Chemical study of Adenocalymma axillarum crude leaf extract and isolated compounds

Abstract Adenocalymma axillarum (K.Schum.) L.G. Lohmann is a liana belonging to the family Bignoniaceae. In traditional medicine, the genus Adenocalymma is used to treat fever, skin ailments, and body, joint, and facial muscle pains, and it is also applied as cosmetic. Biological assays conducted with the A. axillarum crude leaf ethanol extract have indicated leishmanicidal activity and absence of cytotoxicity. This study aimed to analyze the A. axillarum leaf ethanol crude extract by high-performance liquid chromatography-high-resolution mass spectrometry- diode array detector (HPLC-HRMS-DAD) and to evaluate the leishmanicidal and cytotoxic activities of this crude extract, its fractions, and isolated compounds. HPLC-HRMS-DAD analysis of this extract revealed that it consisted mainly of flavonoids, with nine major compounds. Extract purification yielded 4-hydroxy-N-methylproline, 6-β-hydroxyipolamiide, quercetin-3-O-robinobioside, hyperin, isorhamnetin-3-O-robinobioside, and 3'-O-methylhyperin, which were identified by Nuclear Magnetic Resonance. The isolated compounds were inactive against Leishmania amazonensis promastigotes and human lung fibroblast cells.

Synthetic ligustrazine based cyclohexanone and oxime analogs as Anti-Trypanosoma and Anti-Leishmanial agentes

In the present study a series of 34 synthetic ligustrazine-containing α, ß-Unsaturated carbonyl-based compounds and oximes, recognized as anticancer compounds were assessed against protozoa of the Trypanosoma and Leishmania species. Ligustrazine, chemically known as tetramethylpyrazine (TMP), was selected as the core moiety for the synthesis of α, ß-Unsaturated carbonyl-based compounds and these compounds were selected as precursors for the synthesis of new oximes. Some derivates, including 5f and 6i, showed multiple activities against all tested strains. In particular compounds 5f and 8o are the most potent and they are, therefore, potential candidates for trypanosomiasis and leishmaniasis

Increasing putative vector importance of Trichophoromyia phlebotomines (Diptera: Psychodidae)

Despite some phlebotomines being well recognised as vectors of leishmaniasis agents, vector importance of those belonging to the genus Trichophoromyia has not been extensively studied. The present study provides evidence regarding the putative vector role played by some species of Trichophoromyia on leishmanine enzootics, based on literature reports and findings obtained from field experiments conducted in the ecotopes of Pará State, Brazil. The species Th. ubiquitalis, Th. velascoi, Th. auraensis, Th. ininii and Th. brachipyga possess minimal criteria to be included in the list of suspected leishmanine vectors. However, knowledge on man-biting behavior, substantiation of vector competence and determination of epidemiological implications are limited for all of the above mentioned species. Published studies together with present data draw attention to prioritize these phlebotomine species in entomological surveillance programs and studies on experimental susceptibility to Leishmania spp. infection.

Clinical aspects and diagnosis of leishmaniasis in equids: a systematic review and meta-analysis / Aspectos clínicos e diagnóstico da leishmaniose em equídeos: uma revisão sistemática e meta-análise

Abstract Leishmaniases are a group of diseases of zoonotic importance caused by over 20 species of protozoa of the genus Leishmania, in which domestic dogs are considered to be the main reservoir for the disease. However, the involvement of other vertebrates as reservoirs for these parasites has also been investigated. Therefore, the objective of the present study was to carry out a systematic review with meta-analysis on occurrences of leishmaniasis in equids. The case reports described animals with cutaneous symptoms of leishmaniasis (papules, nodules, ulcers or crusts) that regressed spontaneously, located mainly on the head and limbs, from which three species of protozoa were identified in the lesions: Leishmania braziliensis, Leishmania infantum and Leishmania siamensis. In turn, the meta-analysis showed a combined prevalence of 25%, although with high heterogeneity among the studies, which was attributed to the use of different methods for diagnosing the disease. Leishmaniasis in equids is a benign disease but it should be included in the differential diagnosis of cutaneous diseases among these species. Seroepidemiological studies are important in investigating and monitoring suspected exposure of these hosts to the parasite, especially in endemic areas. However, there is also a need to standardize diagnostic methods.

Resumo As leishmanioses são um grupo de doenças de importância zoonótica causadas por mais de 20 espécies de protozoários do gênero Leishmania, sendo o cão doméstico considerado o principal reservatório da doença. No entanto, diversas pesquisas têm investigado o envolvimento de outros vertebrados como reservatórios do parasita. Portanto, o objetivo do presente estudo foi realizar uma revisão sistemática com meta-análise da ocorrência de leishmaniose em equídeos. Os relatos de caso descreviam animais com sintomas cutâneos de leishmaniose (pápulas, nódulos, úlceras, crostas) que regrediam espontaneamente, localizadas principalmente na cabeça e membros, sendo identificadas três espécies do protozoário nas lesões: Leishmania braziliensis, Leishmania infantum e Leishmania siamensis. Por sua vez, a meta-análise evidenciou uma prevalência combinada de 25%, porém com alta heterogeneidade entre os estudos, atribuída às diferenças nos métodos utilizados no diagnóstico da doença. A leishmaniose em equídeos é uma doença benigna, porém deve ser incluída no diagnóstico diferencial de doenças cutâneas nessas espécies. Os estudos soroepidemiológicos são importantes para investigar e monitorar a suspeita de exposição desses hospedeiros ao parasita, principalmente em áreas endêmicas, porém há necessidade de padronização dos métodos de diagnóstico.

Surveillance of phlebotomine fauna and Didelphis marsupialis (Didelphimorphia: Didelphidae) infection in an area highly endemic for visceral leishmaniasis in Colombia / Vigilancia de la fauna flebotomínea e infección de Didelphis marsupialis (Didelphimorphia: Didelphidae) en un área de alta endemia de leishmaniasis visceral en Colombia

Abstract Introduction: The study of the interaction between the parasite, the vector and the mammalian hosts, including man, allows to understand the behavior of the leishmaniases. Objective: To determine the presence of Lutzomyia species and to detect the Leishmania infection in Didelphis marsupialis in an endemic area for visceral leishmaniasis. Materials and methods: Phlebotomine fauna and individuals of D. marsupialis were collected with CDC and Tomahawk™ traps, respectively. The species of Lutzomyia were identified using the Young and Duncan key (1994). Ear and tail biopsies and blood samples from D. marsupialis were taken to identify the Leishmania species by amplifying a fragment of the gene associated with the 70 kD heat shock protein. Results: Seven Lutzomyia species were identified: Lu. evansi, Lu. gomezi, Lu. panamensis, Lu. dubitans, Lu. cayennensis cayennensis, Lu. rangeliana and Lu. trinidadensis. The first three species have epidemiological importance in Colombia because of their implications in the transmission of the Leishmania parasite. Sixty-five tissue samples from 19 D. marsupialis individuals were negative for Leishmania spp. Conclusions: The presence of the Lutzomyia species that have been identified as vectors for Leishmania inside and around houses in the village of El Bledo, in El Carmen de Bolívar represents a risk of infection. Furthermore, the presence of Lu. panamensis is reported for first time in El Carmen de Bolívar in Colombia. Although the lack of detection of Leishmania spp. in D. marsupialis samples may suggest that D. marsupialis does not play an important role in the transmission cycle of Leishmania in this region, it is necessary to carry out further longitudinal studies to confirm this hypothesis.

Resumen Introducción. El estudio de la interacción entre el parásito, el vector y los huéspedes mamíferos, incluido el hombre, permite entender el comportamiento de la leishmaniasis. Objetivo. Determinar la presencia de especies del género Lutzomyia y detectar la infección por Leishmania spp. en Didelphis marsupialis en un área endémica de leishmaniasis visceral. Materiales y métodos. Se recolectaron flebotomíneos y D. marsupialis con trampas CDC y Tomahawk™, respectivamente. Las especies de Lutzomyia se identificaron usando la clave de Young y Duncan, 1994. Se tomaron biopsias de oreja, cola y muestras de sangre de D. marsupialis para diagnosticar Leishmania spp. mediante la amplificación de un fragmento del gen de la proteína de choque térmico de 70 kD. Resultados. Se identificaron siete especies de Lutzomyia: Lu. evansi, Lu. gomezi, Lu. panamensis, Lu. dubitans, Lu. cayennensis cayennensis, Lu. rangeliana y Lu. trinidadensis. Las tres primeras especies son reconocidas como vectores en el país por estar implicadas en la transmisión de Leishmania spp. En total, 65 muestras de tejidos de oreja, cola y de sangre provenientes de 19 D. marsupialis fueron negativas para Leishmania spp. en la PCR-HSP70. Conclusiones. La presencia de flebotomíneos con importancia epidemiológica en la zona evaluada representa un riesgo de transmisión. Asimismo, Lu. panamensis es reportada por primera vez en El Bledo (Carmen de Bolívar). La ausencia de Leishmania spp. en D. marsupialis podría sugerir que esta especie no tiene un papel importante en el ciclo de transmisión de Leishmania en la vereda El Bledo, por lo que es necesario profundizar en estudios longitudinales para corroborar esta hipótesis.

Geographical distribution of Leishmania species in Colombia, 1985-2017 / Distribución geográfica de las especies de Leishmania en Colombia, 1985-2017

Abstract Introduction: Knowledge of the geographical distribution of Leishmania species allows guiding the sampling to little-studied areas and implementing strategies to define risk zones and priority areas for control. Objective: Given that there is no publication that collects this information, the search, review, and compilation of the available scientific literature that has identified species in Colombia is presented in this paper. Materials and methods: A bibliographic search was performed in PubMed, Web of Knowledge, Google Scholar, SciELO and LILACS with the terms "(Leishmania OR Leishmaniasis) AND species AND Colombia", without restrictions on publication year, language or infected organism; records of national scientific events and repositories of theses from Colombian universities were also included. Results: Eighty-six scientific documents published between 1985 and 2017 were found in which the species of Leishmania and their geographical origin were indicated. The species reported, in descending order of frequency, were: Leishmania (Viannia) panamensis, L. (V.) braziliensis, L. (V.) guyanensis, L. (Leishmania) infantum, L. (L.) amazonensis, L. (L.) mexicana, L. (V.) colombiensis, L. (V.) lainsoni and L. (V.) equatorensis; the last three were found with the same frequency. Leishmania species were reported from 29 departments. Conclusion: Information on the distribution of Leishmania species in Colombia is limited; therefore, it is necessary to gather existing data and propose studies that consolidate the distribution maps of Leishmania species in Colombia. This would allow the detection of areas where species have not been identified as well as the comparison of existing parasite and vector distributions.

Resumen Introducción. El conocimiento de la distribución geográfica de las especies de Leishmania permite orientar el muestreo hacia áreas poco estudiadas e implementar estrategias para detectar zonas de riesgo y áreas prioritarias de control. Objetivo. Dado que no existe una publicación que reúna esta información, se planteó la revisión y compilación de la literatura científica disponible de estudios de identificación de especies del país. Materiales y métodos. Se llevó a cabo una búsqueda bibliográfica en PubMed, Web of Knowledge, Google Académico, SciELO y Lilacs con los términos "(Leishmania OR Leishmaniasis) AND especie AND Colombia", así como en memorias de eventos científicos nacionales y repositorios de tesis y trabajos de grado de universidades del país. Resultados. Se encontraron 86 documentos científicos publicados entre 1985 y 2017, en los cuales se informaron la especie de Leishmania y el origen geográfico. Las especies circulantes reportadas, en su orden de frecuencia, fueron: Leishmania (Viannia) panamensis, L. (V.) braziliensis, L. (V.) guyanensis, L. (Leishmania) infantum, L. (L.) amazonensis, L. (L.) mexicana, L. (V.) colombiensis, L. (V.) lainsoni y L. (V.) equatorensis, las últimas tres, con igual frecuencia. Los reportes proceden de 29 departamentos. Conclusión. La información de la distribución de las especies de Leishmania en Colombia es limitada. Por lo tanto, se necesita reunir los datos existentes y plantear trabajos que permitan consolidar el mapa de distribución de las especies en el país, lo cual permitiría detectar las zonas sin información de las especies circulantes y establecer la concordancia entre su distribución y la de los vectores.

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.

Molecular and serological detection of Leishmania spp. in horses from an endemic area for canine visceral leishmaniasis in southeastern Brazil / Detecção molecular e sorológica de infecção por Leishmania (Leishmania) infantum em cavalos na região Sudeste do Brasil

This study aimed to verify the occurrence of Leishmania spp. and Leishmania (Leishmania) infantum in horses from a visceral leishmaniasis endemic area in Brazil. DNA samples from blood and conjunctival swab (CS) were tested by PCR and Indirect Immunofluorescence Antibody Test (IFAT). Although none of the horses was clinically sick, animals infected by Leishmania spp. were found and some could be characterized as infected by L. (L.) infantum. From 40 horses, 100% of the animals were positive by blood PCR, 90% (36/40) by CS PCR, and 2.5% (01/40) in serodiagnosis, by IFAT. Six from these 40 horses were L. (L.) infantum positive by blood PCR. Direct sequencing and analysis of amplicons resulted in a sequence to evolutionary analysis. Results indicate the presence of Leishmania spp. and L. (L.) infantum infecting healthy horses in Brazil. The presence of Leishmania spp. and L. (L.) infantum DNA in asymptomatic horses suggests that they can be important reservoirs of these parasites, a highly relevant finding for the epidemiological surveillance of the diseases they cause.(AU)

O estudo objetivou verificar a ocorrência de Leishmania spp. e Leishmania (Leishmania) infantum em cavalos de uma região endêmica para leishmaniose visceral do Brasil. Amostras de DNA de sangue e suabe conjuntival (SC) foram testadas pela PCR e pela Reação de Imunofluorescência Indireta (RIFI). Embora nenhum cavalo estivesse clinicamente doente, animais infectados por Leishmania spp. e L. (L.) infantum foram encontrados em Ilha Solteira/SP. Dos 40 cavalos, 100% (40/40) foram positivos pela PCR de sangue, 90% (36/40) pela PCR de SC, e 2,5% (01/40) no sorodiagnóstico, pela RIFI. Seis desses 40 cavalos foram positivos para L. (L.) infantum pela PCR de sangue. O sequenciamento direto e a análise dos amplicons resultaram em uma sequência para análise evolutiva. Os resultados indicam a presença de Leishmania spp. e L. (L.) infantum infectando cavalos saudáveis no Brasil. A presença de DNA de Leishmania spp. and L. (L.) infantum em cavalos saudáveis sugere que eles podem ser importantes reservatórios desses parasitas, um achado altamente relevante para a vigilância epidemiológica das doenças que causam.(AU)

Aplicação de planejamento baseado na estrutura do receptor na busca de inibidores de cisteíno-proteases parasitárias (cruzaína (T. cruzi) e PCB (Leishmanioses) / Structure-based virtual screening in the search of parasitic cysteine-proteases inhibitors

Doenças causadas por agentes infecciosos e parasitários são chamadas negligenciadas por não despertarem interesse das indústrias farmacêuticas para o desenvolvimento de novas alternativas terapêuticas. Essas doenças são responsáveis por levar milhões de pessoas à morte todos os anos e afetam principalmente os países pobres e em desenvolvimento. Dentre estas, a doença de Chagas e as leishmanioses, parasitoses causadas por parasitas flagelados pertencentes à família Trypanosomatidae, T. cruzi e Leishmaina sp., respectivamente, se apresentam como um sério problema de saúde pública mundial. Endêmicas em vários países e causando milhões de mortes anualmente, ainda hoje não existem fármacos eficientes e seguros para o tratamento dessas doenças. Este panorama torna eminente a necessidade de pesquisa e desenvolvimento de novos fármacos para essas parasitoses. A busca por agentes quimioterápicos envolve a seleção de vias metabólicas essenciais à sobrevivência dos parasitas. Dentre estas, destacamse cisteíno-proteases presentes nesses tripanossomatídeos, deste modo a cruzaína no T. cruzi, e a CPB2.8 na Leishmania mexicana, se mostram como alvos bioquímicos promissores. A disponibilidade de estruturas cristalográficas da cruzaína e do sequenciamento genômico da CPB2.8, nos permite utilizar estratégias de planejamento de fármacos baseado no receptor (SBDD) na identificação de candidatos a fármacos para essas doenças. Entre as técnicas modernas de SBDD utilizadas, a triagem virtual possibilita identificar promissores candidatos a novos fármacos. Assim neste trabalho, obteve-se por meio da técnica de modelagem comparativa o modelo da enzima CPB2.8 de L. mexicana, visto a indisponibilidade da estrutura cristalográfica no Protein Data Bank (PDB). De modo a refinar o modelo construído realizou-se a simulação por dinâmica molecular de 100ns, apresentando estabilização a partir de 80ns. A simulação por dinâmica molecular foi validada por meio do gráfico de Ramachandran, gráfico de raio de giro, RMSD, gráfico de superfície hidrofóbica. Foram calculados os mapas de interação molecular no programa GRID das seguintes proteínas: cruzaína, CPB2.8, catepsina B e catepsina L, e, posteriormente, foi construído um modelo farmacofórico baseado no sítio ativo das enzimas cruzaína e CPB2.8. O modelo farmacofórico da cruzaína foi validado por curva ROC apresentando valor de AUC 61%. A triagem virtual foi realizada para ambas as proteínas e foram obtidos 369 compostos para a cuzaína e 225 compostos para a CPB2.8. Foi realizado o ancoramento molecular desses compostos obtidos pela triagem virtual a fim de diminuir a quantidade de compostos a serem avaliados experimentalmente

Neglected diseases are caused by parasites and infectious agents and affect mainly people in poor areas being prevalent in 149 countries and causing 534,000 deaths per year. Among neglected diseases we can highlight Chagas Disease and Leishmaniasis, both have a high rate of morbidity and mortality and both are addressed in this project in the search of new drugs against a NTD. Nowadays, the search for new drugs involves the selection of biological pathways essential for parasite survival, in this class of parasites we can suggest the cysteine proteases, a proteases family present in Trypanosoma cruzi and and Leishmania ssp. In order to obtain a new agent against Neglected Disease in this work was obtained the model of the enzyme CPB2.8 of L. mexicana using the comparative modeling technique, due to the unavailability of the crystallographic structure in the Protein Data Bank (PDB). In order to refine the constructed model was performed the molecular dynamics simulation of 100ns, stabilization was achieved from 80ns. Molecular dynamics simulation was validated using the Ramachandran graph, radius of rotation graph, RMSD, hydrophobic surface area graph. The molecular interaction fields were calculated in the GRID program to cruzain, CPB2.8, cathepsin B and cathepsin L. Based on molecular interaction fields generated pharmacophoric models were constructed using information about the active site of the enzymes cruzain and CPB2.8. The pharmacophoric model of cruzain was validated by ROC curve presenting AUC value of 61%. Virtual screening was performed for both proteins and 369 compounds were obtained for cuzain and 225 compounds for CPB2.8. Docking studies of these compounds was performed in order to decrease the amount of compounds to be evaluated experimentally

Seasonality of sand flies (Diptera: Psychodidae) and Leishmania DNA detection in vector species in an area with endemic visceral leishmaniasis

BACKGROUND Leishmaniases are a serious health problem in southeast Brazil, including the city of Belo Horizonte (BH), Minas Gerais state (MG), where there are high rates of incidence and mortality due to visceral leishmaniases. BH is divided into nine sanitary districts (SD) of which one, the Venda Nova SD, was selected for this study because it has high rates of positivity for canine leishmaniasis and high incidence of human leishmaniasis. OBJECTIVES This study aimed to survey the sand fly fauna in Venda Nova SD from August 2011 to July 2013 and perform a descriptive analysis of the vector population. METHODS The sampling was carried out using automatic HP light traps at all covered areas of the Venda Nova SD, in a total of eighteen light traps. Sampled specimens were identified following Galati (2003), and females were submitted to molecular techniques for the detection and identification of Leishmania DNA. A simple environmental description was done for it area and Kernel estimation was used to infer vector density for each study site. FINDINGS A total of 2,427 sand fly specimens belonging to eight species and five genera were collected of which 95.3% were Lutzomyia longipalpis. The seasonal variation curve was delineated by this species. Lu. longipalpis was the most abundant at all collection points and in all months of the study, and exhibited a natural infection rate of 1.01% for Leishmania infantum and 1.77% for Leishmania braziliensis. MAIN CONCLUSIONS The results show the presence and adaptation of Lu. longipalpis to the anthropic environment of BH and reinforces its role as the main vector of L. infantum in the region.

Comparison of LAMP and PCR for molecular mass screening of sand flies for Leishmania martiniquensis infection

BACKGROUND Leishmaniasis caused by Leishmania martiniquensis infection has been reported in human and domestic animals of Martinique Island, Germany, Switzerland, USA, Myanmar and Thailand. The peculiar clinical features of disseminated cutaneous and visceral forms co-existence render the urgent need of specific diagnostic tool to identify the natural sand fly vectors for effective prevention and control strategies. Loop-mediated isothermal amplification (LAMP) of 18S rRNA gene as well as polymerase chain reaction (PCR) of minicircle kinetoplast DNA gene (PCR-mkDNA) have never been applied to detect L. martiniquensis and L. siamensis in sand fly vectors. OBJECTIVE The present study was aimed to validate malachite green-LAMP (MG-LAMP) and PCR-mkDNA techniques to detect L. martiniquensis in sand fly vectors, compared with the conventional PCR of internal transcribed spacer 1 (PCR-ITS1). METHODS We compared the validity of LAMP of 18S rRNA gene and PCR-mkDNA, to PCR-ITS1 in simulation model of L. martiniquensis infection in Sergentomyia gemmea sand flies. Attributable to the sensitivity and specificity, PCR-mkDNA was consecutively applied to detect L. martiniquensis in 380 female sand fly individuals captured in the newly identified affected region of Lamphun Province, Thailand. FINDINGS AND MAIN CONCLUSIONS Results showed that PCR-mkDNA could detect at least one promastigote per sand fly, which was 10-time superior to LAMP and PCR-ITS1. In addition, PCR-mkDNA was more specific, able to differentiate L. martiniquensis from other viscerotropic Leishmania species, such as L. siamensis, L. (L.) donovani, and L. (L.) infantum. Consecutively, mass screening of L. martiniquensis in 380 female sand fly individuals by PCR-mkDNA was implemented in a new affected area of Thailand where a patient with leishmaniasis/HIV co-infection resides; however Leishmania DNA was undetected. In conclusion, PCR-mkDNA is a promising tool for molecular mass screening of L. martiniquensis infection in outbreak areas where several species of Leishmania and sand flies co-exist.

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.

Evaluation of the antiplasmodial and leishmanicidal potential of Myrciaria dubia (Myrtaceae) extract

Abstract INTRODUCTION: Malaria and leishmaniasis are prevalent in tropical regions, which have environmental characteristics that are highly favorable to protozoa and vectors of these diseases; the transmission of these infections in sub-tropical regions, although recognized, represents only a small fraction of cases. Plants are constantly being used in the search for and acquisition of new drugs, and many compounds derived from them have been used to combat various diseases. In this study, we evaluated the action of the dichloromethanolic extract of Myrciaria dubia leaves against the protozoa Plasmodium falciparum, Leishmania amazonensis, Leishmania braziliensis, and Leishmania chagasi through bioassays. METHODS The extract from M. dubia was tested for its anti-P. falciparum activity in an anti-histidine-rich protein II immunosorbent assay. The antileishmanial assays were performed using the resazurin method, while cytotoxicity against human hepatoma (HepG2) strain was determined using the colorimetric MTT [3-(4, 5-dimethyl-2- thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide] method. RESULTS The M. dubia extract presented a half-maximal inhibitory concentration equal to 2.35 (1.05)μg/mL for P. falciparum, 190.73 (6.41) μg/mL for L. amazonensis, and greater than equal to 200µg/mL for L. chagasi and L. braziliensis strains. The cytotoxic concentration for 50% of the cells was above 500μg/mL for HepG2, indicating no toxicity and greater selectivity against parasites. CONCLUSIONS The results obtained indicate the presence of antiplasmodial and leishmanicidal bioactive compounds in the dichloromethanolic extracts of M. dubia leaves, and point towards future studies to elucidate the mechanism of action for each physiological effect.

Recent updates and perspectives on approaches for the development of vaccines against visceral leishmaniasis

Abstract: Visceral leishmaniasis (VL) is one of the most important tropical diseases worldwide. Although chemotherapy has been widely used to treat this disease, problems related to the development of parasite resistance and side effects associated with the compounds used have been noted. Hence, alternative approaches for VL control are desirable. Some methods, such as vector control and culling of infected dogs, are insufficiently effective, with the latter not ethically recommended. The development of vaccines to prevent VL is a feasible and desirable measure for disease control; for example, some vaccines designed to protect dogs against VL have recently been brought to market. These vaccines are based on the combination of parasite fractions or recombinant proteins with adjuvants that are able to induce cellular immune responses; however, their partial efficacy and the absence of a vaccine to protect against human leishmaniasis underline the need for characterization of new vaccine candidates. This review presents recent advances in control measures for VL based on vaccine development, describing extensively studied antigens, as well as new antigenic proteins recently identified using immuno-proteomic techniques.

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.

Correlation between presence of Leishmania RNA virus 1 and clinical characteristics of nasal mucosal leishmaniosis / Correlação entre a presença de Leishmania RNA Vírus 1 e as características clínicas da leishmaniose de mucosa nasal

ABSTRACT INTRODUCTION: Mucosal leishmaniosis (ML) is a severe clinical form of leishmaniosis. Complex factors related to the parasite and the host are attributed to the development of mucosal lesions. Leishmania RNA virus 1 (LRV1) can disrupt immune response, and may be the main determinant of severity of the disease; it should be investigated. OBJECTIVE: To study the existence of clinical differences between patients with ML with endosymbiosis by LRV1 and. those without it. METHODS: A cross-sectional cohort study with clinical evaluation, polymerase chain reaction (PCR) detection of Leishmania, species classification, and search of LRV1 was performed. Only patients with confirmed diagnosis of ML by positive PCR and with nasal mucosa injuries were included in this analysis. RESULTS: Out of 37 patients, 30 (81.1%) were diagnosed with Leishmania braziliensis, five (13.5%) with Leishmania guyanensis, and two (5.4%) with mixed infection of L. braziliensis and L. guyanensis. LVR1 virus was present in 26 (70.3%) of the cases. CONCLUSION: Correlation between clinical phenotype and presence of LRV1 was not observed, although the frequency of the virus is two-fold higher in mucosal lesions than that found in the literature on skin lesions in the same geographical area.

RESUMO Introdução: A leishmaniose de mucosa (LM) é uma forma clínica grave da leishmaniose. Fatores complexos ligados ao parasita e ao hospedeiro são atribuídos ao desenvolvimento das lesões de mucosa. Leishmania RNA Vírus 1 (LRV1) pode subverter a resposta imune, podendo ser o principal determinante da gravidade da doença e deve ser pesquisado. Objetivo: Estudar a existência de diferenças clínicas entre pacientes portadores de LM com endosimbiose por LRV1 e as que não possuem. Métodos: Foi realizado um estudo de coorte histórica com corte transversal com avaliação clínica, detecção da Leishmania por técnica de PCR, classificação da espécie e pesquisa de LRV1. Foram incluídos na análise da pesquisa somente os pacientes com diagnóstico confirmado de LM com PCR positivo, com lesão de mucosa nasal. Resultados: Dos 37 pacientes, 30 (81,1%) foram diagnosticados com L. braziliensis, 5 (13,5%) com L. guyanensis e 2 (5,4%) com infecção mista de L. braziliensis e L. guyanensis. O vírus LVR1 estava presente em 26 casos (70,3%). Conclusão: A correlação entre o fenótipo clínico e a presença do LRV1 não foi constatada, porém a frequência do vírus é duas vezes maior em lesão de mucosa do que encontrado em trabalho, da mesma região, sobre lesão cutânea.

Expression of annexin A1 in Leishmania-infected skin and its correlation with histopathological features

ABSTRACTINTRODUCTION:The aim of this study was quantify annexin A1 expression in macrophages and cluster of differentiation 4 (CD4) + and cluster of differentiation 8 (CD8)+ T cells from the skin of patients with cutaneous leishmaniasis (n=55) and correlate with histopathological aspects.METHODS:Infecting species were identified by polymerase chain reaction-restriction fragment length polymorphism, and expression of annexin A1 was analyzed by immunofluorescence.RESULTS:All patients (n = 55) were infected with Leishmania braziliensis . Annexin A1 was expressed more abundantly in CD163 + macrophages in infected skin (p < 0.0001) than in uninfected skin. In addition, macrophages in necrotic exudative reaction lesions expressed annexin A1 at higher levels than those observed in granulomatous (p < 0.01) and cellular lesions p < 0.05). This difference might be due to the need to clear both parasites and necrotic tissue from necrotic lesions. CD4 + cells in cellular lesions expressed annexin A1 more abundantly than did those in necrotic (p < 0.05) and granulomatous lesions (p < 0.01). Expression in CD8 + T cells followed the same trend. These differences might be due to the pervasiveness of lymphohistiocytic and plasmacytic infiltrate in cellular lesions.CONCLUSIONS:Annexin A1 is differentially expressed in CD163 + macrophages and T cells depending on the histopathological features of Leishmania -infected skin, which might affect cell activation.

Molecular characterization of american Cutaneous Leishmaniasis in the tri-border border area of Assis Brasil, Acre State, Brazil / Caracterização molecular da Leishmaniose Tegumentar americana em área de tríplice fronteira, Assis Brasil, Estado do Acre,Brasil

SUMMARYIn this study, Leishmaniaspecies were identified by Polymerase Chain Reaction (PCR). The epidemiology of patients suspected of having American Cutaneous Leishmaniasis in the municipality of Assis Brasil, Acre State, located in the Brazil/Peru/Bolivia triborder was also investigated. By PCR, the DNA of Leishmaniawas detected in 100% of the cases (37 samples) and a PCR-Restriction Fragment Length Polymorphism (RFLP) of the hsp 70gene identified the species in 32 samples: Leishmania (Viannia) braziliensis (65.6%) , L. (V.) shawi (28.1%) , L. (V.) guyanensis (3.1%) and mixed infection L. (V.) guyanensisand L. (Leishmania) amazonensis(3.1%)This is the first report of L. (V.) shawiand L. (L.) amazonensisin Acre. The two predominant species were found in patients living in urban and rural areas. Most cases were found in males living in rural areas for at least three years and involved in rural work. This suggests, in most cases, a possible transmission of the disease from a rural/forest source, although some patients had not engaged in activities associated with permanence in forestall areas, which indicate a possible sandflies adaptation to the periurban setting.

RESUMOO presente estudo caracterizou as espécies de Leishmaniapela Reação em Cadeia da Polimerase (PCR). Também descreveu os aspectos epidemiológicos de pacientes com suspeita de leishmaniose tegumentar americana do município de Assis Brasil, Estado do Acre, Brasil, localizado na tríplice fronteira Brasil/Peru/Bolívia. A PCR detectou DNA de Leishmaniaem 100% dos casos (37 amostras) e a PCR- Restriction Fragment Length Polymorfism(RFLP) do gene hsp 70identificou as espécies em 32 amostras: Leishmania (Viannia) braziliensis (65,6%) , L. (V.) shawi (28,1%) , L. (V.) guyanensis (3,1%) e infecção mista L. (V.) guyanensise L. (Leishmania) amazonensis(3,1%)Esse é o primeiro registro de L. (V.) shawie L. (L.) amazonensisno Acre. As duas espécies predominantes foram encontradas em indivíduos residentes em áreas rurais e urbanas. O maior número de casos foi notificado entre indivíduos de áreas rurais, sexo masculino, de ocupação rural e tempo de residência maior que três anos. Esses dados sugerem possível transmissão da doença em ambiente rural/florestal na maioria dos casos, no entanto alguns pacientes não tinham envolvimento com atividades relacionadas com a permanência na floresta, indicando possível adaptação de flebotomíneos no ambiente periurbano.

Aplicabilidade da técnica de PCR em tempo real para caracterização de espécies de Leishmania / Aplicability of real time PCR technique for leishmania species characterization

A Leishmaniose Tegumentar Americana (LTA) é causada por protozoários, do gênero Leishmania, envolvidos em um complexo ciclo biológico. No Brasil, sete espécies estão envolvidas com a etiologia da doença, distribuídas em todas as regiões geográficas e responsáveis por diferentes manifestações clínicas. Diante disso, o diagnóstico em conjunto com a identificação da espécie é de grande importância clínico-terapêutica. Este trabalho tem por objetivo avaliar a aplicabilidade da técnica de PCR quantitativa em tempo real (qPCR) para identificação de espécies de Leishmania envolvidas com a etiologia da LTA. Foram realizados ensaios de qPCR para padronização da Temperatura de melting (Tm) utilizando cepas de referência de diferentes espécies de Leishmania. Após o diagnóstico em amostras de sangue de animais domésticos utilizando a qPCR, as amostras positivas foram analisadas através de suas Tm, e os produtos de qPCR foram purificados e sequenciados. Dez amostras previamente caracterizadas por isoenzimas, também foram analisadas através da Tm. Ainda como teste de referência, foi padronizada uma Restriction Fragment Length Polymorphism (RFLP) utilizando as cepas de referência e testada nas amostras. Através da padronização da Tm das espécies, foram criados dois intervalos de análise: 1 (Tm = 78-79,99°C), que compreende: Leishmania (V.) braziliensis, Leishmania (V.) panamensis, Leishmania (V.) lainsoni, Leishmania (V.) guyanensis e Leishmania (V.) shawi; e 2 (Tm = 80-82,2°C), que compreende: Leishmania (V.) naiffi, Leishmania (L.) amazonensis e Leishmania (L.) mexicana. Um total de 223 amostras positivas foi analisado, destas, 58 incluídas no intervalo 1 e 165 no intervalo 2. O sequencimento de 94 destas amostras foi correspondente à L. (V.) braziliensis, L. (V.) panamensis e L. (V.) guyanensis. A RFLP em 173 amostras identificou 167 L. (V.) braziliensis, 05 L. (L.) mexicana e 01 L. (V.) panamensis...

The American Cutaneous Leishmaniasis (ACL) is caused by protozoa of the genus Leishmania, involved in a complex biological cycle. In Brazil, seven species are involved in the etiology of the disease, distributed in all geographic regions and responsible for different clinical manifestations. Therefore, the diagnosis together with the identification of the species is of great clinical and therapeutic importance. This work aims to evaluate the applicability of the technique of real-time quantitative PCR (qPCR) for the identification of Leishmania species involved in the etiology of ACL. qPCR assays for standardizing the melting temperature (Tm) using reference strains of different species of Leishmania were performed. After the diagnosis on blood samples of domestic animals using the qPCR positive samples were analyzed by their Tm and qPCR products were purified and sequenced. Ten samples previously characterized by isoenzymes were also analyzed by Tm. Also as a reference test was standardized as Restriction Fragment Length Polymorphism (RFLP) using the reference strains and tested on samples. Through standardization of Tm species two ranges of analysis were created: 1 (Tm = 78-79,99°C), comprising: Leishmania (V.) braziliensis, Leishmania (V.) panamensis, Leishmania (V.) lainsoni, Leishmania (V.) guyanensis e Leishmania (V.) shawi; and, 2 (Tm = 80-82,2°C), comprising: Leishmania (V.) naiffi, Leishmania (L.) amazonensis e Leishmania (L.) mexicana. A total of 223 positive samples were analyzed, of these, 58 included in the range 1 and 165 in the range 2 to 94 and the sequence of these samples corresponded to L. (V.) braziliensis, L. (V.) panamensis and L. (V.) guyanensis. By RFLP in 173 samples were identified 167 L. (V.) braziliensis, 05 L. (L.) mexicana and 01 L. (V.) panamensis The analysis of Tm of the ten samples characterized by isoenzymes showed 80% agreement (p = 0.6499) between the gold standard (isoenzymes) and intervals developed in this study...

Performance of a real time PCR for leishmaniasis dignosis using a L. (L.) infantum hypothetical protein as target in canine samples

Visceral leishmaniasis represents an important public health issue in different parts of the world, requiring that measures be put in place to control the spread of the disease worldwide. The canine leishmaniasis diagnosis is not easy based on clinical signs, since dogs may not develop the infection with recognizable signs. Thus, the laboratorial diagnosis is essential to ascertain the incidence and prevalence of canine leishmaniasis especially in areas with major control efforts. Although, the diagnosis can be performed by the use of different approaches, the molecular methods such as PCR have become an indispensable tool for leishmaniases diagnosis. A TaqMan assay for real-time PCR (Linj31-qPCR) was developed to determine the parasite occurrence in clinical cases of leishmaniasis. The assay targets an L. (L.) infantum hypothetical protein region. The specificity of the assay was verified by using Leishmania World Health Organization reference strains including parasites belonging to subgenus L. (Leishmania), subgenus L. (Viannia), other Leishmania species and Trypanosoma cruzi. The sensitivity was verified by using isolates of L. (L.) amazonensis and L. (L.) infantum. The usefulness of the assay for diagnosis was ascertained by testing 277 samples from dogs in regions endemic for visceral and/or cutaneous leishmaniasis and from regions in which leishmaniasis was not endemic in São Paulo State, Brazil. Diagnosis of canine visceral leishmaniasis (CVL) was determined on these animals by conventional PCR and three serological tests. The dog samples were divided into four groups. I, dogs with CVL (n = 101); II, dogs with other diseases and without CVL (n = 97); III, dogs with American cutaneous leishmaniasis (n = 7), and, IV, dogs without CVL (n = 72) from areas where leishmaniasis was not endemic as control group. Results indicated that Linj31-qPCR was able to identify parasites belonging to subgenus L. (Leishmania) with no cross-amplification with other parasite subgenera. The Linj31-qPCR detected Leishmania parasites DNA in 98% of samples from Group I. In conclusion this methodology can be used as routine diagnostic tools to detect parasites from subgenus Leishmania.