DC-SIGN receptor is expressed by cells from cutaneous leishmaniasis lesions and differentially binds to Leishmania (Viannia) braziliensis and L. (Leishmania) amazonensis promastigotes

BACKGROUND Dendritic cells (DCs) specific intercellular adhesion molecule (ICAM)-3-grabbing non integrin receptor (DC-SIGN) binds to subgenera Leishmania promastigotes mediating its interaction with DC and neutrophils, potentially influencing the infection outcome. OBJECTIVES In this work, we investigated whether DC-SIGN receptor is expressed in cells from cutaneous leishmaniasis (CL) lesions as well as the in vitro binding pattern of Leishmania (Viannia) braziliensis (Lb) and L. (L.) amazonensis (La) promastigotes. METHODS DC-SIGN receptor was labeled by immunohistochemistry in cryopreserved CL tissue fragments. In vitro binding assay with CFSE-labeled Lb or La promastigotes and RAJI-transfecting cells expressing DC-SIGN (DC-SIGNPOS) or mock-transfected (DC-SIGNNEG) were monitored by flow cytometry at 2 h, 24 h and 48 h in co-culture. RESULTS In CL lesion infiltrate, DC-SIGNPOS cells were present in the dermis and near the epidermis. Both Lb and La bind to DC-SIGNPOS cells, while binding to DC-SIGNNEG was low. La showed precocious and higher affinity to DC-SIGNhi population than to DC-SIGNlow, while Lb binding was similar in these populations. CONCLUSION Our results demonstrate that DC-SIGN receptor is present in L. braziliensis CL lesions and interact with Lb promastigotes. Moreover, the differences in the binding pattern to Lb and La suggest DC-SIGN can influence in a difference way the intake of the parasites at the first hours after Leishmania infection. These results raise the hypothesis that DC-SIGN receptor could participate in the immunopathogenesis of American tegumentary leishmaniasis accounting for the differences in the outcome of the Leishmania spp. infection.

Study on the zoonotic cycle of tegumentary leishmaniasis in an endemic area of a metropolitan region in the Northeastern region of Brazil

ABSTRACT This study was conducted to characterize the transmission cycle of the tegumentary leishmaniasis (TL) in an old colonization area at Pernambuco State, Brazil. The aims were to identify autochthonous cases, sandflies fauna, domestic animals as possible reservoir hosts and the Leishmania species involved in this endemic area. A total of 168 suspected human cases of TL and 272 domestic animals (canine, feline, equine, goat, and sheep) were included. The sandflies were captured and identified by species. Patients were predominantly male and the average age was 37+18.1 years old. Of 85 patients who had skin lesions, 25.6% of them had direct positive smears for TL and 34 isolates were identified as Leishmania (Viannia) braziliensis. The confirmation for TL diagnosed by molecular detection (PCR) was almost three times more sensitive than the direct test [p < 0.001; PR = 2.72] associated with clinical examination. The Kappa test on PCR between two different specimens, biopsy, and skin lesion swab was 60.8% (p < 0.001). More than 200 specimens of sandflies (80 males and 159 females) were captured and identified as Lutzomyia whitmani (99.6%) and Lu. evandroi (0.4%). The detection of L. (V.) braziliensis by Real-Time PCR in the blood of a captured fed female was positive in 59.3% of Lu. whitmani. Of the 272 domestic animals included, 61.76% were male (n = 168). Thirty-six animals (13.2%) had lesions compatible with TL (34 dogs, 1 cat and 1 sheep) and 3 of them, all dogs, had lesions on the snout, showing destruction of cartilage and mucosa. The study suggests the participation of domestic animals as possible reservoirs. However, further studies are necessary to better understand the transmission cycle and take recommended measures in order to control the disease.

First report of Leishmania RNA virus 1 in Leishmania (Viannia) braziliensis clinical isolates from Rio de Janeiro State - Brazil

BACKGROUND Leishmania parasites carry a double-stranded RNA virus (Leishmania RNA virus - LRV) that has been divided in LRV1 and LRV2. OBJECTIVES Leishmania (Viannia) braziliensis clinical isolates were assessed in order to determine LRV presence. METHODS Two-round polymerase chain reaction (PCR and nested PCR) was performed to detect LRV1 or LRV2 in L. (V.) braziliensis clinical isolates (n = 12). FINDINGS LRV1 was detected in three clinical isolates which was phylogenetically related to other sequences reported from other American tegumentary leishmaniasis (ATL) endemic areas of Brazil. Patients infected with L. (V.) braziliensis LRV-negative showed only cutaneous lesions while LRV-positive reported different manifestations. MAIN CONCLUSION Data presented here show for the first time that LRV1 is circulating in L. (V.) braziliensis clinical isolates from Rio de Janeiro State in Brazil.

Persistent cutaneous canine leishmaniasis caused by Leishmania (Viannia) braziliensis in an area with predominance of Nyssomyia neivai in the state of São Paulo, Brazil / Leishmaniose cutânea canina persistente causada por Leishmania (Viannia) braziliensis em uma área com predomínio de Nyssomyia neivai no estado de São Paulo, Brasil

Abstract American cutaneous leishmaniasis (ACL) is a neglected zoonotic disease caused mainly by Leishmania (Viannia) braziliensis, which is endemic throughout Brazil. Canine ACL cases were investigated in a rural area of Monte Mor, São Paulo, where a human ACL case had been confirmed. Dogs were evaluated through clinical and laboratory diagnosis including serology, cytological tissue preparations and PCR on skin lesions, lymph node and bone marrow samples. Entomological investigations on sandflies trapped in the surroundings of the study area were performed for 14 months. Nyssomyia neivai was the predominant phlebotomine species, comprising 94.65% of the captured specimens (832 out of 879). This species was the most abundant in all trapping sites, including human homes and dog shelters. Ny. whitmani, Migonemyia migonei, Pintomyia monticola, Evandromyia cortellezzii, Pi. fischeri and Expapilata firmatoi were also captured. Two of the three dogs examined were positive for anti-Leishmania IgG in ELISA using the antigen Fucose mannose ligand and skin samples were positive for L. (V.) braziliensis in PCR, but all the samples collected were negative for L. (L.) infantum. One of the dogs had a confirmed persistent infection for more than one year.

Resumo A leishmaniose tegumentar Americana (LTA) é uma doença zoonótica negligenciada, causada principalmente por Leishmania (Viannia) braziliensis, sendo endêmica em todo o Brasil. Foram investigados casos de LTA canina em uma área rural da cidade de Monte Mor, São Paulo, onde foi confirmado um caso humano de LTA. Os cães foram avaliados por diagnóstico clínico e laboratorial, incluindo sorologia, esfregaços microscópicos e PCR de amostras em lesões de pele, linfonodos e medula óssea. Também foram realizadas investigações entomológicas durante 14 meses, usando-se armadilhas luminosas para flebotomíneos nas proximidades da área de estudo. Nyssomyia neivai foi a espécie de flebotomíneo predominante com 94,65% dos espécimes capturados (832 de 879). Essa espécie foi a mais abundante em todos os locais de captura, incluindo-se abrigos para humanos e cães. Foram também capturadas as espécies Ny. whitmani, Migonemyia migonei, Pintomyia monticola, Evandromyia cortellezzii, Pi. fischeri e Expapilata firmatoi. Dos três cães examinados, dois apresentaram IgG anti-Leishmania positivo no ELISA, usando-se o antígeno "Fucose mannose ligand", PCR da lesão de pele positivo para L. (V.) braziliensis e negativo em todas amostras para L. (L.) infantum. Um dos cães apresentou infecção persistente por mais de um ano.

Insights into the proteomic profile and gene expression of Lutzomyia longipalpis-derived Lulo cell line

BACKGROUND Lutzomyia longipalpis-derived cell line (Lulo) has been suggested as a model for studies of interaction between sandflies and Leishmania. OBJECTIVES Here, we present data of proteomic and gene expression analyses of Lulo cell related to interactions with Leishmania (Viannia) braziliensis. METHODS Lulo cell protein extracts were analysed through a combination of two-dimensional gel electrophoresis and mass spectrometry and resulting spots were further investigated in silico to identify proteins using Mascot search and, afterwards, resulting sequences were applied for analysis with VectorBase. RESULTS Sixty-four spots were identified showing similarities to other proteins registered in the databases and could be classified according to their biological function, such as ion-binding proteins (23%), proteases (14%), cytoskeletal proteins (11%) and interactive membrane proteins (9.5%). Effects of interaction with L. (V.) braziliensis with the expression of three genes (enolase, tubulin and vacuolar transport protein) were observed after an eight-hour timeframe and compared to culture without parasites, and demonstrated the impact of parasite interaction with the expression of the following genes: LLOJ000219 (1.69-fold), LLOJ000326 (1.43-fold) and LLOJ006663 (2.41-fold). CONCLUSIONS This set of results adds relevant information regarding the usefulness of the Lulo cell line for studies with Leishmania parasites that indicate variations of protein expression.

Increased leishmanicidal activity of alveolar macrophages from mature horses with mild equine asthma / Aumento da atividade leishmanicida de macrófagos alveolares de equinos adultos com asma equina leve

Alveolar macrophages (AMs) are an essential part of defense mechanisms within the lungs and their phagocytic activity is important for organ homeostasis. The phagocytic ability of AMs obtained from bronchoalveolar lavage from 17 mature mixed-breed pleasure horses (8 healthy and 9 diagnosed with mild equine asthma) was studied through assays with Leishmania (Viannia) braziliensis promastigotes, which enabled the calculation of a phagocytic index (PI) and a survival index (SI). Results indicate that phagocytic activity of AMs in asthma affected horses is similar to healthy horses, while leishmanicidal activity is significantly increased in horses with asthma.(AU)

Os macrófagos alveolares (MAs) são uma parte essencial dos mecanismos de defesa dentro dos pulmões e sua atividade fagocítica é importante para a homeostase desse órgão. A capacidade fagocitária dos MAs obtidos do lavado broncoalveolar de 17 equinos adultos, sem raça definida (oito saudáveis e nove com diagnóstico de asma equina leve), foi estudada por meio de ensaios com promastigotas de Leishmania (Viannia) braziliensis. Foi calculado o índice fagocítico e o índice de sobrevivência. Os resultados indicam que a atividade fagocítica de MAs em cavalos com asma é semelhante a cavalos saudáveis, enquanto a atividade leishmanicida está significativamente aumentada em cavalos com essa enfermidade.(AU)

Therapeutic response and safety of the topical, sequential use of antiseptic, keratolytic, and pentamidine creams (3-PACK) on Leishmania (Viannia) braziliensis-infected mice

BACKGROUND Topical treatment of New World cutaneous leishmaniasis can be affected by bacterial coinfection, hyperkeratosis, and transdermal drug delivery. OBJECTIVE The aim of this work was to evaluate the therapeutic response and safety of the topical, sequential use of antiseptic, keratolytic, and pentamidine isethionate (PMD) creams (3-PACK kit) on CL-infected BALB/c mice. METHODS A 0.5% chlorhexidine solution (CGH), 10% salicylic acid (SA), and 3% or 6% PMD were used as antiseptic, keratolytic, and antileishmanial drugs, respectively. During the first seven days, antiseptic, followed by 10% SA gel and PMD cream, were applied topically. Subsequently, treatment was performed only with the antiseptic and PMD creams. Skin irritation, reduction of lesion size (mm2), and parasitic load were observed until 30 days of treatment were completed. FINDINGS The 3-PACK treatment using 6% PMD induced a complete lesion reduction in 3/6 mice and a partial reduction in 1/6 mice, with no parasites observed. In contrast, CGH and SA alone, along with the vehicle, were not effective (p < 0.05). Moderate to severe erythema was observed at the application site. MAIN CONCLUSION The topical 3-PACK using 6% PMD was 67% effective in the treatment of CL by Leishmania (Viannia) braziliensis. Currently, work is ongoing to improve PMD isethionate formulation and to determine a dose-response.

Favorable responses to treatment with 5 mg Sbv/kg/day meglumine antimoniate in patients with American tegumentary leishmaniasis acquired in different Brazilian regions

Abstract INTRODUCTION: Favorable responses in American tegumentary leishmaniasis (ATL) patients to treatment with 5 mg Sbv/kg/day meglumine antimoniate (MA) has been reported in Rio de Janeiro, but little is known regarding the therapeutic response to low doses in patients from other locations. METHODS: A retrospective review of medical records was conducted to compare the therapeutic response to 5 mg Sbv/kg/day MA treatment among 36 patients who acquired ATL in Brazilian states other than Rio de Janeiro (OS group) and 72 patients from Rio de Janeiro (RJ group). RESULTS: One course of 5 mg Sbv/kg/day MA cured 72.8% of 81 cutaneous (CL) and 66.6% of 27 mucosal (ML) leishmaniasis-infected patients: 70% in the CL/RJ group, 81% in the CL/OS group, 50% in the ML/RJ group, and 80% in the ML/OS group. After up to two additional treatment courses at the same dose, 88.9% and 85.2% of the CL and ML patients were cured, respectively. Adverse events were observed in 40% of patients in the CL/RJ group, 57% of the CL/OS group, 58% of the ML/RJ group, and 80% of the ML/OS group. No significant differences were observed in the cure rates or adverse effects between the RJ and OS groups. No patients required permanent discontinuation of treatment due to adverse events. CONCLUSIONS: Patients with ATL acquired in both RJ and OS may respond to low-dose MA. While high-dose MA should remain the standard treatment for ATL, low-dose MA might be preferred when toxicity is a primary concern.

Evaluación de formulaciones de ketoconazol para uso tópico en leishmaniasis cutánea por Leishmania(Viannia) / Evaluation of ketoconazole formulations for topical use in cutaneous leishmaniasis caused by Leishmania (Viannia)

RESUMEN Se evaluó la efectividad de diversas formulaciones farmacéuticas de ketoconazol en modelos experimentales de leishmaniasis cutánea (LC) en ratones BALB C. Fueron preparadas formulaciones tópicas tipo gel, lipogel y crema conteniendo potenciadores de la permeación y diferentes concentraciones de ketoconazol. Se determinó la estabilidad, la toxicidad y la actividad anti-Leishmania in vitro. Además, se evaluó in vivo la efectividad de las formulaciones aplicadas tópicamente en ratones con LC infectados con Leishmania (Viannia) braziliensis. Las formulaciones tipo crema fueron evaluadas adicionalmente en ratones infectados con L. (V.) panamensis. Los sistemas evaluados mantuvieron in vitro la actividad del ketoconazol contra los parásitos; sin embargo, ninguna de las formulaciones fue efectiva en curar las lesiones de LC en los ratones. El tratamiento tópico con miltefosina (utilizado como control) curó las lesiones. Se concluye que las formulaciones que contienen ketoconazol diseñados en este estudio, no fueron efectivos contra la LC en los ratones infectados.

ABSTRACT The effectiveness of various pharmaceutical formulations of ketoconazole was evaluated in experimental models of cutaneous leishmaniasis (LC) in BALB C mice. Topical gel, lipogel, and cream formulations containing permeation enhancers and different concentrations of ketoconazole were prepared. Stability, toxicity and anti-Leishmania activity were determined in vitro. In addition, the effectiveness of topically applied formulations in LC-infected mice infected with Leishmania (Viannia) braziliensis was evaluated in vivo. Cream formulations were additionally evaluated in mice infected with L. (V.) panamensis. The systems evaluated maintained in vitro the activity of ketoconazole against parasites; however, none of the formulations were effective in curing LC lesions in mice. Topical treatment with miltefosine (used as a control) cured the lesions. It is concluded that the ketoconazole-containing formulations designed in this study were not effective against LC in infected mice.

Structural insights into leishmanolysins encoded on chromosome 10 of Leishmania (Viannia) braziliensis

BACKGROUND Leishmanolysins have been described as important parasite virulence factors because of their roles in the infection of promastigotes and resistance to host's defenses. Leishmania (Viannia) braziliensis contains several leishmanolysin genes in its genome, especially in chromosome 10. However, the functional impact of such diversity is not understood, but may be attributed partially to the lack of structural data for proteins from this parasite. OBJECTIVES This works aims to compare leishmanolysin sequences from L. (V.) braziliensis and to understand how the diversity impacts in their structural and dynamic features. METHODS Leishmanolysin sequences were retrieved from GeneDB. Subsequently, 3D models were built using comparative modeling methods and their dynamical behavior was studied using molecular dynamic simulations. FINDINGS We identified three subgroups of leishmanolysins according to sequence variations. These differences directly affect the electrostatic properties of leishmanolysins and the geometry of their active sites. We identified two levels of structural heterogeneity that might be related to the ability of promastigotes to interact with a broad range of substrates. MAIN CONCLUSION Altogether, the structural plasticity of leishmanolysins may constitute an important evolutionary adaptation rarely explored when considering the virulence of L. (V.) braziliensis parasites.

Disseminated cutaneous leishmaniasis caused by Leishmania braziliensis in Southern Brazil

ABSTRACT The authors report a case of disseminated cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis, in a 55 years old patient with 1,119 lesions distributed throughout the body. The patient resides in Sabáudia municipality, North of Paraná State, Southern Brazil, where there was no previous report of this form of leishmaniasis. Treatment with meglumine antimoniate was successful, although the diagnosis was made only five months later.

Detection of Leishmania (Viannia) braziliensis by PCR in samples collected by scraping the lesion edges from patients of an endemic area

American cutaneous leishmaniasis (ACL) is an infectious disease caused by Leishmania. Their diagnosis is performed in samples collected from the lesion biopsies, which has to be performedby physicians. For simplifying the sample collection, this study proposes a minimally invasive procedure, by scraping the lesion edges. Laboratory diagnosis by PCR was performed and compared with the microscopic examination, by analyzing 28 samples collected from patients with suspicion of ACL. Sample collected from the lesion edge with a sterile toothpick was divided into two aliquots. One aliquot was analyzed under direct microscopy, and the second by PCR, by using two primer pairs (one for Leishmania genus and other for L. (V.) braziliensis). Of 28 samples, 27 (96.43 %) showed concordant results in both methodologies (eight positive and 20 negative). The PCR methodologyis an invaluable tool: (i) to determine the Leishmania species; (ii) to provide an alternative procedure of sample collection, when an authorized professional is not available in the respective health service;and (iii) to propose a minimally invasive procedure for collecting biological material...

GENOTYPE CHARACTERIZATION OF Leishmania (Viannia) braziliensis ISOLATED FROM HUMAN AND CANINE BIOPSIES WITH AMERICAN CUTANEOUS LEISHMANIASIS / Caracterização genotípica de isolados de Leishmania (Viannia) braziliensis provenientes de biopsias de humanos e cães com leishmaniose tegumentar americana

Introduction: American tegumentary leishmaniasis (ATL) can be caused by Leishmania (Viannia) braziliensis complex. The evolution of ATL initially results in lesions and can develop into disseminated or diffuse forms. The genetic diversity of L. (V.) braziliensis in some endemic areas of Brazil has been poorly studied, such as in the state of São Paulo. This study analyzed the genetic diversity of L. (V.) braziliensis isolates collected from patients and dogs with LTA from the state of São Paulo. Methods: Leishmaniasis diagnosis was determined by PCR. The 132 biopsies were collected in different regions of Sao Paulo State, Brazil (36 municipalities). The genetic characterization of L. (V.) braziliensis isolates was tested by RFLP-PCR using DNA extracted from biopsies. The primer set amplified a specific region of Leishmania internal transcribed spacers of the ribosomal DNA locus. Results: Of the 132 samples, 52 (40%) were completely genotyped by RFLP-PCR (44 from human patients and eight from dogs). The results showed nine distinct patterns. The majority of the genotyped samples were from Sorocaba (30), and the others were distributed among 14 other municipalities. The first pattern was more frequent (29 samples), followed by pattern 2 (nine samples) and pattern 3 (three samples). Patterns 4, 6, 7, 8 and 9 were composed of two samples each and pattern 5 of one sample. Conclusion: These results suggest that polymorphic strains of L. (V.) braziliensis circulate in the state of São Paulo. These data agree with studies from other regions of Brazil, showing great variability among the natural populations of endemic foci. .

Introdução: A leishmaniose tegumentar americana (LTA) é causada pelo sub-gênero Leishmania (Viannia) braziliensis. A evolução da LTA resulta com a evolução das lesões iniciais. A diversidade genética de L. (V.) braziliensis em algumas áreas endêmicas brasileiras, como no estado de São Paulo, é pouco conhecida. Assim, este estudo teve como objetivo analisar a variabilidade genética de isolados de L. (V.) braziliensis coletados de biopsias de pacientes e cães com LTA no estado de São Paulo. Métodos: O diagnóstico da leishmaniose foi realizado por PCR. As 132 biópsias analisadas foram coletadas em diferentes regiões do Estado de São Paulo, Brasil (36 municípios). A caracterização genética de L. (V.) braziliensis foi realizada por RFLP-PCR utilizando DNA extraído das biopsias. O conjunto de iniciadores utilizado amplificou a região ITS de Leishmania. Resultados: Das 132 amostras analisadas, 52 (40%) foram completamente genotipadas por RFLP-PCR (44 de pacientes e oito de cães). Os resultados mostraram nove padrões distintos. A maioria das amostras genotipadas foi de Sorocaba (30), e as demais foram distribuídas entre 14 outros municípios. O primeiro padrão foi mais frequente (29 amostras), seguido pelo padrão 2 (nove amostras), padrão 3 (três amostras). Padrões 4, 6, 7, 8 e 9 foram compostos de duas amostras de cada um e o padrão 5, com uma amostra. Conclusão: Estes resultados sugerem que cepas polimórficas de L. (V.) braziliensis circulam no estado de São Paulo. Estes dados são concordantes com estudos em outras regiões do Brasil, mostrando grande variabilidade destas populações naturais de focos endêmicos. .

Caracterização da infecciosidade de roedores silvestres e sinantrópicos como hospedeiros reservatórios envolvidos no ciclo zoonótico da leishmaniose tegumentar associada à Leishmania (Viannia) braziliensis / Characterization of infectivity of wild and synanthropic rodents as reservoir hosts involved in the zoonotic cycle of American Cutaneous Leishmaniasis associated with Leishmania (Viannia) braziliensis

Uma das lacunas relacionadas com a ecoepidemiologia da LTA associada à Leishmania (Viannia) braziliensis está relacionada à identificação de hospedeiros reservatórios e flebotomíneos que mantém o ciclo de transmissão. Amaraji, município da Zona da Mata de Pernambuco, apresenta importante incidência da LTA. Este estudo objetivou caracterizar a infecciosidade de roedores silvestres e sinantrópicos à L. (V.) braziliensis como reservatórios envolvidos na manutenção do ciclo zoonótico na região, através do diagnóstico de infecção natural por L. (V.) spp detectado por qPCR (Reação em Cadeia da Polimerase quantitativa); xenodiagnósticos utilizando Lutzomyia longipalpis ou Lutzomyia whitmani em roedores infectados; e, avaliação da interrupção de exposição à transmissão. Estudo experimental realizado entre maio/2012 e agosto/2014, capturou-se 638 roedores pertencentes a 11 diferentes espécies, com predominância de Nectomys squamipes 38,3 por cento (245/638), e, Rattus rattus 23,2 por cento (148/638). Foram marcados com microchips 603 animais, e, realizadas 394 recapturas. Foram obtidos DNA de pele e sangue dos roedores a cada captura/recaptura. Em 176 (29,2 por cento) roedores detectou-se infecção. Foram realizados 51 xenodiagnósticos (46 L. whitmani; 5 L. longipalpis), onde infectaram-se 72,58 por cento (1400/1929) dos flebotomíneos. Não foram identificadas diferenças quanto às espécies vetoras. Roedores foram infectivos aos vetores independentemente da carga parasitária da infecção natural. Foi verificada diminuição da carga parasitária dos roedores em laboratório. A infecção natural por L. (Viannia.) spp detectada nos roedores, indicam que N. squamipes e N. lasiurus atuam como reservatórios primários e, R. rattus, como reservatório secundário no ciclo de transmisssão da LTA na região.

One of the gaps related to ACL (American Cutaneous Leishmaniasis) eco-epidemiology associated with Leishmania (Viannia) braziliensis is related to identification of reservoir hosts and sand flies that keeps the transmission cycle. Amaraji, unicipality of Zona da Mata of Pernambuco state, show a significant incidence of ACL. This study aimed to characterize the infectivity of wild and synanthropic rodents to L. (V.) braziliensis as reservoirs involved in maintaining the zoonotic cycle in the region, through the diagnosis of natural infection with L. (V.) spp detected by qPCR quantitative Reaction Polymerase Chain); xenodiagnoses using fed sandflies (Lutzomyia longipalpisand Lutzomyia whitmani) on infected rodents; and evaluation of exposure to interrupt transmission. Experimental study conducted between may/2012 and august/2014 was captured 638 rodents of 11 various species, with a redominance of Nectomys squamipes 38.3% (245/638), and Rattus rattus 23.2% (148/638). They were markwith microchips 603 rodents, and performed 394 recaptures. DNA samples were obtained from skin and blood of rodents every capt ure / ecapture. In 176 (29.2%) was detected rodents infection. 51 xenodiagnosis were performed (46 use L. whitmaniand 5 use L. longipalpis), where an infected 72.58% (1400/1929) of sand flies. No differences were identified as the vector species. Rodents were infectious to vectors regardless of the load parasite of infection. It was observed decrease in parasite load of laboratory rodents. Natural infection by L. (V.) spp in rodents indicate that N. squamipes and N. lasiurus act as primary reservoirs and R. rattus as secondary reservoir in transmission cycle of LTA in region.

Caracterização genotípica de isolados de Leishmania obtidos de hospedeiros portadores de leishmaniose tegumentar americana / Genotipic characterization of Leishmania isolated obtained from patients hosts of cutaneous leishmaniasis

As leishmanioses constituem um grupo de zoonoses causadas por protozoários do gênero Leishmania. Causam variadas manifestações clínicas e são de grande importância epidemiológica. Constituem um sério problema de saúde pública mundial e estão entre as sete prioridades da Organização Mundial de Saúde. Nas Américas, Leishmania (Viannia) braziliensis é a espécie mais prevalente na forma tegumentar da doença, com diferentes formas clínicas. No Brasil, a leishmaniose tegumentar americana (LTA) é uma doença com diversidade de agentes, reservatórios e vetores, apresentando diferentes padrões de transmissão. Considerável variabilidade genética encontrada nestes parasitas explicaria as adaptações nos diferentes ambientes geográficos. A relevância do quadro sintomatológico e a possível influência genotípica são fatores que devem ser estudados, pois podem contribuir para a melhoria no diagnóstico, tratamento, prognostico e no desenvolvimento de ações epidemiológicas. O objetivo deste estudo foi de analisar a variabilidade genética de isolados de L. (V.) braziliensis isoladas de amostras clínicas de diferentes regiões do Estado de São Paulo. Foram analisadas 132 amostras de origem humana e canina de casos confirmados por diagnóstico molecular de LTA por L.(V.) braziliensis distribuídas em 36 municípios. As amostras foram ensaiadas por duas metodologias de genotipagem: a PCR-LSSP e a PCR-RFLP. DNA de dezenove amostras foram amplificados e genotipados pela PCR-LSSP. Os resultados mostraram que os isolados apresentaram características genéticas distintas. A seguir, amostras de DNA de cinquenta e duas amostras foram amplificadas e genotipadas pela PCR-RFLP, onde se revelaram nove padrões genéticos distintos. Esses resultados sugerem que no Estado de São Paulo circulam isolados de L. (V.) braziliensis polimórficos. Estes dados corroboram com estudos em outras regiões do Brasil, que mostram uma grande variabilidade destas populações naturais de focos endêmicos.

The leishmaniases are a group of zoonoses caused by protozoa of the genus Leishmania, presenting with various clinical manifestations and having a wide epidemiological diversity. They are a serious public health problem worldwide and are among the seven priorities of the World Health Organization. In the Americas, Leishmania (Viannia) braziliensis is the most prevalent species of the cutaneous form of the disease, causing different clinical forms. In Brazil, American cutaneous leishmaniasis (ACL) has a wide variety of agents, reservoirs and vectors, with different transmission patterns. Considerable genetic variability found in these parasites could explain the adaptations to different geographical environments. The significance of symptomatology and possible genotypic influence are factors that must be studied as they can contribute to improving diagnosis, treatment, prognosis and development of epidemiological surveillance measures. The objective of this study was to analyze the genetic variability of isolated of L. (V.) braziliensis isolated from clinical samples from different regions of São Paulo.We analyzed 132 samples of human and canine cases of confirmed ACL by molecular diagnosis by L. (V) braziliensis distributed in 36 municipalities. The samples were tested by two genotyping methods: LSSP-PCR and PCR-RFLP. DNA of nineteen samples was amplified and genotyped by PCRLSSP. The results showed high levels of polymorphism. Next, the fifty-two DNA samples were amplified and genotyped by PCR-RFLP and produced 9 distinct genetic patterns. These results suggest that polymorphic L. (V.) braziliensis isolates have been circulating in the state of São Paulo. These data corroborate studies in other regions of Brazil that show a great variability of these natural populations in endemic areas.

APPLICABILITY OF kDNA-PCR FOR ROUTINE DIAGNOSIS OF AMERICAN TEGUMENTARY LEISHMANIASIS IN A TERTIARY REFERENCE HOSPITAL / Aplicacao do kDNA-PCR para diagnostico de rotina de leishmaniose tegumentar americana em um hospital de referencia

SUMMARY This study evaluated the applicability of kDNA-PCR as a prospective routine diagnosis method for American tegumentary leishmaniasis (ATL) in patients from the Instituto de Infectologia Emílio Ribas (IIER), a reference center for infectious diseases in São Paulo - SP, Brazil. The kDNA-PCR method detected Leishmania DNA in 87.5% (112/128) of the clinically suspected ATL patients, while the traditional methods demonstrated the following percentages of positivity: 62.8% (49/78) for the Montenegro skin test, 61.8% (47/76) for direct investigation, and 19.3% (22/114) for in vitro culture. The molecular method was able to confirm the disease in samples considered negative or inconclusive by traditional laboratory methods, contributing to the final clinical diagnosis and therapy of ATL in this hospital. Thus, we strongly recommend the inclusion of kDNA-PCR amplification as an alternative diagnostic method for ATL, suggesting a new algorithm routine to be followed to help the diagnosis and treatment of ATL in IIER. .

RESUMO Este estudo avaliou a aplicabilidade do kDNA-PCR como método de rotina para diagnóstico de leishmaniose tegumentar americana (ATL) no Instituto de Infectologia Emílio Ribas (IIER), São Paulo, SP, Brasil. O método kDNA-PCR detectou DNA de Leishmania em 87,5% (112/128) dos pacientes com suspeita de ter leishmaniose e, os métodos tradicionais apresentaram as seguintes porcentagens de positividade: 62,8% (49/78) para o teste de Montenegro, 61,8% (47/76) para a pesquisa direta e 19,3% (22/114) para cultura in vitro. O método molecular confirmou a doença em amostras negativas ou inconclusivas pelos métodos laboratoriais tradicionais e, mostrou-se capaz de auxiliar na identificação de infecções causadas pela espécie Leishmania (V.) braziliensis. Além disso, a revisão dos prontuários médicos confirmou a importância do método PCR-RFLP no diagnóstico final de ATL, prognóstico e escolha do tratamento. Assim, recomendamos a inclusão do PCR como método diagnóstico de ATL na rotina hospitalar, e sugerimos um fluxograma para solicitação de exames laboratoriais. .

Sandflies (Diptera: Psychodidae) in rural and urban environments in an endemic area of cutaneous leishmaniasis in southern Brazil

The high proportion of cases of cutaneous leishmaniasis reported amongst residents in the city of Bandeirantes, in the state of Paraná, Brazil, led the authors to investigate the phlebotomine fauna in both urban and rural environments. The sandflies were captured with automatic light traps from 07:00 pm-07:00 am fortnightly in 11 urban peridomiciles from April 2008-March 2009 and monthly in three ecotopes within four rural localities from April 2009-March 2010. In one of these latter localities, sandfly capture was conducted with white/black Shannon traps during each of three seasons: spring, summer and fall. A total of 5,729 sandflies of 17 species were captured. Nyssomyia neivai (46.7%) and Nyssomyia whitmani (35.3%) were the predominant species. In this study, 3,865 specimens were captured with automatic light traps: 22 (0.083 sandflies/trap) in the urban areas and 3,843 (26.69 sandflies/trap) in the rural areas. Ny. neivai was predominant in urban (68.2%) and rural (42.8%) areas. A total of 1,864 specimens were captured with the white/black Shannon traps and Ny. neivai (54.5%) and Ny. whitmani (31.4%) were the predominant species captured. The small numbers of sandflies captured in the urban areas suggest that the transmission of Leishmania has occurred in the rural area due to Ny. neivai and Ny. whitmani as the probable vectors.

Uso de técnicas moleculares na detecção de DNA parasitário e no estudo da variabilidade genética em diferentes fragmentos teciduais de cães naturalmente infectados por Leishmania (Viannia) braziliensis / Use of molecular techniques in parasite DNA detection and genetic variability study in different tissue fragments from naturally infected dogs by Leishmania (Viannia) braziliensis

A leishmaniose tegumentar americana (LTA), em cães, costuma estar associada à resposta humoral baixa ou mesmo negativa, o que inviabiliza os métodos sorológicos convencionais, como ferramenta única no diagnóstico. [...] Métodos convencionais de diagnóstico parasitológico, não têm sido capazes de detectar a presença do parasito em outros sítios anatômicos diferentes da lesão cutânea, em cães naturalmente infectados por Leishmania (Viannia) braziliensis. Diante dos questionamentos sobre o papel do cão doméstico no ciclo de transmissão da LTA e sobre o valor de métodos diagnósticos aplicados na rotina, principalmente em áreas de sobreposição da forma tegumentar e visceral, faz-se necessário a avaliação desses animais sob diversos aspectos. O presente estudo teve como objetivo empregar a PCR específica associada à hibridização molecular e a técnica da reação em cadeia da polimerase com primer único em condições de baixa estringência (LSSP-PCR) visando detectar a presença de DNA parasitário e investigar a variabilidade genética de populações parasitárias presentes em diferentes tecidos de cães naturalmente infectados por L. (V.) braziliensis. Os animais foram selecionados entre os cães sororeatores para Leishmania procedentes de cidades do estado do Rio de Janeiro e encaminhados para eutanásia ao Laboratório de Pesquisa Clínica em Dermatozoonoses em Animais Domésticos do Instituto de Pesquisa Clínica Evandro Chagas da Fundação Oswaldo Cruz. Durante a necropsia, foram obtidas amostras de lesão cutânea, pele íntegra (região escapular e abdominal), baço, fígado e linfonodos (poplíteo e cervical)...

In dogs, American tegumentary leishmaniasis (ATL) is usually associated to a low humoral responseor even to negative results which turns not unfeasible the conventional serological methods. [...] Conventional methods ofparasitological diagnosis have failed to detect the presence of the parasite anatomic sites others thanthe cutaneous lesion, in dogs naturally infected by Leishmania (Viannia) braziliensis. Regarding thequestions on the rule of domestic dogs in the transmission cycle of ATL and, on the applicability ofdiagnostic methods mainly in areas where both visceral and tegumentary disease are found, theevaluation of these animals under different aspects is needed. The objective of this project is to applyspecific PCR assays associated to molecular hybridization and the technique of Low-StringencySpecific-Single Primer – PCR (LSSP-PCR) in order to detect the presence of parasite DNA andevaluate the genetic variability of parasite populations found in different tissues of dogs naturallyinfected by Leishmania (V.) braziliensis...

Tratamento intralesional da leishmaniose cutânea com antimoniato de meglumina no instituto de pesquisa clínica Evandro Chagas, Fiocruz, Rio de Janeiro (2002 a julho 2011) / Intralesional treatment of cutaneous leishmaniasis with meglumine antimoniate at the institute of clinical research Evandro Chagas, Fiocruz, Rio de Janeiro (2002 to July 2011)

A leishmaniose tegumentar americana é uma doença que acomete a pele e as mucosas das vias aero digestivas superiores. Os antimoniais pentavalentes vêm sendo empregados há muitas décadas como fármacos de primeira linha para o seu tratamento. Pacientes com poucas lesões cutâneas, com impossibilidade de receber medicação parenteral regular ou com sinais de toxicidade importante ao antimonial por via sistêmica, podem ser submetidosao tratamento intralesional com antimoniato de meglumina. Objetivos:descrever a eficácia e a segurança do antimoniato de meglumina administrado por via intralesional, para o tratamento da leishmaniose cutânea. Método: Foi realizado um estudo descritivo, retrospectivo, tipo série de casos, de pacientes atendidos no Laboratório de Vigilância em Leishmanioses do Instituto de Pesquisa Clínica Evandro Chagas – FIOCRUZ, de 2002 até julho de 2011, que tivessem sido tratados para leishmaniose cutânea com aplicação intralesional de antimoniato de meglumina, após tratamento sistêmico com o mesmo fármaco...

American tegumentary leishmaniasis is a disease that affects the skin and mucous membranes of the upper aerodigestive tract. Pentavalent antimonial compounds have been used for decades as a first-line drugs for its treatment.Patients with few skin lesions, with inability to receive parenteral medication regularly or with signs of significant toxicity to antimony systemically, may be subjected to treatment with intralesional meglumine antimoniate. Objectives: Todescribe the efficacy and safety of meglumine antimoniate administered intralesionally, for the treatment of cutaneous leishmaniasis. Methods: We conducted a descriptive, retrospective case series of patients treated at the Leishmaniasis Surveillance Laboratory, Evandro Chagas Clinical Research Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil, from 2002 until July2011, which had been treated for cutaneous leishmaniasis with intralesional meglumine antimoniate after systemic treatment with the same drug...

Ensaio clínico fase III para leishmaniose tegumentar americana forma cutânea. Equivalência entre esquemas de alta e baixa dose de antimoniato de meglumina / Phase III clinical trial for american cutaneous leishmaniasis. Equivalence between high and low dose schemes of meglumine antimoniate

Neste estudo comparamos esquemas de alta e baixa dose de antimoniato demeglumina (AM) para o tratamento da forma cutânea de leishmaniose tegumentar americana, em pacientes oriundos do estado do Rio de Janeiro. OBJETIVO:Comparar a eficácia representada pela cura imediata (epitelização em 120 dias),tardia (cicatrização em 360 dias) e definitiva (ausência de reativação ou lesão mucosa em 720 dias) e toxicidade (clínica, laboratorial e eletrocardiográfica) com duas diferentes doses de tratamento com AM para leishmaniose cutânea (LC) e comparar os critérios de cura clínica aqui adotados com aqueles estabelecidos pelo Ministério da Saúde. MÉTODO: Ensaio clínico de não inferioridade, controlado,randomizado, cego e de fase III, com 60 pacientes com LC alocados em dois grupos de tratamento: (A) 20mg Sb5+/kg/dia por 20 dias e (B) 5mg Sb5+/kg/dia por 30 dias administrados por via intramuscular. RESULTADOS: Pacientes dos grupos A e B apresentaram, respectivamente: Cura imediata 90,0% e 86,7%, com tempo médio de epitelização de 58,7 e 54,9 dias; cura tardia por intenção de tratar 76,7% e73,3%; e cura tardia por análise de protocolo 84,6% e 75,9%. Dos 53 pacientes que apresentaram epitelização em até 120 dias, 44 (83,4%)evoluíram para cura tardia...

In this study, we compare schemes of high and low dose of meglumine antimoniate(MA) for the treatment of cutaneous form of American tegumentary leishmaniasis in patients from Rio de Janeiro. OBJECTIVE: To compare the efficacy represented by immediate healing ( epithelialization in 120 days) , late healing (scarring within 360days ) and final healing (no reactivation or mucosal lesion in 720 days) and clinical ,laboratory and electrocardiographic toxicity with two different schemes of treatment with MA for cutaneous leishmaniasis (CL ) and compare the clinical cure criteria adopted here with those established by the Ministry of Health. METHOD :Randomized, controlled, blind, phase III clinical trial, of non-inferiority with 60patients presenting CL divided into two treatment groups: (A) 20 mg Sb5+ / kg / day for 20 days and (B) 5 mg Sb5+ / kg / day for 30 days administered intramuscularly.RESULTS : Patients in groups A and B presented, respectively : immediate healingof 86.7 % and 90.0 % , with a mean time of epithelialization of 58.7 and 54.9 days;late healing by intention to treat of 76.7 % and 73.3%; and late healing by protocol analysis of 84.6 % and 75.9 % .From 53 patients who presented epithelialization within 120 days , 44 (83.4 %) had late healing...