Natural infection of Lutzomyia longipalpis (Lutz & Neiva, 1912) by Leishmania infantum in a municipality with a high incidence of visceral leishmaniasis in the Brazilian Midwest

ABSTRACT Background: Here, Leishmania presence in sand flies from Três Lagoas, Mato Grosso do Sul, Brazil, after visceral leishmaniasis (VL) was investigated. Methods: In April 2022, two light traps were deployed within and around the residence for two days post-VL case report. Results: A total of 120 Lutzomyia longipalpis were collected. Suprapyloric flagellates were found in a female sand fly with eggs and residual blood during midgut dissection. Sequencing of ITS1 and cytb fragments confirmed Leishmania infantum DNA and identified Homo sapiens as the blood source, respectively. Conclusions: This study emphasizes the importance of monitoring sand flies in VL endemic areas.

Polymerase chain reaction using conjunctival swab samples for detecting Leishmania DNA in dogs / Reação em cadeia da polimerase usando amostras de Swab de conjuntiva para detecção de DNA de Leishmania em cães

Abstract The dog is the main domestic reservoir of Leishmania and font of infection for the vector, constituting an important host for the transmission of the parasite to humans. Non-invasive collection of swab samples for leishmaniasis diagnosis has been a promising alternative. This study analyzed the positivity of polymerase chain reaction (PCR) for the diagnosis of canine leishmaniasis in conjunctiva samples. DNA extraction was performed using SDS 20% and PCR was performed using 13A/13B primers that amplify 120-bp of Leishmania kDNA. Of the 77 dogs analyzed, 50 (64.93%) had ocular changes: 25 (32.47%) dogs had periocular lesion, 41 (53.25%) dogs had purulent eye discharge, and 17 (22.08%) dogs had both signals. PCR was positive in 35 dogs (45.45%), and there was no significant difference between dogs with and without ocular signals (p=0.4074). PCR positivity was significant higher in dogs without periocular injury (p=0.0018). Conjunctive PCR, a less invasive, fast, and painless collection technique, is indicated to complement the diagnosis, especially in dogs without periocular injury, independent of the presence of purulent eye discharge.

Resumo O cão é o principal reservatório doméstico de Leishmania e também fonte de infecção para o vetor, constituindo um importante hospedeiro para a transmissão do parasita ao homem. A coleta não invasiva de amostras em swab para diagnóstico das leishmanioses tem sido uma alternativa promissora. Este estudo analisou a positividade da reação em cadeia da polimerase (PCR) para o diagnóstico de leishmaniose canina em amostras de conjuntiva. A extração do DNA foi realizada com SDS 20%. A PCR foi realizada com primers 13A/13B que amplificam 120-pb do kDNA de Leishmania. Dos 77 cães analisados, 50 (64,93%) tiveram alterações oculares; 25 (32,47%) cães tiveram uma lesão periocular; 41 (53,25%) tiveram secreção ocular purulenta e 17 (22,08%) cães tiveram ambos os sinais. A PCR foi positiva em 35 cães (45,45%) e não houve diferença significativa em cães com e sem sinais oculares (p = 0,4074). A positividade da PCR foi significativamente maior em cães sem lesão periocular (p = 0,0018). PCR em conjuntiva, uma técnica de coleta menos invasiva, rápida e indolor, é indicada para complementar o diagnóstico, principalmente em cães sem lesão periocular, independentemente da presença de secreção ocular purulenta.

Sandfly fauna and behavior (Diptera: Psychodidae) in municipalities of the Mesoregion North Pioneer of Paraná, Brazil

Abstract The occurrence of cutaneous leishmaniasis (CL) in the municipalities of Jaboti, Japira, Pinhalão, and Tomazina in the North Pioneer of the State of Paraná, where this disease is endemic, prompted the investigation of sandfly fauna, their population changes throughout the year, and their behavior in and around human accommodation. In these municipalities, the collection of sandflies was conducted using Falcão traps from 7 pm to 6 am once a month. 32,994 sandflies, 18,442 males and 14,552 females, were collected and represented by the species Brumptomyia brumpti, Brumptomyia cunhai, Evandromyia correalimai, Evandromyia cortelezzii, Expapillata firmatoi, Micropygomyia ferreirana, Migonemyia migonei, Nyssomyia neivai, Nyssomyia whitmani, Pintomyia fischeri, Pintomyia monticola, Pintomyia pessoai, and Psathyromyia bigeniculata. Ny. neivai (SISA = 0.9808) predominated in Japira (46.9%), Pinhalão (53.1%), and Tomazina (38.9%), while Ny. whitmani (SISA = 0.9423) predominated in Jaboti (53.8%). Most sandflies were captured in domestic animal shelters, with a peak in September. The high number of sandflies collected in domestic animal shelters, species richness, and presence of species involved in the epidemiology of CL reveal the need for permanent entomological surveillance in the municipalities mentioned. The use of phlebotomine control measures in these municipalities should be considered in conjunction with the anthropogenic actions that have resulted in changes to sandfly fauna and behavior.

The sand fly species (diptera: psychodidae) in an urban environment of Mato Grosso do Sul, Brazil

The purpose of this study was to search for Lutzomyia longipalpis and Lutzomyia cruzi in the urban area of the Água Clara Municipality, Mato Grosso do Sul State, Brazil. Two CDC traps were placed in the intradomicile and the peridomicile areas in four residences once a month, for three consecutive nights during 24 months. Were collected 11 species of sand flies totaling 3,418 specimens, 2,762 males and 656 females. Lu. longipalpis was the dominant species and there was no statistical difference between the peridomicile and intradomicile areas. Lu. cruzi was not found. The high capture rate for Lu. longipalpis in the urban area of Água Clara is cause for concern regarding necessary preventive measures to avoid future cases of visceral leishmaniasis.

PCR sensitivity of peripheral blood of dogs co-infected with Leishmania spp. and Ehrlichia spp. in endemic area of Brazil

Abstract INTRODUCTION Peripheral blood of 400 dogs infected with Leishmania and Ehrlichia were analyzed using polymerase chain reaction (PCR), and clinical signs were characterized. METHODS PCR and parasitological tests were conducted. RESULTS PCR was positive for Leishmania in 84.75%, and parasitological tests showed that 63.25% and 31.75% were positive for Leishmania and Ehrlichia, respectively. All animals showed more than three clinical signs. PCR results were negative for Leishmania in 15.25% of the samples. CONCLUSIONS Conventional PCR of peripheral blood can be used for diagnosing canine visceral leishmaniasis in combination with other techniques, especially in uncertain cases that need species identification.

Genetic variability of populations of Nyssomyia neivai in the Northern State of Paraná, Brazil

ABSTRACT The genetic study of sandfly populations needs to be further explored given the importance of these insects for public health. Were sequenced the NDH4 mitochondrial gene from populations of Nyssomyia neivai from Doutor Camargo, Lobato, Japira, and Porto Rico, municipalities in the State of Paraná, Brazil, to understand the genetic structure and gene flow. Eighty specimens of Ny. Neivai were sequenced, 20 from each municipality, and 269 base pairs were obtained. A total of 27 haplotypes and 28 polymorphic sites were found, along with a haplotypic diversity of 0.80696 and a nucleotide diversity of 0.00567. Haplotype H5, with 33 specimens, was the most common among the four populations. Only haplotypes H5 and H7 were present in all four populations. The population from Doutor Camargo showed the highest genetic diversity, and only this population shared haplotypes with those from the other municipalities. The highest number of haplotypes was sheared with Lobato which also had the highest number of unique haplotypes. This probably occurred because of constant anthropic changes that happened in the environment during the first half of the twentieth century, mainly after 1998. There was no significant correlation between genetic and geographical distances regarding these populations. However, the highest genetic and geographical distances, and the lowest gene flow were observed between Japira and Porto Rico. Geographical distance is a possible barrier between these municipalities through the blocking of haplotype sharing.

NEW PRIMERS FOR DETECTION OF Leishmania infantum USING POLYMERASE CHAIN REACTION / Novos iniciadores para detecção de Leishmania infantum pela reação em cadeia da polimerase

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.

RESUMO Leishmania infantum causa leishmaniose visceral (LV) no Novo Mundo. O diagnóstico de LV é confirmado por testes parasitológicos e sorológicos, os quais nem sempre são sensíveis ou específicos. Nosso objetivo foi desenhar novos iniciadores para realizar uma Reação em Cadeia da Polimerase (PCR) para detecção de L. infantum. Sequências do DNA do minicírculo do cinetoplasto (kDNA) foram obtidos do GenBank, e os iniciadores FLC2/RLC2 foram desenhados. Amostras de DNA de L. infantum, Leishmania amazonensis, Leishmania braziliensis, Leishmania guyanensis, Leishmania naiffi, Leishmania lainsoni, Leishmania panamensis, Leishmania major e Trypanosoma cruzi foram usados para padronizar a PCR. PCR com iniciadores FLC2/RLC2 amplificou um fragmento de 230 pb e detectou 0,2 fg de DNA de L. infantum.Das espécies de parasitos analisadas, somente DNA de L. infantum foi amplificado. Após sequenciamento, o fragmento foi analisado no GenBank, que mostrou homologia com L. infantum. Em análises de amostras de sangue e lesão de cão com suspeita clínica de LV, a PCR detectou DNA de L. infantum. Em amostras de lesão de humanos e cães com leishmaniose cutânea, a PCR foi negativa. A PCR padronizada com os iniciadores FLC2/RLC2 mostrou alta sensibilidade e especificidade, sendo técnica promissora para o diagnóstico de LV.

DETECTION OF Leishmania (Viannia) IN Nyssomyia neivai AND Nyssomyia whitmani BY MULTIPLEX POLYMERASE CHAIN REACTION, IN SOUTHERN BRAZIL / Detecção de Leishmania (Viannia) em Nyssomyia neivai e Nyssomyia whitmani por Multiplex Reação em Cadeia da Polimerase, no sul do Brasil

Sandflies transmit pathogens of leishmaniasis. The natural infection of sandflies by Leishmania (Viannia) was assessed in municipalities, in the state of Paraná, in Southern Brazil. Sandflies were collected with Falcão and Shannon traps. After dissection in search of flagellates in digestive tubes and identification of the species, female sandflies were submitted to the Multiplex Polymerase Chain Reaction (multiplex PCR) for detection of the fragment of the kDNA of Leishmania (Viannia) and the fragment from the IVS6 cacophony gene region of the phlebotomine insects. The analysis was performed in pools containing seven to 12 guts from females of the same species. A total of 510 female sandflies were analyzed, including nine Migonemyia migonei, 17 Pintomyia fischeri, 216 Nyssomyia neivai, and 268 Nyssomyia whitmani. Although none of the females was found naturally infected by flagellates through dissection, the fragment of DNA from Leishmania (Viannia) was shown by multiplex PCR in one sample of Ny. neivai (0.46%) and three samples of Ny. whitmani (1.12%). It was concluded that Ny. neivai and Ny. whitmani are susceptible to Leishmania infection, and that multiplex PCR can be used in epidemiological studies to detect the natural infection of the sandfly vector, because of its sensitivity, specificity and feasibility.

Flebotomíneos transmitem os patógenos das leishmanioses. Foi avaliada a infecção natural de flebotomíneos por Leishmania (Viannia) em municípios do Estado do Paraná, sul do Brasil. Os flebotomíneos foram coletados com armadilhas de Falcão e Shannon. Após dissecação para pesquisa de flagelados no tubo digestório e identificação das espécies, as fêmeas de flebotomíneos foram submetidas a Multiplex Reação em Cadeia da Polimerase (multiplex PCR) para a detecção do fragmento do kDNA de Leishmania (Viannia) e do fragmento do gene IVS6 da cacofonia de flebotomíneos. A análise foi realizada em pools contendo sete a 12 tubos digestórios de fêmeas da mesma espécie. Um total de 510 fêmeas foram analisadas, incluindo nove Migonemyia migonei, 17 Pintomyia fischeri, 216 Nyssomyia neivai e 268 Nyssomyia whitmani. Embora nenhuma fêmea tenha sido encontrada naturalmente infectada com flagelados pela dissecação, o fragmento de DNA de Leishmania (Viannia) foi mostrado por multiplex PCR em uma amostra de Ny. neivai (0,46%) e três amostras de Ny. whitmani (1,12%). Conclui-se que Ny. neivai e Ny. whitmani são suscetíveis à infecção por Leishmania, e que multiplex PCR, devido à sua sensibilidade, especificidade e viabilidade, pode ser utilizada em estudos epidemiológicos para a detecção da infecção natural do inseto vetor.

Sandfly frequency in a clean and well-organized rural environment in the state of Paraná, Brazil / Frequência de flebotomíneos em ambiente rural com boas condições de limpeza e organização, no estado do Paraná, Brasil

INTRODUCTION: Sandflies caught in Santa Juliana Farm in Sarandi, State of Paraná, Brazil, were assessed in terms of their fauna, seasonality, and frequency in the homes and in shelters of domestic animals around the homes, as well as in the nearby forest. METHODS: In Santa Juliana Farm, there are no records of cases of ACL, differing from other relatively clean and organized areas where surveys of sandflies have been conducted in Paraná. Samples were collected with Falcão light traps, fortnightly from 22:00 to 02:00 hours, from November 2007 to November 2008. RESULTS: A total of 4,506 sandflies were captured, representing 13 species, predominantly Nyssomyia whitmani (71.8 percent). More sandflies were collected in the forest (52.6 percent) than outside the forest (residences and pigsty) (47.4 percent). However, Ny. whitmani was collected in greater numbers outside (38.3 percent) than inside the forest (33.5 percent). Most sandflies were collected in the warmer months and during periods with regular rainfall. CONCLUSIONS: The results suggest that cleaning and organization around the houses could reduce sandfly population in peridomicile. Constructing shelters for animal at a distance of approximately 100m from domiciles is recommended to prevent the invasion of sandflies, as this farm has an area of preserved forest, with wild animals and sandflies present to maintain the enzootic cycle of Leishmania.

INTRODUÇÃO: Relatam-se os resultados de coletas de flebotomíneos na Fazenda Santa Juliana, município de Sarandi, Estado do Paraná, Brasil, onde se verificou a fauna, a sazonalidade e a frequência desses insetos nos ambientes florestal e extraflorestal. MÉTODOS: O trabalho foi realizado nesta fazenda pela inexistência de registro de casos de LTA e por diferir de outras áreas onde foram realizadas pesquisas com flebotomíneos, no Paraná, e pela limpeza e organização do peridomicílio. As coletas foram realizadas com sete armadilhas luminosas de Falcão, quinzenalmente das 22 às 2 horas, de novembro de 2007 a novembro de 2008. RESULTADOS: Foram capturados 4.506 flebotomíneos de 13 espécies, com predomínio de Nyssomyia whitmani (71,8 por cento). Coletou-se maior proporção de flebotomíneos no ambiente florestal (52,6 por cento) do que no extraflorestal (residências e chiqueiro) (47,4 por cento), entretanto a proporção de Ny. whitmani coletada foi maior no ambiente extraflorestal (38,3 por cento) do que no florestal (33,5 por cento). A maioria dos flebotomíneos foi coletada nos meses mais quentes e com quedas regulares de chuva. CONCLUSÕES: Os resultados sugerem que a limpeza e a organização do peridomicílio podem auxiliar a reduzir a população de flebotomíneos no domicílio e no peridomicílio, recomendando-se que a construção de abrigos de animais guarde a distância aproximada de 100m das residências, para evitar a invasão desses insetos, uma vez que nesta fazenda existe uma área de mata de preservação, com a presença de animais silvestres e flebotomíneos, que mantem o ciclo enzoótico de Leishmania.

Mobilidade populacional e produção da leishmaniose tegumentar americana no Estado do Paraná, sul do Brasil / Population mobility and production of American tegumentary leishmaniasis in the State of Paraná, southern Brazil

São escassas as informações sobre o papel da mobilidade populacional na manutenção da leishmaniose tegumentar americana no estado do Paraná. Avalia-se a mobilidade populacional como fator de risco para esta endemia em três mesorregiões do Paraná, utilizando dados gerados na Universidade Estadual de Maringá, no período de 1987 a 2004. Foram notificados 1.933 casos, predominando os casos migrantes (54,4 por cento). Os municípios com maior número de casos notificados foram Maringá (358), Doutor Camargo (108) e Terra Boa (105). Os casos rurais foram predominantemente autóctones (89,8 por cento), enquanto os urbanos, na maioria (84,8 por cento) migrantes (p<0,0001). Para os casos rurais autóctones, não houve predomínio entre os sexos (p=0,127); para os casos urbanos migrantes, prevaleceu o sexo masculino (p<0,0001). Os casos migrantes foram na maioria relacionados com a mobilidade intra e intermunicipal. A mobilidade populacional parece ser uma variável importante na epidemiologia desta doença no Estado do Paraná.

Information on the role of population mobility in maintaining American tegumentary leishmaniasis in the State of Paraná is scarce. Population mobility was evaluated as a risk factor for this endemic disease in three mesoregions of Paraná, using data built up at the State University of Maringá, covering 1987 to 2004. A total of 1,933 cases were notified, mostly among migrants (54.4 percent). The municipalities with the greatest numbers of cases notified were Maringá (358), Doutor Camargo (108) and Terra Boa (105). The rural cases were predominantly autochthonous (89.8 percent), while the urban cases were mostly among migrants (84.8 percent) (p < 0.0001). Among the rural autochthonous cases, there was no difference between the sexes (p = 0.127), whereas among the urban migrant cases, men predominated (p < 0.0001). The migrant cases were mostly related to mobility within and between municipalities. Population mobility seems to be an important variable in the epidemiology of this disease in the State of Paraná.

Investigation of natural infection by Leishmania in sandflies of Paraná State, Southern Brazil

The purpose of this work was to verify the occurrence of Leishmania in naturally infected sandflies. The insects were collected with Falcão, Shannon and HP light-traps, in Doutor Camargo and Maringá municipalities between November 2004 and October 2005. Of the 11,033 sandflies collected in Doutor Camargo, 2,133 surviving females were dissected, particularly those of the Nyssomyia neivai species (86.87 percent). In Maringá, 136 sandflies were collected, of which 79 N. whitmani females and 1 Migonemyia migonei female were dissected. The dissected insects were identified and stored in the pools of 10 specimens. The PCR was carried out on 1,190 females of N. neivai and 190 of N. whitmani from Doutor Camargo, and on 30 of N. whitmani from Maringá, using the primers MP1L/MP3H. The natural infection by Leishmania in sandflies was not confirmed by either of the methods used. The results suggested the low natural infection rate of sandflies by Leishmania in these areas, corroborating other studies carried out in endemic areas of ACL.

Procurou-se verificar a ocorrência de flebotomíneos naturalmente infectados por Leishmania. Estes insetos foram coletados com armadilhas luminosas de Falcão, Shannon e HP, nos municípios de Doutor Camargo e Maringá, de novembro de 2004 a outubro de 2005. Coletaram-se 11.033 flebotomíneos em Doutor Camargo e destes 2.133 fêmeas foram dissecadas, especialmente de Nyssomyia neivai (86,87 por cento). Em Maringá coletaram-se 136 flebotomíneos, dos quais foram dissecadas 79 fêmeas de N. whitmani e 1 Migonemyia migonei. Parte das fêmeas dissecadas foi armazenada em pools de 10 espécimes. A PCR, utilizando os iniciadores MP1L/MP3H, foi realizada em 1.190 fêmeas de N. neivai e 190 de N. whitmani de Doutor Camargo e 30 N. whitmani de Maringá. Não se constatou a infecção natural por Leishmania pelos métodos utilizados. Estes resultados indicam que é baixa a taxa de infecção natural de flebotomíneos por Leishmania, corroborando outros trabalhos realizados em áreas endêmicas de LTA, inclusive no Paraná.