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.

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.

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.

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.

Leishmaniasis Cutánea Difusa por Leishmania (Viannia) brasiliensis. A propósito de un caso / Diffuse Cutaneous Leishmaniasis by Leishmania (Viannia) brasiliensis. About a case

La leishmaniasis cutánea o mucocutánea presenta variedad de formas clínicas, siendo la más frecuente la úlcera moldurada. Una forma poco frecuente es la llamada leishmaniasis cutánea difusa caracterizada por presentar pápulas, tubérculos, nódulos, placas e infiltración. Inicialmente localizadas, pero con tendencia a la progresión, pudiendo llegar a ser diseminadas. Es una forma aún no comunicada en Paraguay. Presentamos el caso de una mujer adulta mayor, con placas y nódulos en ambos miembros inferiores, y cuyo frotis y anatomía patológica confirmaron el diagnóstico de leishmaniasis. Clínicamente clasificada como leishmaniasis cutánea difusa, la PCR y HRM demostraron ser producida por Leishmania (Viannia) brasiliensis.

Cutaneous or mucocutaneous leishmaniasis presents a variety of clinical forms, the most common being a molded ulcer. A rare form is the so-called diffuse cutaneous leishmaniasis characterized by presenting papules, tubers, nodules, plaques and infiltration; initially located but with a tendency to progression, and may become widespread. It is a form not reported in Paraguay. We present the case of an older adult woman, with plaques and nodules on both lower limbs, and whose smear and pathological anatomy confirmed the diagnosis of leishmaniasis. Clinically classified as diffuse cutaneous leishmaniasis, PCR and HRM were shown to be produced by Leishmania (Viannia) brasiliensis.

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.

First report of treatment failure in a patient with cutaneous leishmaniasis infected by Leishmania (Viannia) naiffi carrying Leishmania RNA virus: a fortuitous combination?

Abstract We report the case of a 32-year-old man from Rio de Janeiro, who was infected in the Amazon region of Brazil by Leishmania (Viannia) naiffi. Generally, patients with L. naiffi cutaneous leishmaniasis exhibit a good therapeutic response to either pentavalent antimonials or pentamidine. However, after pentamidine treatment, this patient's infection evolved to therapeutic failure. To understand this clinical outcome, we investigated the presence of the Leishmania RNA virus (LRV) in parasites isolated from the cutaneous lesion; herein, we discuss the possible association between a poor response to pentamidine therapy and the presence of the LRV.

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.

First description of Leishmania (Viannia) infection in Evandromyia saulensis, Pressatia sp. and Trichophoromyia auraensis (Psychodidae: Phlebotominae) in a transmission area of cutaneous leishmaniasis in Acre state, Amazon Basin, Brazil

Studies on the sandfly fauna to evaluate natural infection indexes are still limited in the Brazilian Amazon, a region with an increasing incidence of cutaneous leishmaniasis. Here, by using a multiplex polymerase chain reaction directed to Leishmania kDNA and hybridisation, we were able to identify L. (Viannia) subgenus in 12 out of 173 sandflies captured in the municipality of Rio Branco, Acre state, revealing a positivity of 6.94%. By sequencing the Leishmania 234 bp-hsp70 amplified products from positive samples, infection by L. (V.) braziliensis was confirmed in five sandflies: one Evandromyia saulensis, three Trichophoromyia auraensis and one Pressatia sp. The finding of L. (Viannia) DNA in two Ev. saulensis corresponds to the first record of possible infection associated with this sandfly. Moreover, our study reveals for the first time in Brazil, Th. auraensis and Pressatia sp. infected by L. (Viannia) 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.

Cell accumulation and antileishmanial effect of exogenous and endogenous protoporphyrin IX after photodynamic treatment / Acumulación celular y efecto anti- Leishmania de la protoporfirina IX exógena y endógena después del tratamiento fotodinámico

Introduction: Photodynamic therapy (PDT) using 5-aminolevulinic acid-induced protoporphyrin IX (ALA-PpIX) constitutes an interesting alternative for cutaneous leishmaniasis treatment. Objective: To evaluate the production of PpIXbased on the administration of ALA and MAL and the effect of ALA-PDTat cellular level on non-infected and infected THP-1 cells using Leishmania ( Viannia ) panamensis or Leishmania ( Leishmania ) infantum (syn Leishmania chagasi ) parasites. Materials and methods: Protoporphyrin IX (PpIX) production and mitochondrial colocalization were evaluated by confocal microscopy. Cell toxicities were evaluated after treatment with the compounds, followed by light irradiation (597-752 nm) at 2.5 J/cm 2 fluency using a colorimetric MTT assay for THP-1 cells and a standard microscopic analysis of parasites. Results were expressed as compound concentration activity against 50% of cells or parasites (CC 50 or IC 50 ). Results: ALA or MAL induced an endogenous PpIX with a red fluorescence localized mainly in the mitochondria inside human cells. ALA and MAL-PDT induced a similar range of toxicities on THP-1 cells (CC 50 0.16±0.01mM and 0.33±0.019 mM, respectively) without any apparent inhibition of intracellular parasites in the infected cells as compared to untreated controls. Exogenous PpIX-PDT was toxic to THP-1 cells (CC 50 0.00032±0.00002 mM), L. (L.) infantum (IC 50 0.003±0.0001 mM) and L. (V.) panamensis (IC 50 0.024±0.0001 mM) promastigotes. Conclusions: Despite the effectiveness of exogenous PpIX on promastigotes and the production of PpIX by human infected cells, treatment with ALA or MAL before irradiation was unable to completely destroy L. (L.) infantum or L. (V.) panamensis intracellular amastigotes.

Introducción. El tratamiento fotodinámico con ácido 5-aminolevulínico como inductor de la protoporfirina IX (ALA-PpIX) constituye una alternativa interesante en el tratamiento de la leishmaniasis cutánea. Objetivo. Evaluar la producción de protoporfirina IX (PpIX) a partir de la administración de ALA o MAL y el efecto de la PDT con ALA a nivel celular en células THP-1 no infectadas e infectadas con Leishmania ( Viannia ) panamensis o Leishmania ( Leishmania ) infantum (syn. Leishmania chagasi ). Materiales y métodos. La producción de protoporfirina IX y su ‘colocalización´ mitocondrial se evaluaron mediante microscopía ‘confocal´. Se evaluó la toxicidad celular después del tratamiento con los compuestos y la aplicación de irradiación de luz (597-752 nm) en una fluencia de 2,5 J/cm 2 mediante el empleo de la prueba colorimétrica con metil-tiazol-tetrazolio (MTT) en las células, y de métodos microscópicos estándar en los parásitos. Los resultados se expresaron como la concentración del compuesto activo en el 50 % de las células o parásitos (CC 50 o CI 50 ). Resultados. El ácido aminolevulínico o el metil-5-aminolevulinato indujeron la protoporfirina IX endógena en células humanas, y se observó fluorescencia de color rojo en las mitocondrias. La actividad del ácido aminolevulínico y del metil-5-aminolevulinato utilizados con terapia fotodinámica fue similar en las células THP-1 (CC 50 0,16±0,01 mM y 0,33±0,019 mM, respectivamente) y, aparentemente, no inhibió los parásitos en las células infectadas, en comparación con los controles. El tratamiento exógeno con protoporfirina IX y terapia fotodinámica fue tóxico para las células THP-1 (CC 50 0,00032 ±0,00002 mM) y para los promastigotes de L. (L .) infantum (IC 50 0,003±0,0001 mM) y L. ( V .) panamensis (CI 50 0,024±0,0001 mM). Conclusiones. A pesar de la ‘fotoactividad´ del tratamiento con protoporfirina IX en promastigotes y de su producción después del tratamiento con ácido aminolevulínico y metil-5-aminolevulinato en las células infectadas con Leishmania , no se observó daño en los amastigotes presentes en las células de L. ( L .) infantum o L . ( V .) panamensis .

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. .