Campomanesia xanthocarpa (Mart.) O. Berg essential oil induces antileishmanial activity and remodeling of the cytoplasm organelles.

Leishmaniasis is a neglected disease that affects millions of people worldwide. This study aimed to analyze antileishmanial activity of Campomanesia xanthocarpa leaf essential oil (EO) on promastigote and amastigote forms of Leishmania amazonensis, cytotoxicity in murine macrophages and sheep erythrocytes. The essential oil (EO) was analyzed by gas chromatography/mass spectrophotometry. The main and most abundant compounds were sesquiterpene hydrocarbons (71.22%) such as trans-caryophyllene (7.87%), bicyclogermacrene (11.28%), and δ-cadinene (8.34%). The IC50 for promastigote and amastigote forms of L. amazonensis was 70 µg mL-1 and 6 µg mL-1, respectively. C. xanthocarpa EO was not cytotoxic for murine macrophages (CC50 1860 µg mL-1) and sheep erythrocytes (1.5%), presenting high selectivity index for protozoan (310). C. xanthocarpa EO induced effects on the morphology and ultrastructure of this parasite. The high activity for intracellular amastigote forms, low toxicity to murine macrophages, and erythrocytes, suggest that C. xanthocarpa EO is promising for the treatment of leishmaniasis.

Filter paper performance in PCR for cutaneous leishmaniasis diagnosis

Abstract INTRODUCTION: The objective of this study was to evaluate the performance of filter paper (FP) for lesion scraping collection in a polymerase chain reaction (PCR) for cutaneous leishmaniasis (CL) diagnosis. METHODS: Lesion scrapings from 48 patients were collected and analyzed for PCR. RESULTS: PCR with FP detected up to three Leishmania braziliensis promastigotes. Considering the direct search by microscopy or PCR of samples collected in STE buffer as standards, the sensitivity of PCR with FP was 100%. CONCLUSIONS: FP can be useful for CL diagnosis in remote regions, allowing high sensitivity in the detection of the parasite by PCR.

Potential of Pluronics® P-123 and F-127 as nanocarriers of anti-Leishmania chemotherapy.

Leishmaniasis is a neglected disease and drugs approved for its treatment often lead to abandonment, failure of therapy and even death. Photodynamic therapy (PDT) has been shown to be a promising, non-invasive and selective for a target region without requiring high-cost technology. Usually, it is employed a photosensitizing agent (PS) incorporated into nanoparticles (NP). Pluronics® P-123 and F-127 micelles are very interesting aqueous NP promoting efficient and selective delivery and less adverse effects. This study aimed to detect the activity of Pluronics® P-123 and F-127 themselves since there is a scarcity of data on these NP activities without drugs incorporation. This study evaluated, in vitro, the activity of Pluronics® against promastigotes and amastigotes of Leishmania amazonensis and also their cytotoxicities. Additionally, the determination of the mitochondria membrane potential in promastigotes, internalization of these Pluronics® in the parasite membrane and macrophages and its stability in the culture medium was evaluated. Results showed that Pluronics® did not cause significant damage to human red cells and promastigotes. The P-123 and F-127 inhibited the survival rate of L. amazonensis amastigotes, and also presented loss of mitochondrial membrane potential on promastigotes. The Pluronics® showed low cytotoxic activity on J774A.1 macrophages, while only P-123 showed moderate cytotoxicity for BALB/c macrophages. The stability of P-123 and F-127 in culture medium was maintained for ten days. In conclusion, the NP studied can be used for incorporating potent leishmanicidal chemotherapy, due to their selectivity towards macrophages, being a promising system for the treatment of cutaneous leishmaniasis.

6,7-Dehydroroyleanone diterpene derived from Tetradenia riparia essential oil modulates IL-4/IL-12 release by macrophages that are infected with Leishmania amazonensis.

The treatment of cutaneous leishmaniasis in associated with several adverse effects and therapeutic failure, resulting in patients' abandonment of treatment. Research on new drugs with leishmanicidal potential from medicinal plants is essential. The anti-Leishmania activity of Tetradenia riparia essential oil (TrEO) and its derivatives, such as the diterpene 6,7-dehydroroyleanone (TrROY), and the immunomodulatory effects of TrEO have been reported. However, few studies have investigated the effects of TrROY. The present study evaluated the modulation of cytokine production by murine macrophages that were infected with Leishmania amazonensis (6 parasites/macrophage) and treated with TrROY (0.1, 1, and 100 µg/ml). Cytokine levels were measured by flow cytometry. The results were analyzed using Student's t test at a 95% confidence interval. Microscopic counting was performed to evaluate the inhibitory effects of TrROY on intracellular infection. TrROY modulated the production of cytokines that are essential for the immune defense response to Leishmania, with a decrease in interleukin-4 (IL-4) levels and an increase in IL-12 levels. A TrROY concentration of 0.1 µg/ml was chosen for the subsequent experiments. This dose was chosen because it modulated IL-4/IL-12 release by murine macrophages that were infected with Leishmania and because it presented no cytotoxic effects. TrROY (0.1 µg/ml) induced a 31% reduction of the rate of infection in murine macrophages compared with untreated cells. TrROY may be a promising leishmanicidal agent. Further in vitro and in vivo studies should be conducted to evaluate the anti-Leishmania and immunomodulatory activity of TrROY.

Alterations induced in the ileum of mice upon inoculation with different species of Leishmania: a preliminary study

Abstract INTRODUCTION: Leishmania species cause skin, mucosal, and disseminated lesions. We studied the effects of three Leishmania species on ileal morphology in mice. METHODS: BALB/c mice were intraperitoneally inoculated with Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis, and Leishmania (Leishmania) major (4 animals/group). After 72h, the ilea were collected and histologically processed. RESULTS: Following inoculation, the goblet cell and intraepithelial lymphocyte populations increased, while Paneth cell number and crypt width decreased. In addition, enterocyte size, villi height, and mucosa, submucosa, and muscular tunic thickness increased. CONCLUSIONS: Leishmania modified the quantity of cells in and morphology of mice ilea.

Contribution of photodynamic therapy in wound healing: A systematic review.

OBJECTIVE: We researched articles that used photodynamic therapy (PDT) for skin wound healing in humans. METHODS: The systematic review was conducted through scientific articles that investigated the action of PDT on wound healing in humans, published from July 2005 to March 2017, in the data bases PubMed and LILACS. RESULTS: The main types of wound described in selected articles in this review were chronic ulcer and non-melanoma skin cancer. For accomplishing the PDT, second generation of photosensitizing agents with laser or light emitting diode were used. The studies demonstrated that PDT contribute in several ways to the wound healing process: leading to cellular death; reducing or increasing inflammation; stimulating fibroblasts proliferation and, consequently, of collagen and elastin; raising transforming growth factor beta and metalloproteinases. Based on this, PDT provided good results in wound healing process, acting in several steps and accelerating tissue repair. CONCLUSIONS: PDT improved healing in many wound models in humans, revealing itself as a promising therapeutic modality for stimulating wound healing and remodelling.

Antileishmanial Activity of a Calophyllum brasiliense Leaf Extract.

Calophyllum brasiliense (Clusiaceae) is a tree that grows mainly in the Atlantic Forest in Brazil. Its leaves possess antibacterial activity, cytotoxic activity against certain tumor cell lines, and antimicrobial activity in BALB/c mice infected with Leishmania (Leishmania) amazonensis.Aiming to identify ultrastructural changes and DNA fragmentation in Leishmania (Viannia) braziliensis, promastigotes were treated with a concentration of the dichloromethane extract and coumarin (-) mammea A/BB from C. brasiliense leaves that inhibited 50 % of the parasites (IC50), and were evaluated by transmission and scanning electron microscopy. Ultrastructural changes showed different levels of mitochondrial alterations, including mitochondrial swelling and a reduction in the density of the mitochondrial matrix. Induced DNA fragmentation, as observed by TUNEL, suggested that the extract and coumarin (-) mammea A/BB induced apoptosis-like cell death. These results suggest that the combination of C. brasiliense extract and coumarin (-) mammea A/BB can be considered a promising candidate for the development of new antiprotozoal agents, because of its significant leishmanicidal activity.

Clinical, laboratory, and therapeutic characteristics of American tegumentary leishmaniasis in the 15 th state health division, northwest Paraná State, Southern Brazil

Abstract INTRODUCTION American tegumentary leishmaniasis (ATL) is an endemic disease in many regions of Brazil; however, only few reports on the actual epidemiological conditions are available. Here, we aimed to assess the clinical, epidemiological, and laboratory characteristics of ATL patients and their treatment in the 15th Regional Health Division of Paraná State, Maringá, Brazil. METHODS This epidemiological study included patients diagnosed with ATL from January 2010 to September 2014, from the 15th Regional Health Division database. RESULTS A total of 220 cases aged 3-84 years (85% male and 60.9% with up to 8 years of schooling) were included. The cases were classified as having the cutaneous form (n=183; 83.2%), mucosal form (n=26; 11.8%), mucocutaneous form (n=11; 5%), and relapses (n=21; 9.6%). Diagnosis was made via laboratory test results in 197 (89.5%) patients, and 172 (78.2%) completed the treatment within the study period. With regard to patients with the cutaneous form, 134 (95%) were cured, 131 (97.8%) were treated with Glucantime(r), and 47 (36.7%) received dosage of >15 and <20mg Sb5+/kg/day. Among the cases with mucosal involvement, 87.1% were cured and most were treated with <20mg Sb5+/kg/day. Thus, the cure rate was 93.6%. CONCLUSIONS During the study period in the 15th Regional Health Division of Paraná State, ATL cases had a good response to treatment with a low rate of relapse or treatment failure, although a high percentage of mucosal or mucocutaneous form cases was also noted.

Prevalence and predictors associated with intestinal infections by protozoa and helminths in southern Brazil.

Approximately 2 billion people are infected with soil-transmitted helminths worldwide, mainly in tropical and subtropical areas. This research aimed to investigate the prevalence and predictors associated with parasitic infections in primary health care. A cross-sectional study was performed with a large random sample to identify the prevalence and predictors associated with parasitic infections in primary health care in Marialva, southern Brazil, from April 2011 to September 2013. Stool samples from 775 individuals were analyzed for the presence of protozoan cysts, helminth eggs, and larvae. The overall prevalence of intestinal parasites was 13.94 %, and the prevalence of protozoa and helminths was 15.1 and 2.9 %, respectively. The predictor variables that were associated with intestinal parasites were male gender odds ratio (OR) 1.60, 95 % confidence interval (CI 1.10-2.40) and the absence of a kitchen garden (OR 2.28, 95 % CI, 1.08-4.85). Positive associations were found between Giardia duodenalis and individuals aged ≤18 with high risk (OR 19.0, 95 % CI 2.16-167.52), between Endolimax nana and the absence of a kitchen garden (p < 0.01), and between Trichuris trichiura and the presence of a kitchen garden (p = 0.014). Polyparasitism was present in 27.27 % of infected individuals. Our findings confirmed a relatively low prevalence in primary care, compared to international standards, despite the rare publications in the area. As variables, male gender and the absence of a kitchen garden stood out as important predictors. It is highly relevant that the health conditions of the population comply with consistent standards.

Antileishmanial and immunomodulatory effects of the essential oil from Tetradenia riparia (Hochstetter) Codd.

Cutaneous leishmaniasis usually presents therapeutic resistance to antimonials, and the existing therapies for leishmaniasis have many adverse effects and toxicity. Natural products may be regarded as possible candidates for alternative leishmaniasis treatment. The plant Tetradenia riparia has shown promise for the treatment of infectious diseases in folk medicine. We evaluated the antileishmanial activity of an essential oil from T. riparia (TrEO) and the modulatory effects of TrEO on cytokine modulation by peritoneal fluid cells that were infected with L. (L.) amazonensis. Peritoneal fluid cells were infected with Leishmania and incubated with TrEO (30 ng/mL) for 3, 6, and 24 h. Cytokines were screened using semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) and flow cytometry. Antileishmanial activity was evaluated at 24 h by microscopic counting and quantitative PCR (qPCR). TrEO treatment induced the death of 50% of Leishmania amastigotes (indicated by microscopic counting) and 91% of the parasite load (indicated by qPCR). TrEO inhibited some of the most critical cytokines for parasite growth and the establishment of infection, including granulocyte-macrophage colony-stimulating factor, interleukin-4 (IL-4), IL-10, and tumour necrosis factor. The parasite inhibited interferon-γ and IL-12, and TrEO blocked this inhibition, indicating that these cytokines are critical for activating mechanisms associated with the death and elimination of the parasite. These results suggest that TrEO may be an alternative leishmaniasis therapy when considering its antileishmanial and immunomodulatory activity.

Diagnostic methods to cutaneous leishmaniasis detection in domestic dogs and cats

Abstract: Cutaneous leishmaniasis is caused by different species of Leishmania. In domestic animals such as dogs and cats, the diagnostic consists of clinical, epidemiological and serological tests, which changes among countries all around the world. Because of this diversity in the methods selected, we propose this systematic literature review to identify the methods of laboratory diagnosis used to detect cutaneous leishmaniasis in domestic dogs and cats in the Americas. Articles published in the last 5 years were searched in PubMed, ISI Web of Science, LILACS and Scielo, and we selected 10 papers about cutaneous leishmaniasis in dogs and cats in the Americas. In Brazil, often the indirect immunofluorescence and enzyme immunoassay (ELISA) have been applied. Other countries like United States and Mexico have been using antigenic fractions for antibodies detections by Western blot. ELISA and Western blot showed a higher sensitivity and efficacy in the detection of leishmaniasis. Analysis of sensibility and specificity of the methods was rarely used. Although confirmatory to leishmaniasis, direct methods for parasites detection and polymerase chain reaction showed low positivity in disease detection. We suggested that more than one method should be used for the detection of feline and canine leishmaniasis. Serological methods such as Western blot and enzyme immunoassay have a high efficacy in the diagnosis of this disease.

Antileishmanial activity of the essential oil from Tetradenia riparia obtained in different seasons

The herbaceous shrub Tetradenia riparia has been traditionally used to treat inflammatory and infectious diseases. Recently, a study showed that T. riparia essential oil (TrEO) obtained in summer has antileishmanial effects, although these results could be influenced by seasonal variation. This study evaluated the activity of the TrEO obtained in different seasons against Leishmania (Leishmania) amazonensis, in vitro and in vivo. The compounds in the TrEO were analysed by gas chromatography-mass spectrometry; terpenoids were present and oxygenated sesquiterpenes were the majority compounds (55.28%). The cytotoxicity and nitric oxide (NO) production were also tested after TrEO treatment. The TrEO from all seasons showed a 50% growth inhibitory concentration for promastigotes of about 15 ng/mL; at 30 ng/mL and 3 ng/mL, the TrEO reduced intracellular amastigote infection, independently of season. The TrEO from plants harvested in summer had the highest 50% cytotoxic concentration, 1,476 ng/mL for J774.A1 macrophages, and in spring (90.94 ng/mL) for murine macrophages. NO production did not change in samples of the TrEO from different seasons. The antileishmanial effect in vivo consisted of a reduction of the parasite load in the spleen. These results suggest that the TrEO has potential effects on L. (L.) amazonensis, consonant with its traditional use to treat parasitic diseases.

Antileishmanial activity of the essential oil from Tetradenia riparia obtained in different seasons.

The herbaceous shrub Tetradenia riparia has been traditionally used to treat inflammatory and infectious diseases. Recently, a study showed that T. riparia essential oil (TrEO) obtained in summer has antileishmanial effects, although these results could be influenced by seasonal variation. This study evaluated the activity of the TrEO obtained in different seasons against Leishmania (Leishmania) amazonensis, in vitro and in vivo. The compounds in the TrEO were analysed by gas chromatography-mass spectrometry; terpenoids were present and oxygenated sesquiterpenes were the majority compounds (55.28%). The cytotoxicity and nitric oxide (NO) production were also tested after TrEO treatment. The TrEO from all seasons showed a 50% growth inhibitory concentration for promastigotes of about 15 ng/mL; at 30 ng/mL and 3 ng/mL, the TrEO reduced intracellular amastigote infection, independently of season. The TrEO from plants harvested in summer had the highest 50% cytotoxic concentration, 1,476 ng/mL for J774.A1 macrophages, and in spring (90.94 ng/mL) for murine macrophages. NO production did not change in samples of the TrEO from different seasons. The antileishmanial effect in vivo consisted of a reduction of the parasite load in the spleen. These results suggest that the TrEO has potential effects on L. (L.) amazonensis, consonant with its traditional use to treat parasitic diseases.

NEW PRIMERS FOR DETECTION OF Leishmania infantum USING POLYMERASE CHAIN REACTION.

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.

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.

Evaluation of the reference value for the Montenegro skin test

The Montenegro skin test (MST) has good clinical applicability and low cost for the diagnosis of American tegumentary leishmaniasis (ATL). However, no studies have validated the reference value (5mm) typically used to discriminate positive and negative results. We investigated MST results and evaluated its performance using different cut-off points.

Antileishmanial activity of essential oil and 6,7-dehydroroyleanone isolated from Tetradenia riparia.

Tetradenia riparia plant is used as a traditional medicine in Africa for the treatment of inflammatory and infectious diseases as like parasitic. Therapy for leishmaniasis caused by Leishmania (Leishmania) amazonensis specie often fails, and the conventional drugs are toxic, expensive, require a long period of treatment, and adverse effects are common. The alternative therapies using natural products are inexpensive and have few or any adverse reaction. These reasons are sufficient to investigate the new natural therapeutic for leishmaniasis. We evaluated the potential of the essential oil (TrEO) and 6,7-dehydroroyleanone (TrROY) isolated from T. riparia on L. (L.) amazonensis promastigote and amastigote forms, cytotoxicity on human erythrocytes and murine macrophages, nitric production and inducible nitric oxide synthase (iNOS) mRNA expression. TrEO was the most effective to promote the Leishmania promastigote death. After 72 h incubation, the lethal dose of TrEO and TrROY that promoted 50% Leishmania death (LD50) were 0.8 µg/mL and 3 µg/mL, respectively. TrEO and TrROY were not cytotoxic to human erythrocytes, but TrROY was toxic to murine macrophages resulting in a low selectivity index. The transmission electronic microscopy showed that TrEO (0.03 µg/mL) was able to modify the promastigote ultrastructures suggesting autophagy as chromatin condensation, blebbing, membranous profiles and nuclear fragmentation. Infected-macrophages treated with TrEO (0.03 µg/mL) or TrROY (10 µg/mL) had an infection index decreased in 65 and 48%. TrEO did not induce iNOS mRNA expression or nitrite production in macrophages infected with Leishmania. TrROY and mainly TrEO promoted the Leishmania death, and TrROY showed loss toxicity to erythrocytes cells. Other compounds derived from T. riparia and the essential oil could be explored to develop a new alternative treatment for leishmaniasis.

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.

Diagnosis of American cutaneous leishmaniasis by enzyme immunoassay using membrane antigens of Leishmania (Viannia) braziliensis.

This study evaluated the reactivity of membrane antigens of Leishmania (Viannia) braziliensis for the diagnosis of ACL by enzyme immunoassay (EIA). Promastigotes of L. (V.) braziliensis were grown in medium 199 and lysed in a sonicator. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting showed that specific proteins of L. (V.) braziliensis (apparent molecular weights 36 kDa and 48-56 kDa) were recognized by sera from ACL patients. These proteins were eluted from the SDS-PAGE and tested in EIA-IgG with sera from ACL patients, healthy individuals, patients with toxoplasmosis, paracoccidioidomycosis, syphilis, tuberculosis, leprosy, and Chagas disease. The EIA-IgG with membrane antigens allowed us to distinguish patients with ACL from healthy individuals and patients with other diseases (P < 0.0001), and showed a sensitivity of 93.3% and specificity of 90.8%, not including Chagas disease patients. 2D-SDS-PAGE followed by Western blotting was performed to improve the characterization of the antigens, and showed a component with isoelectric points near the acid pH side and apparent molecular weights of 48-56 kDa. The results showed good sensitivity and specificity of EIA-IgG with membrane antigens, indicating their potential use for diagnosis of ACL, as well as seroepidemiological surveys and follow-up of clinically cured patients.

Antileishmanial activity of amides from Piper amalago and synthetic analogs

Two natural amides isolated from the chloroform extract of Piper amalago L., Piperaceae, leaves, a hydrogenated derivative and seven synthetic analogs were tested against the promastigote and intracellular amastigote forms of Leishmania amazonensis. The antileishmanial activity was evaluated in terms of growth inhibitory concentration for 50% of protozoa (IC50). The cytotoxicity toward the J774A1 macrophages was evaluated in terms of the cytotoxic concentrations for 50% of macrophages (CC50). The ability to induce nitric oxide production was also investigated for all compounds. The saturated amide 7-(1,3-benzodioxol-5-yl)-1-(1-pyrrolidinyl)-1-heptanone was obtained by hydrogenation of the natural compound N-[7-(3',4'-methylenedioxyphenyl)-2(Z),4(Z)-heptadienoyl]pyrrolidine. Synthetic amides were prepared by addition of the appropriate amine to the corresponding acyl chloride. The natural compound, N-[7-(3',4'-methylenedioxyphenyl)-2(E),4(E)-heptadienoyl]pyrrolidine, was the most active of all tested compounds against the promastigote and intracellular amastigote forms with IC50 values of 15 µM and 14.5 µM, respectively. None of the compounds modulated the production of nitric oxide.