GENETIC DIVERSITY OF Lutzomyia (Nyssomyia) intermedia IN AN ENDEMIC AREA OF AMERICAN CUTANEOUS LEISHMANIASIS, STATE OF PARANÁ, BRAZIL / Diversidad genética de Lutzomyia (Nyssomyia) intermedia en un área endémica de leishmaniasis cutánea americana del estado de Paraná, Brasil

ABSTRACT Lutzomyia intermedia (Diptera: Psychodidae) features as one of the main vectors that are involved in the transmission of American cutaneous leishmaniasis (ACL) in the Neotropical region. However, genetic studies involving this taxon are still incipient and important for understanding the level of variability of different populations, their role, and implications as vectors. The aim of this study was to determine the level of genetic diversity of L. intermedia present in the Ribeira River Valley, an area of ACL transmission in the state of Paraná, Brazil, through the Random amplified polymorphic DNA (RAPD). Two municipalities were chosen to collect sand flies: Cerro Azul (new transmission area of the ACL) and Adrianópolis (endemic area of the ACL). The insects were captured in the house, in the peridomicile and in the wild (forest). Two of the used markers made it possible to estimate the polymorphism of the studied populations, resulting in 40 genotypes, most of them from peridomicile. The dendrogram generated by the analysis with the primer A10 showed different degrees of similarity, suggesting that there may be gene flow in the studied populations. The Principal Coordinate Analysis (PCO) with the A2 primer, was useful in grouping L. intermedia according to its ecological and geographical origin. There was no distinction between the lineages composing the L. intermedia complex. The results of this study, with the record of great genotypic diversity in L. intermedia, may contribute to explain the maintenance of the life cycle of Leishmania braziliensis (Kinetoplastida: Trypanosomatidae) in the region.

RESUMEN Lutzomyia intermedia (Diptera: Psychodidae) es uno de los principales vectores que participan en la transmisión de leishmaniasis cutánea americana (LCA) en la región Neotropical. A pesar de que aún los estudios genéticos que involucran a este taxón son incipientes, tienen una gran importancia para comprender el nivel de variabilidad de las diferentes poblaciones y sus implicaciones en su papel vectorial. El objetivo de este estudio fue determinar el nivel de diversidad genética de L. intermedia presente en el Valle del Río Ribeira, área de transmisión de LCA en el estado de Paraná, Brasil, mediante RAPD (ADN polimórfico amplificado aleatoriamente). Los flebótomos fueron recolectados en los municipios Cerro Azul (nueva área de transmisión de LCA) y Adrianópolis (área endémica de LCA), donde fueron capturados en ambientes residenciales, en el peridomicilio y en el bosque. Dos de los marcadores utilizados permitieron estimar el polimorfismo en las poblaciones estudiadas con la obtención de 40 genotipos, la mayoría de ellos en el peridomicilio. El dendrograma generado por el análisis con el cebador A10 mostró diferentes grados de similitud, lo que sugiere que puede haber flujo gènico en las poblaciones. El Análisis de Coordenadas Principales (PCO) con el cebador A2 fue útil para agrupar L. intermedia según su origen ecológico y geográfico. No hubo distinción entre los linajes que componen el complejo L. intermedia. Los resultados de este estudio, con el registro de gran diversidad genotipica en L. intermedia, pueden contribuir a explicar el mantenimiento del ciclo biológico de Leishmania braziliensis (Kinetoplastida: Trypanosomatidae) en la región.

Guinea Pigs Naturally Infected by Leishmania enriettii: Clinical Analyses, Parasite Isolation and Identification

Abstract Leishmania enriettii has only been found in Curitiba metropolitan region, southern Brazil were it was first observed in Cavia porcellus from the vivarium of Instituto de Biologia e Pesquisas Tecnológicas (IBPT - today named TECPAR) by Medina, 1944. Despite more than a half century from its discovery and several research articles on this species, the natural clinical signs in guinea pigs and the parasite genetic variability is still unclear. The aims of this study were to describe the clinical features, investigate the potential wild reservoirs and, in addition, we intended to understand the polymorphism trait of the species. We analyzed 26 naturally infected guinea pigs from eight Paraná state cities. All animals showed lesions compatible with leishmaniosis, such as skin nodules or ulcers on body extremities. Direct examination of the lesion samples obtained by fine-needle aspiration or punch biopsy was conducted followed by isolation and identification of parasite DNA by random amplification of polymorphic DNA (RAPD)-PCR. Through the direct exam, a large number of intracellular amastigote forms were observed in the lesions. Different strains of the parasite, isolated from the 26 animals, were grouped in 5 clusters of approximately 65% similarity. We looked for L. enriettii in other potential reservoir hosts but the parasite was not observed. These results confirm that distinct strains of L. enriettii circulate in guinea pigs from Paraná state, more specifically in the Atlantic forest region, where we believe it serves as the center for dispersion of the species.

Clinical aspects and relevance of molecular diagnosis in late mucocutaneous leishmaniasis patients in Paraná, Brazil

The aim of the present work was to study the clinical aspects and relevance of molecular diagnosis in late mucocutaneous leishmaniasis patients in Parana, Brazil. Twenty one suspected cases of mucocutaneous leishmaniasis (MCL) in patients from the endemic areas of leishmaniasis were assessed. Different methods used in diagnosing the disease and the polymerase chain reaction (PCR) technique were compared in order to establish the sensitivity of each method. Out of the 21 patients analyzed, 14.3 percent presented other etiologies such as vasculitis, syphilis, and paracoccidioidomycosis, with all tests negative for leishmaniasis. Out of the remaining 15 patients, 6.7 percent cases were confirmed for leishmaniasis by direct examination; 46.67 percent were positive for culture, which allowed isolating and identifying the parasite and - with the PCR technique - it was possible to diagnose 100 percent MCL patients for all the three repetitions of exams. The PCR optimized for the present work proved to be an auxiliary method for diagnosing leishmaniasis applicable in the patients carrying MCL due to Leishmania (Viannia) braziliensis and did not need culture to be performed, resulting in a faster diagnosis.

Casos alóctones de leishmaniose visceral canina no Paraná, Brasil: implicações epidemiológicas / Allochthonous cases of canine visceral leishmaniasis in Paraná, Brazil: epidemiological implications

O presente trabalho teve como objetivo pesquisar a ocorrência de leishmaniose visceral em cães com sinais clínicos compatíveis, procedentes de clínicas veterinárias das diferentes regiões do Estado do Paraná e em caso positivo verificar a autoctonia dos mesmos. Dos animais com suspeita clínica e epidemiológica de LV, foi coletada amostra de sangue para realização de hemograma, provas bioquímicas, sorologia, cultura do parasito, PCR e RAPD. Os cães portadores de quadro clínico, como febre, hiperqueratose, onicogrifose e emagrecimento exagerado, tiveram gânglio poplíteo e/ou medula óssea puncionados e o conteúdo inoculado em meio de cultivo NNN. A extração de DNA do parasito em sangue e cultura foi realizada pelo método fenol/clorofórmio. A amplificação de DNA do protozoário foi feita por PCR e RAPD. Dos 24 animais analisados, o parasito foi isolado em 19 cães. As técnicas moleculares permitiram identificar 14 isolados como L. (Leihmania) infantum e cinco como L. (Viannia) braziliensis. A análise epidemiológica dos casos permitiu determinar o local de transmissão e definir que todos os cães com LV diagnosticados eram casos alóctones, ou seja eram importados de regiões endêmicas. Sugere-se que a migração dos cães de regiões endêmicas para regiões indenes poderá permitir a instalação de novos focos, desde que o agente encontre ecótopo adequado e vetor específico (Lutzomyia longipalpis).

The south region is the only area in Brazil that does not present autochthonous cases of visceral leishmaniasis (VL), however, in the state of Parana, dogs and humans have been found showing a VL compatible clinical profile. In view of this problem, the present work aimed at isolating and identifying the parasite and determining the cases autochthony. All animals clinically suspect of VL were clinically evaluated, and had samples of their blood collected for hemoculture (NNN culture medium), serology, PCR and RAPD-PCR, hemogram, and biochemical assays. The dogs presenting VL clinical profile had their lymph nodes and/or bone marrow punctured and their content inoculated in NNN culture medium. The protozoan isolated was identified by PCR and PCR-RAPD. Strains of Leishmania were isolated in 19 out of the 24 studied animals. Fourteen isolates were identified as L. (Leishmania) infantum, and five were L. (Viannia) braziliensis. In the epidemiological analysis, it was possible to determine that all dogs with L. (L.) infantum being allochthonous cases. Leishmaniasis is a zoonose that has the domestic dog as reservoir, the migration ofsuch animals can disseminate the parasite to other regions, provided the agent finds an adequate ecotope and a specific vector (Lutzomyia longipalpis).

Phenetic analysis of Panstrongylus megistus Burmeister, 1835 (Hemiptera: Reduviidae: Triatominae) in the State of Paraná-Brazil

Panstrongylus megistus is an important Chagas Disease vector and is said to be one of the species that might replace Triatoma infestans as the main vector of that disease in Brazil. The different degrees of P. megistus domiciliation in Brazil and its epidemiological relevance draw forth the need for the development of genetic studies that make it possible to analyze and understand the interchange of individual and gene fluxes among different populations. Thus, the present work aimed at studying the genetic variability of P. megistus in the State of Paraná - south of Brazil- and at comparing it with populations of the same species from five other states in Brazil (SP, MG, SC, RS, SE). In order to attain the proposed objective, 25 populations were studied using fifteen isoenzymatic systems (6PGD, G6PD, ME¹, ME², ICD, PGM, GPI, GOT¹, GOT², NP¹, NP², DIA, MPI, F, and MDH). The phenetic analysis allowed the individuation of 22 electromorphs and five zymodemes. The G6PD enzyme was the only polymorphic one presenting four electromorphs for the studied populations, all of them described for the State of Paraná-BR. The P. megistus populations from other states grouped with those from Paraná-BR, evidencing a low genetic variability in that species. Despite the existing geographic barriers, sub-samples - away from one another by at most 570km - were grouped in one and the same zymodeme. The epidemiological implications of such results are discussed in the present work.

Panstrongylus megistus é um importante vetor da Doença de Chagas e é apontado como uma das espécies com potencial para substituir Triatoma infestans como principal vetor desta doença no Brasil. Os diferentes graus de domiciliação por P. megistus - no Brasil - e sua importância epidemiológica evocam a necessidade de estudos com bases genéticas que possibilitem analisar e compreender os intercâmbios de indivíduos e os fluxos gênicos entre as distintas populações. Assim, o presente trabalho tem como objetivo estudar a variabilidade genética de P. megistus no Estado do Paraná e compará-los com populações da mesma espécie de cinco estados do Brasil (SP, MG, SC, RS, SE). Para atingir o objetivo proposto, 25 populações foram estudadas empregando quinze sistemas isoenzimáticos (6PGD, G6PD, ME¹, ME², ICD, PGM, GPI, GOT¹, GOT², NP¹, NP², DIA, MPI, FH e MDH). A análise fenética permitiu a individualização de 22 eletromorfos e quatro zimodemas. A enzima G6PD foi a única polimórfica que apresentou quatro eletromorfos para as populações estudadas, todas descritas para o Estado do Paraná. As populações de P. megistus procedentes dos outros estados agruparam-se com as do Paraná, demonstrando haver baixa variabilidade genética na espécie. Apesar das barreiras geográficas existentes, sub-amostras - distantes entre si por até 570 km - ficaram reunidas num mesmo zimodema. As implicações epidemiológicas destes resultados são discutidas no presente trabalho.

Comparison of serological and parasitological methods for cutaneous leishmaniasis diagnosis in the state of Paraná, Brazil

We evaluated the effectiveness of serological and parasitological methods for cutaneous leishmaniasis (CL) diagnosis in patients from the central region of Paraná state, southern Brazil. Five groups were compared: clinical diagnosis, parasitological diagnosis, communicants, inhabitants of a non-endemic area and carriers of other etiologies. Two antigens were prepared from promastigotes of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis for indirect immunofluorescence assay, ELISA and immunoblotting. The parasitological approaches detected 79.3 percent of the patients with a clinical diagnosis; the parasites were identified by PCR as L. (V.) braziliensis. Serological methods showed 95 percent sensitivity for homologous antigens. Immunoblotting revealed specific proteins for diagnosis of CL and detected 96.6 percent of the patients when L. (V.) braziliensis was used as an antigen, and 83.3 percent with L. (L.) amazonensis. This study demonstrated the importance of differential diagnosis for leishmaniasis; the association of two or more indirect methods increased diagnosis sensitivity.

Estudo das características epidemiológicas e clínicas de 332 casos de leishmaniose tegumentar notificados na regiäo norte do Estado do Paraná de 1993 a 1998 / Epidemiological and clinical study of 332 cases of cutaneous leishmaniasis in the north of Parana State from 1993 to 1998

Casos de leishmaniose tegumentar no norte do estado do Paraná têm sido assinalados desde os anos 50, mas no final dos anos 80, houve um surto epidêmico. Para conhecer o perfil epidemiológico da leishmaniose cutânea na regiäo Norte do Paraná foi feito um levantamento de casos, entre 1993 e 1998. Foram notificados 316 casos da doença em 35 municípios paranaenses. Dezesseis casos eram importados de outros estados e do Paraguai. Os indivíduos do sexo masculino (61,2 por cento), na faixa etária de 15 a 49 anos (70,8 por cento) representaram a maioria dos casos de leishmaniose tegumentar. Sessenta e sete porcento dos pacientes apresentaram lesöes únicas, 31 por cento lesöes múltiplas e 2 por cento lesöes de mucosas. Num total de 367 lesöes analisadas 47,7 por cento estavam localizadas nos membros inferiores, 26,7 por cento nos membros superiores e 16 por cento na face. Trinta e duas cepas de Leishmania isoladas foram identificadas, com base em 13 isoenzimas, como pertencentes à espécie Leishmania (Viannia) braziliensis

Raillietina (Raillietina) guaricana n. sp. (Cestoda-Davaineidae), Parasite of wild rats from the environmental protection area of Guaricana, Paraná, Brazil

Raillietina (Raillietina) guaricanae n. sp. is described from the wild rats Oryzomys intermedius, O. nigripes and O. ratticeps, captured in the Environmental Protection Area of Guaricana, from November 1988 to December 1989. Raillietina (Railietina) guaricanae n. sp. is closely related to the Neotropical mammalian Raillietina, however it differs by the fewer number of rostellar hooks, and tests different number of eggs capsules and host species. The number of known species of Raillietina (Raillietina), parasites of mammals in the Neotropical Region, is increased to four

Flebótomos da área do reservatório da Hidroelétrica de Itaipu, Estado do Paraná, Brasil (Diptera, Psychodidae) / Sand-flies at the area of Itaipu Hydroeletric plant, state of Paraná, Brazil

Durante os anos de 1979 a 1986 foram feitas capturas intre e extradomiciliares e também com isca humana, em diversas localidades de 6 municípios situados ao longa da margem brasileira do reservatório da hidroelétrica de Itaipu. Foram coletadas 8 espécies de Lutzomyia, sendo que duas, já incriminadas como transmissoras da leishmaniose tegumentar, L. intermedia e L. whitmani, säo abundantes na área

Larvas de Anophelas (subgênero Kerteszia) Theobald 1905 encontradas em criadouros artificiais, no Estado do Paraná, Brasil / Larvas of the Anopheles (sub-genus Kerteszia) Theobald 1905, found in artificial breeding pens, in the State of Paraná, Brazil

Discute-se encontro de larvas de duas espécies de anofelinos do subgênero Kerteszia - An. (Ker.) cruzii e An. (Ker.) bambusicolus - em criadouros artificiais, no Estado do Paraná, Brasil