SPATIAL DISTRIBUTION OF PHLEBOTOMINAE IN PUERTO IGUAZU-MISIONES, ARGENTINA-BRAZIL-PARAGUAY BORDER AREA

SUMMARY The first Argentinian autochthonous human case of visceral leishmaniasis (VL) was confirmed in Posadas (Misiones) in 2006. Since then, the disease has increased its incidence and geographical distribution. In the 2006-2012 period, 107 human cases were detected (11 deaths). The presence of Lutzomyia longipalpis was detected in peridomiciles in Puerto Iguazú urban area in 2010; some of these findings were associated with households where cases of canine VL had already been reported. The objective of this study was to ascertain the abundance and spatial distribution of Lu. longipalpis in Puerto Iguazú City, on the Argentina-Brazil-Paraguay border. Lu. longipalpis proved to be exclusively urban and was found in 31% of the households sampled (n = 53), 67% of which belonged to areas of low abundance, 20% to areas of moderate abundance and 13% to areas of high abundance. Nyssomyia whitmani was the only species found both in urban and peri-urban environments, and Migonemyia migonei was registered only on the outskirts of the city. Due to the fact that Puerto Iguazú is considered to be at moderate risk at the moment, it is necessary to intensify human and canine case controls, as well as take integrated prevention and control measures regarding the environment, vectors and reservoirs on the Argentina-Brazil-Paraguay border area.

PHLEBOTOMINE SANDFLIES IN RURAL LOCATIONS IN THE STATE OF PARANA, SOUTHERN BRAZIL

SUMMARYThis study reports the fauna and frequency of sandflies in domestic animal shelters, residences and other ecotopes in rural areas of the municipality of Bandeirantes, Paraná State. Sandflies were collected twice in eight rural villages by using Falcon traps from 8pm to 6am in 2008. In these localities 4,790 sandflies were collected, which were represented by ten sandfly species, prevailing of Nyssomyia neivai and Nyssomyia whitmani species. It was observed that animal shelters are the domestic ecotopes where there is the greatest frequency of these insects. The localities where the collections were made had the environmental characteristics that allow the persistence of transmission of parasites from the American tegumentary leishmaniasis. Although the fauna and the behavior of sandflies species are similar in different localities, the method of controlling these insects should be adjusted to the environmental characteristics of each one of the most diverse endemic areas of American tegumentary leishmaniasis in the municipalities of Paraná State.

DISTINCT CELLULAR MIGRATION INDUCED BY Leishmania infantum chagasi AND SALIVA FROM Lutzomyia longipalpis IN A HEMORRHAGIC POOL MODEL

Recruitment of a specific cell population after Leishmania infection can influence the outcome of the disease. Cellular migration in response to Leishmania or vector saliva has been reported in air pouch model, however, cellular migration induced by Leishmania associated with host's blood and vector saliva in this model has not been described. Herein we investigated cellular migration into air pouch of hamster after stimulation with combination of L. chagasi and host's blood and Lutzomyia longipalpis saliva. Migration induced by saliva was 3-fold more than those induced by L. chagasi alone. Additionally, L. chagasi associated with blood and saliva induced significantly even more leukocytes into air pouch than Leishmania alone. L. chagasi recruited a diverse cell population; however, most of these cells seem to have not migrated to the inflammatory exudate, remaining in the pouch lining tissue. These results indicate that L. chagasi can reduce leukocyte accumulation to the initial site of infection, and when associated with vector saliva in the presence of blood components, increase the influx of more neutrophils than macrophages, suggesting that the parasite has developed a strategy to minimize the initial inflammatory response, allowing an unlimited progression within the host. This work reinforces the importance of studies on the salivary components of sand fly vectors of leishmaniasis in the transmission process and the establishment of the infection.

EVIDENCE OF Leishmania (Leishmania) infantum INFECTION IN DOGS FROM JUIZ DE FORA, MINAS GERAIS STATE, BRAZIL, BASED ON IMMUNOCHROMATOGRAPHIC DUAL-PATH PLATFORM (DPP®) AND PCR ASSAYS

In Brazil, domestic dogs are branded as the primary reservoir for zoonotic visceral leishmaniasis, due to the clear positive correlation observed between human and canine infection rates. This study aimed to carry out a serological survey of canine visceral leishmaniasis (CVL) in dogs housed at a public kennel in the municipality of Juiz de Fora, Minas Gerais State, Brazil, using the immunochromatographic TR DPP® CVL rapid test. Additionally, conventional and/or real time PCR assay was used to detect and confirm L. infantum infection in the DPP positive dogs only. Of the 400 dogs studied, most did not present clinical signs for CVL (p < 0.05), and fifteen (3.8%) were seropositive in the DPP test. There was no statistically significant difference between the DPP seropositive dogs and the clinical signs of the disease (p > 0.05). Both conventional and real time PCR tests confirmed L. infantum infection in nine (75.0%) of the twelve DPP seropositive dogs that remained alive during the follow-up period. This study is the first seroepidemiologic survey of CVL held in the city of Juiz de Fora, and the results reinforce the idea that this disease is currently in a process of expansion and urbanization in Brazil. Furthermore, this study highlights the use of the DPP test as an alternative for diagnosing CVL in large and mid-sized cities, due to its ease of implementation.

FIRST REPORT ON OTOTOXICITY OF MEGLUMINE ANTIMONIATE

Introduction: Pentavalent antimonials are the first drug of choice in the treatment of tegumentary leishmaniasis. Data on ototoxicity related with such drugs is scarcely available in literature, leading us to develop a study on cochleovestibular functions. Case Report: A case of a tegumentary leishmaniasis patient, a 78-year-old man who presented a substantial increase in auditory threshold with tinnitus and severe rotatory dizziness during the treatment with meglumine antimoniate, is reported. These symptoms worsened in two weeks after treatment was interrupted. Conclusion: Dizziness and tinnitus had already been related to meglumine antimoniate. However, this is the first well documented case of cochlear-vestibular toxicity related to meglumine antimoniate.

IDENTIFICATION OF Leishmania infantum IN PUERTO IGUAZÚ, MISIONES, ARGENTINA

 The emergence of zoonotic visceral leishmaniasis (ZVL) in Latin America is a growing public health problem. The urbanization of ZVL has been observed in different countries around the world, and there are a growing number of reports drawing attention to the emergence of this infection in new locations, as well as its increase in previously established areas of endemicity. In the city of Posadas, Misiones province, Northeastern Argentina, the transmission of ZVL associated with canines and Lutzomyia longipalpis was first reported in 2006. In the city of Puerto Iguazú, also in Misiones province, the first human case of ZVL was reported in February 2014. From 209 surveyed dogs, 15 (7.17%) were identified as positive by serological and/or parasitological methods. Amplification was observed in 14 samples and in all cases the species implicated was Leishmania infantum. To the authors’ knowledge, this is the first molecular characterization of L. infantum from dogs in this area.

PHLEBOTOMINE FAUNA (DIPTERA: PSYCHODIDAE) IN AN AREA OF FISHING TOURISM IN CENTRAL-WESTERN BRAZIL

The aim of this study was to identify behavioral aspects of the sandfly fauna of a fishing tourism area in the municipality of Bonito (MS). Monthly captures were undertaken from December 2009 to November 2010, using automatic CDC type light traps, from 18h00 to 06h00, in a forested area, a savannah area, peridomiciles and animal shelters near peridomiciliary areas. Nyssomyia whitmani was the most frequent out of a total of 6,699 specimens collected, belonging to 16 species, followed by Psathyromyia bigeniculata and Lutzomyia longipalpis, found in all the environments investigated, though in their greatest numbers in the animal shelters. Ny. whitmani exhibited its highest frequencies during the dry months, coincident with the fishing season, when the risk of transmission of cutaneous leishmaniasis for tourists and inhabitants increases. Noteworthy was the finding of two species naturally infected by flagellates: Ny. whitmani and Pa. bigeniculata. The local population and visiting tourists should be warned of the threat posed by leishmaniasis and the health authorities alerted to the need for adopting environmental sanitary measures, especially regarding such animal shelters as they seem to provide favorable conditions to the proliferation, maintenance and breeding opportunities of phlebotomines.

NEW PRIMERS FOR DETECTION OF Leishmania infantum USING POLYMERASE CHAIN REACTION

SUMMARY Leishmania infantum causes visceral leishmaniasis (VL) in the New World. The diagnosis of VL is confirmed by parasitological and serological tests, which are not always sensitive or specific. Our aim was to design new primers to perform a Polymerase Chain Reaction (PCR) for detecting L. infantum. Sequences of the minicircle kinetoplast DNA (kDNA) were obtained from GenBank, and the FLC2/RLC2 primers were designed. Samples of DNA from L. infantum, Leishmania amazonensis, Leishmania braziliensis, Leishmania guyanensis, Leishmania naiffi, Leishmania lainsoni, Leishmania panamensis, Leishmania major and Trypanosoma cruzi were used to standardize the PCR. PCR with FLC2/RLC2 primers amplified a fragment of 230 bp and the detection limit was 0.2 fg of L. infantum DNA. Of the parasite species assayed, only L. infantum DNA was amplified. After sequencing, the fragment was aligned to GenBank sequences, and showed (99%) homology with L. infantum. In the analysis of blood samples and lesion biopsy from a dog clinically suspected to have VL, the PCR detected DNA from L. infantum. In biopsy lesions from humans and dogs with cutaneous leishmaniasis, the PCR was negative. The PCR with FLC2/RLC2 primers showed high sensitivity and specificity, and constitutes a promising technique for the diagnosis of VL.

PERFORMANCE OF CONVENTIONAL PCRs BASED ON PRIMERS DIRECTED TO NUCLEAR AND MITOCHONDRIAL GENES FOR THE DETECTION AND IDENTIFICATION OF Leishmania spp.

In visceral leishmaniasis, the detection of the agent is of paramount importance to identify reservoirs of infection. Here, we evaluated the diagnostic attributes of PCRs based on primers directed to cytochrome-B (cytB), cytochrome-oxidase-subunit II (coxII), cytochrome-C (cytC), and the minicircle-kDNA. Although PCRs directed to cytB, coxII, cytC were able to detect different species of Leishmania, and the nucleotide sequence of their amplicons allowed the unequivocal differentiation of species, the analytical and diagnostic sensitivity of these PCRs were much lower than the analytical and diagnostic sensitivity of the kDNA-PCR. Among the 73 seropositive animals, the asymptomatic dogs had spleen and bone marrow samples collected and tested; only two animals were positive by PCRs based on cytB, coxII, and cytC, whereas 18 were positive by the kDNA-PCR. Considering the kDNA-PCR results, six dogs had positive spleen and bone marrow samples, eight dogs had positive bone marrow results but negative results in spleen samples and, in four dogs, the reverse situation occurred. We concluded that PCRs based on cytB, coxII, and cytC can be useful tools to identify Leishmania species when used in combination with automated sequencing. The discordance between the results of the kDNA-PCR in bone marrow and spleen samples may indicate that conventional PCR lacks sensitivity for the detection of infected dogs. Thus, primers based on the kDNA should be preferred for the screening of infected dogs.

THE PROCESS OF Leishmania INFECTION - DISEASE AND NEW PERSPECTIVES OF PALEOPARASITOLOGY

Species of the genus Leishmania (Kinetoplastida, Trypanosomatidae) are causative agents of leishmaniasis, a complex disease with variable clinical spectrum and epidemiological diversity, constituting, in some countries, a serious public health problem. The origin and evolution of leishmaniasis has been under discussion regarding some clinical and parasitological aspects. After the introduction of paleoparasitology, molecular methods and immunodiagnostic techniques have been applied allowing the recovery of parasite remains, as well as the diagnosis of past infections in humans and other hosts. The dating of archaeological samples has allowed the parasitological analysis in time and space. This manuscript presents the state of the art of leishmaniasis and prospects related to paleoparasitology studies and their contribution to the evolutionary and phylogenetic clarification of parasites belonging to the genus Leishmania, and the leishmaniasis caused by them.

Leishmania mexicana: aspectos taxonómicos y rareza de la infección humana en Três Braços, Bahia, Brasil

Del estúdio de 51 stocks de Leishmania aislados de pacientes humanos de leishmaniasis cutaneomucosa en Três Braços, Bahia, Brasil, los autores describen, en detalle, el análisis de los dos únicos stocks de L. mexicana, identificando uno de ellos como L. mexicana amazonensis. El otro aislado permanece en posición taxonómica no definida pues considerándosele como un miembro de L. mexicana, encuéntranse dificultades para su identificación subespecífica. Evaluan también los parâmetros biológicos e isoenzimáticos y discuten el papel de los anticuerpos monoclonales en la tipificación de éstos stocks. Los autores remarcan la rareza de la transmisión de parásitos del complejo L. mexicana en esta región, aun cuando estudien epidemiológicamente por mais de 8 anos la infección humana.

Glucantime resistant Leishmania promastigotes are sensitive to pentostam

Growth inhibition in vitro tests were used to study the susceptibility to pentostam of different Leishmania strains involved in cutaneous and mucocutaneos leishmaniasis - one glucantime sensitive strain, three naturally glucantime resistant strains and one glucantime resistant line developed by in vitro drug exposure. Contrasting with the high degree , of glucantime resistance, all strains were sensitive to pentostam. These differences suggest that there is some relationship between chemical structure and in vitro activity for these antimonial compounds. These data justify a clinical re-evaluation to compare therapeutic efficacy of glucantime and pentostam in the treatment of leishmaniasis.

Estudo epidemiológico sobre Leishmaniose tegumentar (LT) no município de Corguinho, Mato Grosso do Sul: estudos na população humana

Com o objetivo de se conhecer a prevalência da infecção por leishmaniose tegumentar americana, e caracterização da população atingida, em área de transmissão recente, na localidade de Boa Sorte, município de Corguinho, MS, procedeu-se ao estudo, no período de março/91 a março/94, tia população residente, compreendendo 150 habitantes. Destes, 12 apresentavam lesões suspeitas de leishmaniose tegumentar. Em 8 deles foi possível a confirmação por meio de exames parasitológicos. Apenas um paciente apresentou a forma mucosa, os demais manifestaram as formas cutâneas, sendo: ulcerada (3), úlcero-verrucosa (1), úlcero- vegetante (1), placa-infiltrada (1) e lesão nodular com exuberante adenopatia regional (1). Os pacientes responderam bem ao tratamento com antimoniato de N-metil- glucamina (glucantime), 10 a 20mg Sbv/kg/dia durante 20 dias, com cicatrização das lesões e raros efeitos colaterais. O parasito isolado de todos os pacientes foi identificado como Leishmania (Viannia) braziliensis, através de anticorpos monoclonais. O teste de Montenegro aplicado em 150 moradores revelou 32 reagentes. Destes, 6 eram portadores da doença, 21 mostraram seqüelas sugestivas da parasitose e 5 não apresentaram sitiais da infecção. A faixa etária atingida pela parasitose compreendia de 22 a 78 anos com predomínio de homens (75%). A transmissão até o momento revelou-se de caráter extradomiciliar.

The FML (Fucose Mannose Ligand) of Leishmania donovani: a new tool in diagnosis, prognosis, transfusional control and vaccination against human kala-azar

The Fucose-Mannose Ligand (FML) of Leishmania donovani is a complex glycoproteic fraction. Its potential use as a tool for diagnosis of human visceral leishmaniasis was tested with human sera from Natal, Rio Grande do Norte, Brazil. The FML-ELISA test, showed 100% sensitivity and 96% specificity, identifying patients with overt kala-azar (p < 0.001, when compared to normal sera), and subjects with subclinical infection. More than 20% apparently healthy subjects with positive reaction to FML developed overt kala-azar during the following 10 months. In the screening of human blood donnors, a prevalence of 5% of sororeactive subjects was detected, attaining 17% in a single day. The GP36 glycoprotein of FHL is specifically reconized by human kala-azar sera. The immunoprotective effect of FML on experimental L. donovanii infection was tested in swiss albino mice. The protection scheemes included three weekly doses of FML, supplemented or not with saponin by the subcutaneous or intraperitoneal routes and challenge with 2x 10(7) amastigotes of Leishmania donovani. An enhancement of 80.0 % in antibody response (p<0.001) and reduction of 85.5 % parasite liver burden (p<0.001) was detected in animals immunized with FML saponin, unrespectivety of the immunization route.

Inquérito canino em foco recente de leishmaniose tegumentar no Município de Sabará, região metropolitana de Belo Horizonte

Em 1992, foi obsewada lesão cutânea por Leishmania em um cão do município de Sabará, Minas Gerais, onde já haviam sido registrados casos humanos de leishmaniose tegumentar. O parasita foi caracterizado como pertencente ao subgênero Viannia, ao qual pertence a Leishmania braziliensis, principal espécie encontrada tia região sudeste do Brasil. Com o objetivo de determinar o papel do cão no ciclo de transmissão da doença, foi realizado um inquérito canino na área. Foram examinados 631 cães, sendo a soroprevalência para leishmaniose igual a 3,2%. Foi observada a proximidade ou presença no mesmo domicílio de cães e pessoas doentes. Este aspecto fala a favor da transmissão domiciliar ou peridomiciliar, com o cão infectado podendo atuar como fator de risco nesta área periurbana. Entretanto, a baixa soroprevalência encontrada deve-se provavelmente ao pequeno papel deste animal na transmissão da doença neste foco recente da doença.