients in the lowlands of Larkana district and Sibi city in Sindh and Balochistan provinces, respectively. The remaining six, distributed in two zymodemes (five and one), isolated from the highland of Quetta city, Balochistan, were identified as L. (L.) tropica. The same result at species level was obtained by the Cyt b sequencing for all the stocks examined. No clear-cut association between the clinical features (wet or dry type lesions) and the Leishmania species involved was found. Leishmania (L.) major was highly polymorphic compared with L. (L.) tropica. This difference may be explained by the fact that humans may act as a sole reservoir of L. (L.) tropica in anthroponotic cycles; however, many wild mammals can be reservoirs of L. (L.) major in zoonotic cycles.

Sixteen Leishmania stocks, 15 isolated from patients with cutaneous (CL), mucocutaneous (MCL), or recurrent cutaneous leishmaniasis, plus one from a dog with CL in Salta and Corrientes Provinces, Argentina, were studied by multilocus enzyme electrophoresis. Thirteen of the stocks from humans were grouped in two zymodemes; nine termed as KMS 1, four as KMS 2, and assigned to Leishmania (Viannia) braziliensis. Two additional stocks from CL cases expressed a KMS 4 enzyme profile, corresponding to L. (V.) guyanensis. Although the parasites from the dog were also assigned to L. (V.) braziliensis, its zymodeme, KMS 3, was not expressed in any of the current human isolates. The characterization of Leishmania from a dog was done for the first time in Argentina. The importance of the intraspecific polymorphism in the induction of clinical forms and in the host-reservoir concept is briefly discussed, based on the zymodeme data of isolates from humans and dogs. The presence of L. (V.) guyanensis was confirmed in the country.

Leishmaniasis is a vector-borne protozoan infection affecting over 350 million people around the world. In Argentina cutaneous leishmaniasis is endemic in nine provinces and visceral leishmaniasis is spreading from autochthonous transmission foci in seven provinces. However, there is limited information about the diversity of the parasite in this country. Implementation of molecular strategies for parasite typing, particularly multilocus sequence typing (MLST), represents an improved approach for genetic variability and population dynamics analyses. We selected six loci as candidates implemented in reference strains and Argentinean isolates. Phylogenetic analysis showed high correlation with taxonomic classification of the parasite. Autochthonous Leishmania (Viannia) braziliensis showed higher genetic diversity than L. (Leishmania) infantum but low support was obtained for intra-L. braziliensis complex variants suggesting the need of new loci that contribute to phylogenetic resolution for an improved MLST or nested-MLST scheme. This study represents the first characterization of genetic variability of Leishmania spp. in Argentina. Fil: Marco, Jorge Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta. Sede Regional Oran. Instituto de Investigación de Enfermedades Tropicales; Argentina Fil: Barroso, Paola Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta. Sede Regional Oran. Instituto de Investigación de Enfermedades Tropicales; Argentina Fil: Locatelli, Fabricio M.. Kochi University. Kochi Medical School. Departament of Parasitology; Japón Fil: Cajal, S. Pamela. Universidad Nacional de Salta. Sede Regional Oran. Instituto de Investigación de Enfermedades Tropicales; Argentina Fil: Hoyos, Carlos Lorenzo. Universidad Nacional de Salta. Sede Regional Oran. Instituto de Investigación de Enfermedades Tropicales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Nevot, Cecilia. Kochi University. Kochi Medical School. Departament of Parasitology; Japón Fil: Lauthier, Juan José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina Fil: Tomasini, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina Fil: Juarez, Marisa. Universidad Nacional de Salta. Sede Regional Oran. Instituto de Investigación de Enfermedades Tropicales; Argentina Fil: Estevez, J. Octavio. Veterinaria Del Oeste; Argentina Fil: Korenaga, Masataka. Kochi University. Kochi Medical School. Departament of Parasitology; Japón Fil: Nasser, Julio. Universidad Nacional de Salta. Sede Regional Oran. Instituto de Investigación de Enfermedades Tropicales; Argentina Fil: Hashiguchi, Yoshihisa. Kochi University. Kochi Medical School. Dept.of Parasitology; Japón. Universidad Central del Ecuador y Proyecto Prometeo. Centro de Biomedicina; Ecuador Fil: Ruybal, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones En Microbiología y Parasitología Médica; Argentina

Natural infection of sand flies with Leishmania parasites was surveyed in an Amazonian area in Ecuador where leishmaniasis is endemic. Seventy-one female sand flies were dissected and one was positive for Leishmania protozoa. The species of this sand fly was identified as Lutzomyia (Lu.) tortura on the basis of morphologic characteristics. Analysis of the cytochrome b gene sequence identified the parasite as L. (Viannia) naiffi. We report the distribution of L. (V.) naiffi in Ecuador and detection of a naturally infected sand fly in the Ecuadorian Amazon and natural infection of Lu. tortura with Leishmania parasites in the New World.

American tegumentary leishmaniasis (ATL) is a group of zoonotic diseases caused by kinetoplastid flagellates of the genus Leishmania. A total of 66 patients diagnosed as positive ATL cases from northwest Argentina were included in this study. Leishmania stocks were isolated in vitro and analyzed over promastigote cultures sown on FTA through nested PCR and sequence of cytochrome b (cyt b). The molecular analysis resulted in the incrimination of L. (Viannia) braziliensis as the predominant species in the studied area, identifying two genotypes of L. (V.) braziliensis, 24 cases of Ab-1 cyt b and 41 cases of Ab-2 cyt b. One L. (V.) guyanensis strain was obtained from a traveler from the Brazilian Amazon. The prevalence of different genotypes was in agreement with previous studies, suggesting the necessity for new systems to study the genetic diversity in more detail. Most of the cases typified in this study were registered in the area of Zenta Valley (Orán, Hipólito Yrigoyen, and Pichanal cities), pointing a link between genotype and geographical origin of the sample. Sex and age distribution of the patients indicate that the transmission was predominantly associated with rural areas or rural activities, although the results might not exclude the possibility of peri-urban transmission. This work represents, so far, the largest isolation and molecular characterization of ATL cases in Argentina Fil: Locatelli, Fabricio M.. Kochi University. Kochi Medical School. Dept.of Parasitology; Japón. Universidad Nacional de Salta. Sede Regional Oran. Instituto de Investigación de Enfermedades Tropicales; Argentina Fil: Cajal, S. Pamela. Universidad Nacional de Salta. Instituto de Investigaciones en Enfermedades Tropicales; Argentina Fil: Barroso, Paola Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta. Sede Regional Oran. Instituto de Investigación de Enfermedades Tropicales; Argentina Fil: Lauthier, Juan José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina Fil: Mora, Maria Celia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina Fil: Juarez, Marisa. Universidad Nacional de Salta. Instituto de Investigaciones en Enfermedades Tropicales; Argentina Fil: Kato, Hirotomo. Hokkaido University; Japón Fil: Nasser, Julio R.. Universidad Nacional de Salta. Instituto de Investigaciones en Enfermedades Tropicales; Argentina Fil: Hashiguchi, Yoshihisa. Universidad Central del Ecuador y Proyecto Prometeo; Ecuador. Kochi University. Kochi Medical School. Dept.of Parasitology; Japón Fil: Korenaga, Masataka. Kochi University. Kochi Medical School. Dept.of Parasitology; Japón Fil: Marco, Jorge Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta. Sede Regional Oran. Instituto de Investigación de Enfermedades Tropicales; Argentina

Background The diagnosis of the leishmaniases poses enormous challenges in Argentina. The Polymorphism-Specific PCR (PS-PCR) designed and validated in our laboratories has been proven effective for typifying the Leishmania genus from cultured material. Here we evaluated the performance of this method in the diagnosis of American tegumentary leishmaniasis (ATL) and the rapid identification of Leishmania spp. directly from clinical specimens. Methods A total of 63 patients from northwestern Argentina, with cutaneous or mucocutaneous lesions, underwent an ATL diagnosis protocol which included clinical examination, Leishmanin skin test, and microscopic examination of dermal smears. In addition, we performed PS-PCR on DNA directly extracted from the specimens scraped from the lesions. Results Out of the 63 patients, 44 were classified as ATL cases and 19 as non-ATL cases. The diagnostic sensitivity of the microscopic analysis of dermal smears and PS-PCR individually were 70.5% and 81%, respectively. When performing both tests in parallel, this parameter increased significantly to 97.6% (p = 0.0018). The specificities, on the other hand, were 100%, 84.2%, and 83.3% for the combination, respectively (p > 0.05). Using the PS-PCR analysis we successfully identified the Leishmania spp. in 31 out of the 44 ATL cases. Twenty-eight (90.3%) cases were caused by L. (V.) braziliensis, two (6.5%) by L. (V.) guyanensis, and one (3.2%) by L. (V.) panamensis. Conclusions The efficacy of the ATL diagnosis was significantly improved by combining the dermal smear examination with a PS-PCR analysis. Our strategy allowed us to reach the diagnosis of ATL with high accuracy regarding the species of the etiological agent in 70.5% of the cases. Moreover, we diagnosed two cases of the disseminated cutaneous form caused by L. (V.) braziliensis and a cutaneous case due to L. (V.) panamensis infection, both findings reported for the first time in Argentina.

Sand flies from the Andean areas of Ecuador and Peru were examined for Leishmania infections by using our recently established molecular mass screening method. Leishmanial minicircle DNA-positive sand flies were detected in 3 of 192 and 1 of 462 samples from Ecuador and Peru, respectively. Sand fly species were identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the 18S ribosomal RNA (rRNA) gene, and the positive flies were Lutzomyia (Lu.) ayacuchensis and Lu. peruensis, respectively. Furthermore, cytochrome b and mannose-phosphate isomerase gene sequence analyses identified the parasites from Ecuador and Peru as Leishmania (Leishmania) mexicana and L. (Viannia) peruviana, respectively. Thus, the mass screening method was confirmed to be a powerful tool for sand fly research.

Activation of innate immunity using adjuvants that activate Toll-like receptor 4 pathways have great potential for improving the protection induced by parasite vaccines. We investigated protective and therapeutic effects of a vaccine against leishmaniasis containing a combination of an adjuvant synthetic lipid A-analogue, ONO-4007 and Leishmania amazonensis antigens. ONO-4007 was co-injected with soluble and membrane-enriched L. amazonensis-amastigote antigens into BALB/c mice that had either already been infected with 1 x 10(6) L. amazonensis promastigotes (immunotherapy study) or before challenge with the same infectious dose (immunoprophylaxis study). Sixty percent of mice vaccinated before infectious challenge controlled their Leishmania infections - defined by the absence of footpad-swelling and negative Leishmania cultures - compared to 0% of controls, and 40% of mice vaccinated after infection resolved their infections compared to 0% of controls. Protective immunity in both immunoprophylaxis and immunotherapy models was associated with increased protein production of IL-12 and IFN-gamma. These data suggest that vaccination with a combination of ONO-4007 and amastigote antigens of L. amazonensis may be useful for the prevention and treatment of leishmaniasis, and that the protective immunity induced is associated with the production of type-1 cytokines.

The surveillance of prevalent Leishmania and sand fly species in endemic areas is important for prediction of the risk and expansion of leishmaniasis. In this study, we developed a polymerase chain reaction (PCR)-based method for detection of Leishmania minicircle DNA within individual sand flies. Using this method, we detected minicircle DNA in 6 (3.3%) of 183 sand flies, while 5 (3.5%) of 143 were positive for Leishmania promastigotes in the same areas by microscopic examination. The species were identified as Leishmania (Leishmania) mexicana by nucleotide sequencing of the cytochrome b gene. Additionally, all the Leishmania-positive sand flies were identified as Lutzomyia ayacuchensis by the restriction enzyme digestion of the PCR-amplified 18S ribosomal RNA gene fragments. Since this combined method is relatively easy and can process a large number of samples, it will be a powerful tool for the rapid identification of prevalent sand fly and Leishmania species as well as monitoring the infection rate in sand fly populations in endemic areas. Fil: Kato, Hirotomo. Yamaguchi University; Japón Fil: Uezato, Hiroshi. University of the Ryukyus; Japón Fil: Katakura, Ken. Hokkaido University; Japón Fil: Calvopina, Manuel. Kochi University. Kochi Medical School; Japón Fil: Marco, Jorge Diego. Kochi University. Kochi Medical School; Japón. 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. Kochi University. Kochi Medical School; Japón. 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: Gomez, Eduardo. Universidad Católica de Guayaquil; Ecuador Fil: Mimori, Tatsuyuki. Kumamoto University; Japón Fil: Korenaga, Masataka. Kochi University. Kochi Medical School; Japón Fil: Iwata, Hiroyuki. Yamaguchi University; Japón Fil: Nonaka, Shigeo. University ok the Ryukyus; Japón Fil: Hashiguchi, Yoshihisa. Kochi University. Kochi Medical School; Japón

Recently, two techniques, polymerase chain reaction (PCR) amplification and sequencing of cytochrome b gene (cyt b gene sequencing) and polymorphism-specific PCR (PS-PCR) were recommended for Leishmania species identification. Before this study, however, the accuracy of these methods had not been tested against the multilocus enzyme electrophoresis, the current gold standard technique on this task. Therefore, a trial was done for the first time to compare the results obtained by these techniques, using 17 Argentinean Leishmania stocks in independent assays. For all the stocks examined, the same results at species level were obtained by the three techniques. Among them, 14 were assigned to L. (Viannia) braziliensis, and three to L. (V.) guyanensis. The two techniques, cyt b gene sequencing and PS-PCR, were able to distinguish between all the proven species responsible for leishmaniases in Argentina. Thus, both techniques were validated and could be used independently for the species designation of Leishmania parasites in the country. Fil: Marco, Jorge Diego. Kochi University. Kochi Medical School; Japón Fil: Uezato, Hiroshi. Universidad de Ryukyus. Okinawa; Japón Fil: Mimori, Tatsuyuki. Universidad de Kumamoto; Japón 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. Kochi University. Kochi Medical School; Japón Fil: Korenaga, Masataka. Kochi University. Kochi Medical School; Japón Fil: Nonaka, Shigeo. Universidad de Ryukyus, Okinawa.; Japón Fil: Basombrío, Miguel Ángel Manuel. 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: Taranto, Nestor Juan. Instituto de Enfermedades Tropicales. Sede Regional Oran. Universidad Nacional de Salta; Argentina Fil: Hashiguchi, Yoshihisa. Kochi University. Kochi Medical School; Japón