Detection and identification of Leishmania species within naturally infected sand flies in the Andean areas of Ecuador by a polymerase chain reaction

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; JapónFil: Uezato, Hiroshi. University of the Ryukyus; JapónFil: Katakura, Ken. Hokkaido University; JapónFil: Calvopina, Manuel. Kochi University. Kochi Medical School; JapónFil: 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; ArgentinaFil: 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; ArgentinaFil: Gomez, Eduardo. Universidad Católica de Guayaquil; EcuadorFil: Mimori, Tatsuyuki. Kumamoto University; JapónFil: Korenaga, Masataka. Kochi University. Kochi Medical School; JapónFil: Iwata, Hiroyuki. Yamaguchi University; JapónFil: Nonaka, Shigeo. University ok the Ryukyus; JapónFil: Hashiguchi, Yoshihisa. Kochi University. Kochi Medical School; Japó

Leishmania isoenzyme polymorphisms in Ecuador: Relationships with geographic distribution and clinical presentation

Background: Determinants of the clinical presentation of the leishmaniases are poorly understood but Leishmania species and strain differences are important. To examine the relationship between clinical presentation, species and isoenzyme polymorphisms, 56 Leishmania isolates from distinct presentations of American tegumentary leishmaniasis (ATL) from Ecuador were analyzed. Methods: Isolates were characterized by multilocus enzyme electrophoresis for polymorphisms of 11 isoenzymes. Patients were infected in four different ecologic regions: highland and lowland jungle of the Pacific coast, Amazonian lowlands and Andean highlands. Results: Six Leishmania species constituting 21 zymodemes were identified: L. (Viannia) panamensis (21 isolates, 7 zymodemes), L. (V.) guyanensis (7 isolates, 4 zymodemes), L. (V.) braziliensis (5 isolates, 3 zymodemes), L. (Leishmania) mexicana (11 isolates, 4 zymodemes), L. (L.) amazonensis (10 isolates, 2 zymodemes) and L. (L.) major (2 isolates, 1 zymodeme). L. panamensis was the species most frequently identified in the Pacific region and was associated with several clinical variants of cutaneous disease (CL); eight cases of leishmaniasis recidiva cutis (LRC) found in the Pacific highlands were associated with 3 zymodemes of this species. Mucocutaneous leishmaniasis found only in the Amazonian focus was associated with 3 zymodemes of L. braziliensis. The papular variant of CL, Uta, found in the Andean highlands was related predominantly with a single zymodeme of L. mexicana. Conclusion: Our data show a high degree of phenotypic variation within species, and some evidence for associations between specific variants of ATL (i.e. Uta and LRC) and specific Leishmania zymodemes. This study further defines the geographic distribution of Leishmania species and clinical variants of ATL in Ecuador

Andean Leishmaniasis in Ecuador caused by Infection with Leishmania Mexicana and L. Major-Like Parasites

Between 1986 and 1988, epidemiologic studies were carried out in a small rural community in an Andean region of Ecuador, where cutaneous leishmaniasis is highly endemic. A total of 25 human cases, positive for Leishmania parasites by culture and/or smear, were examined. Fourteen of the cases were in infants less than one year of age, suggesting intradomiciliary transmission of the disease. Clinically, many of these cases were similar to descriptions of "uta," a form of cutaneous leishmaniasis which occurs in Andean regions of Peru and is reportedly caused by L. peruviana. Of the 11 positive cultures obtained from human cases in the present study, eight were identified by molecular characterization as L. mexicana and three were identified as L. major-like. Two additional isolates of L. mexicana were also made from an infected dog and from a sand fly, Lutzomyia ayacuchensis, living in the region, thus implicating the latter species as possible reservoir and vector, respectively, of L. mexicana in this highland community. The significance and validity of recent isolates of L. major-like parasites from the New World are also discussed

Molecular Epidemiology for Vector Research on Leishmaniasis

Leishmaniasis is a protozoan disease caused by the genus Leishmania transmitted by female phlebotomine sand flies. Surveillance of the prevalence of Leishmania and responsive vector species in endemic and surrounding areas is important for predicting the risk and expansion of the disease. Molecular biological methods are now widely applied to epidemiological studies of infectious diseases including leishmaniasis. These techniques are used to detect natural infections of sand fly vectors with Leishmania protozoa and are becoming powerful tools due to their sensitivity and specificity. Recently, genetic analyses have been performed on sand fly species and genotyping using PCR-RFLP has been applied to the sand fly taxonomy. In addition, a molecular mass screening method has been established that enables both sand fly species and natural leishmanial infections to be identified simultaneously in hundreds of sand flies with limited effort. This paper reviews recent advances in the study of sand flies, vectors of leishmaniasis, using molecular biological approaches

Distribution of Lutzomyia ayacuchensis, the vector of Andean-type cutaneous leishmaniasis, at different altitudes on the Andean slope of Ecuador

Distribution of the vector species is a major risk factor for the endemicity of leishmaniasis. In the present study, the vertical distribution of Lutzomyia (Lu.) ayacuchensis, the vector of Leishmania (Leishmarzia) mexicana in the Ecuadorian Andes, was surveyed at different altitudes (300-2500 m above sea level) of the Andean slope. The vector species Lu. ayacuchensis was identified at an altitude of 650 m and a higher areas, and higher distribution ratio of the species was observed at higher altitudes. In addition, high ratios of L. (L.) mexicana infection were detected in higher areas, but none in lower populations of sand flies. Since an association between sand fly populations and vector competence is suggested in Lu. ayacuchensis, haplotype analysis was performed on the species from different altitudes of the study areas; however, no apparent difference Was observed among populations. These results suggested that Lu. ayacuchensis in Andean slope areas of Ecuador has the potential to transmit L. (L.) mexicana and spread leishmaniasis in these areas. (C) 2014 Elsevier B.V. All rights reserved

Genetic divergence in populations of Lutzomyia ayacuchensis, a vector of Andean-type cutaneous leishmaniasis, in Ecuador and Peru

Haplotype and gene network analyses were performed on mitochondrial cytochrome oxidase I and cytochrome b gene sequences of Lutzomyia (Lu.) ayacuchensis populations from Andean areas of Ecuador and southern Peru where the sand fly species transmit Leishmania (Leishmania) mexicana and Leishmania (Viannia) peruviana, respectively, and populations from the northern Peruvian Andes, for which transmission of Leishmania by Lu. ayacuchensis has not been reported. The haplotype analyses showed higher intrapopulation genetic divergence in northern Peruvian Andes populations and less divergence in the southern Peru and Ecuador populations, suggesting that a population bottleneck occurred in the latter populations, but not in former ones. Importantly, both haplotype and phylogenetic analyses showed that populations from Ecuador consisted of clearly distinct clusters from southern Peru, and the two populations were separated from those of northern Peru. Crown Copyright (C) 2014 Published by Elsevier B.V. All rights reserved

マイクロアレイ カイセキ ニ ヨル サイボウナイ キセイ ゲンチュウ カンセンジ ノ シュクシュ サイボウ イデンシ ノ ドウタイ

鞭毛虫類であるリーシュマニア原虫とマクロファージ細胞を用いて、経時的に細胞内に侵入、増殖したLeishmania amazonensis のアマスティゴート型原虫を、顕微鏡で観察するとともに、宿主細胞の動態解析をcDNA マイクロアレイでおこなった。マウス・マクロファージ細胞株（J774A.1）の未感染細胞、原虫感染初期、感染成立期の感染宿主細胞および、原虫からTotal RNA を分離した。それらのサンプルをオリゴマイクロアレイ(Agilent Technologies)(4 X 44K)１色法を用いて、約41,252 個のプローブについて、解析した