Macrogeographic genetic structure of Lutzomyia longipalpis complex populations using Next Generation Sequencing.

Lutzomyia longipalpis is the main vector of Leishmania infantum, the causative agent of visceral leishmaniasis in the Neotropical realm. Its taxonomic status has been widely discussed once it encompasses a complex of species. The knowledge about the genetic structure of insect vector populations helps the elucidation of components and interactions of the disease ecoepidemiology. Thus, the objective of this study was to genotypically analyze populations of the Lu. longipalpis complex from a macrogeographic perspective using Next Generation Sequencing. Polymorphism analysis of three molecular markers was used to access the levels of population genetic structure among nine different populations of sand flies. Illumina Amplicon Sequencing Protocol® was used to identify possible polymorphic sites. The library was sequenced on paired-end Illumina MiSeq platform. Significant macrogeographical population differentiation was observed among Lu. longipalpis populations via PCA and DAPC analyses. Our results revealed that populations of Lu. longipalpis from the nine municipalities were grouped into three clusters. In addition, it was observed that the levels of Lu. longipalpis population structure could be associated with distance isolation. This new sequencing method allowed us to study different molecular markers after a single sequencing run, and to evaluate population and inter-species differences on a macrogeographic scale.

Os produtos naturais são uma alternativa para o tratamento da candidose oral? Uma revisão de ensaios clínicos / Are natural products an alternative for the treatment of oral candidosis? A review of clinical trials / ¿Los productos naturales son una alternativa para el tratamiento de la candidiasis oral? Una revisión de los ensayos clínicos

Introdução: a candidose oral é uma doença oportunista que acomete principalmente pacientes imunossuprimidos e usuários de prótese dentária total. Apesar da eficácia terapêutica dos antifúngicos utilizados atualmente para o tratamento desta doença, essas drogas apresentam muitos efeitos adversos relacionados à dose. Além disso, tem-se observado um aumento da resistência microbiana para esses agentes. Baseado nisso, uma fonte de busca promissora de princípios ativos alternativos para o tratamento da candidose oral são os produtos naturais. Objetivo: revisar a literatura sobre ensaios clínicos com produtos naturais para o tratamento da candidose oral, a fim de responder se existe evidência que os produtos naturais podem ser utilizados como tratamento alternativo para esta doença. Material e método: foi realizada uma síntese de todos os ensaios clínicos com produtos naturais para tratamento da candidose indexados banco de dados Pubmed. Resultado: seguindo os critérios de inclusão e exclusão entraram nesta revisão 6 estudos. Os estudos avaliaram a eficácia dos produtos naturais quanto aos parâmetros clínicos e micológicos e grau de satisfação do paciente. Conclusão: apesar da pouca quantidade de ensaios clínicos nesta temática, há evidência de que os produtos naturais podem ser usdos para tratar a candidose oral, especialmente alho, mamona, româ e melaleuca(AU)

Introduction: oral candidiasis is an opportunistic disease that mainly affects immunosuppressed patients and users of total dental prosthesis. Despite the therapeutic efficacy of currently used antifungals for the treatment of this disease, these drugs have many dose related adverse effects. In addition, an increase in microbial resistance has been observed for these agents. Based on this, a promising source of alternative active principles for the treatment of oral candidosis are the natural products. Objective: to review the literature on clinical trials with natural products for the treatment of oral candidosis in order to answer if there is evidence that natural products can be used as an alternative treatment for this disease. Material and method: a synthesis of all clinical trials with natural products for the treatment of candidiasis indexed Pubmed database was performed. Results: following the inclusion and exclusion criteria, 6 studies were included in this review. The studies evaluated the efficacy of the natural products regarding clinical and mycological parameters and degree of patient satisfaction. Conclusion: despite the limited amount of clinical trials on this subject, there is evidence that natural products can be used to treat oral candidosis, especially garlic, castor oil, pomegranate, and melaleuca(AU)

Introducción: la candidose oral es una enfermedad oportunista que afecta principalmente a los pacientes inmunosuprimidos ya los usuarios de prótesis dentales totales. A pesar de la eficacia terapéutica de los antifúngicos utilizados actualmente para el tratamiento de esta enfermedad, estos medicamentos presentan muchos efectos adversos relacionados con la dosis. Además, se ha observado un aumento de la resistencia microbiana para estos agentes. En base a esto, una fuente de búsqueda prometedora de principios activos alternativos para el tratamiento de la candidiasis oral son los productos naturales. Objetivo: revisar la literatura sobre ensayos clínicos con productos naturales para el tratamiento de la candidiasis oral, a fin de responder si existe evidencia que los productos naturales pueden ser utilizados como tratamiento alternativo para esta enfermedad. Material y método: se realizó una síntesis de todos los ensayos clínicos con productos naturales para el tratamiento de la candidose indexada base de datos Pubmed. Resultado: siguiendo los criterios de inclusión y exclusión entraron en esta revisión 6 estudios. Los estudios evaluaron la eficacia de los productos naturales en cuanto a los parámetros clínicos y micológicos y grado de satisfacción del paciente. Conclusión: a pesar de la poca cantidad de ensayos clínicos en esta temática, hay evidencia de que los productos naturales pueden ser utilizados para tratar la candidose oral, especialmente ajo, ricino, granada y melaleuca(AU)

Epidemiologia da leishmaniose visceral no município de Fortaleza, Ceará / Epidemiology of visceral leishmaniasis in Fortaleza, Ceará, Brazil

O objetivo do trabalho foi analisar os dados relacionados à transmissão da leishmaniose visceral (LV), no município de Fortaleza, e discutir a respeito da distribuição do vetor, reservatório doméstico e casos humanos ocorridos no período de 2009 a 2013. O presente estudo é do tipo descritivo realizado por meio de levantamento de dados secundários. A correlação entre casos humanos, caninos e número de flebotomíneos foi feita pelo teste de correlação de Spearman, com nível de significância de 5%. No período de 2009 a 2013 foram confirmados 941 casos e 55 óbitos. A letalidade média no período foi de 5,84%. Na distribuição por sexo, houve uma maior proporção de casos no sexo masculino em todos os anos analisados. A faixa etária de 1 a 4 anos apresentou a maior porcentagem de casos, destacando-se o ano de 2010 com 31,5% dos casos. A distribuição média de casos por regional demonstrou um predomínio nas regionais I, V e VI. Em relação aos flebotomíneos, a espécie encontrada em maior abundância foi Lutzomyia (Lutzomyia) longipalpis, seguido de Lutzomyia (Lutzomyia) migonei e Lutzomyia (Lutzomyia) lenti. Segundo os registros da Secretaria Municipal de Saúde de Fortaleza, 39.626 cães foram soro reagentes para LV nos anos de 2009 a 2013, destes 14.313 foram eutanasiados. O ano de 2013 destacou-se com 17.808 cães soro reagentes, sendo 1.273 da SER (Secretaria Executiva Regional) III, 2.572 da SER V e 1.909 da SER VI. Não foi observada correlação significativa entre o número de flebotomíneos capturados e o número de casos caninos de LV(p>0,05). Houve correlação negativa entre casos humanos e caninos (r=-0,0388) e correlação positiva entre casos humanos e número de flebotomíneos (r=0,7469). Os achados criam perspectivas para a identificação de outros fatores que podem influenciar a incidência de casos humanos e caninos, como a participação de outros possíveis reservatórios e vetores na cadeia de transmissão da leishmaniose visceral no município de Fortaleza.(AU)

The objective was to analyze the data relating to the transmission of visceral leishmaniasis (LV) in Fortaleza municipality, and discuss about the vector distribution, domestic reservoir and human cases from 2009 to 2013.This study is descriptive conducted through a survey of secondary data. The correlation between human and canine cases and sandflies number was taken by Spearman correlation test, with 5% significance level. In the period 2009 to 2013, 941 cases and 55 deaths were confirmed. The mortality rate for the period was 5.84%. There was a higher proportion of cases among men in all the years analyzed. The age group 1-4 years old had the highest percentage of cases, highlighting the year 2010 with 31.5% of the cases. The average distribution of cases by SER demonstrated a predominance in SER I, V and VI. With regard to the vector species found in greater abundance was Lutzomyia (Lutzomyia) longipalpis, followed by Lutzomyia (Lutzomyia) migonei and Lutzomyia (Lutzomyia) lenti. According to the records, 39,626 dogs were serum reagents for LV in the years 2009-2013, these 14,313 were killed. The year 2013 stood out with 17,808 dogs serum reagents, and 1,273 of SER III, 2572 of SER V and 1909 of SER VI. There was no significant correlation between the number of sand flies and the number of canine VL cases (p?γτ;0.05). There was a negative correlation between human and canine cases (r = -0.0388) and correlation was observed between human cases and number of sand flies (r= 0.7469).Our findings create prospects for the identification of other factors that may influence the incidence of human and canine cases, such as the participation of other possible reservoirs and vectors in the transmission of the visceral leishmaniasis in Fortaleza.(AU)

A new whole mitochondrial genome qPCR (WMG-qPCR) with SYBR Green® to identify phlebotomine sand fly blood meals.

Phlebotomine sand flies are blood-feeding insects of marked medical and veterinary significance. Investigations on the biology of these insects hold great importance for both ecological and epidemiological purposes. The present work describes a new approach for real-time PCR (qPCR) with SYBR Green®, named WMG-qPCR, to identify phlebotomine blood meals. The novelty of the assay was to design primers based on the Whole Mitochondrial Genome (WMG) of the potential hosts (human, dog, cat, brown rat and chicken) aiming to amplify through qPCR the regions of mitochondrial DNA (mtDNA) which are less conserved among all species. Initially, the best method for mtDNA extraction to be applied in WMG-qPCR was determined. Afterwards, amplification specificities were accessed by cross-reaction assays with mtDNA samples from all animal species, besides phlebotomine DNA. Finally, the selected primers were also tested for their limit of DNA detection through standard curves constructed by serial dilution of blood DNA obtained for each target animal species. The WMG-qPCR was able to detect as low as 10pL of blood, equivalent to 26, 84, 130, and 320fg DNA of cat, human, dog and rat, respectively. The assay was also capable to amplify as low as 5pL of chicken blood (5pg DNA). In conclusion, WMG-qPCR seems to be a promising tool to identify phlebotomine blood meals, with high species-specificity and sensitivity. Furthermore, as no supplementary techniques are required, this new approach presents minimized costs and simplified technical-training requirements for execution.

Molecular identification of Lutzomyia migonei (Diptera: Psychodidae) as a potential vector for Leishmania infantum (Kinetoplastida: Trypanosomatidae).

Visceral leishmaniasis (VL) in Brazil is caused by the protozoan Leishmania infantum. This parasite is transmitted by the bite of a female sand fly. The most important sand fly species in VL transmission is Lutzomyia longipalpis. In Fortaleza, the capital of Ceará State, Brazil, the simultaneous occurrence of Lutzomyia migonei and L. longipalpis was detected in localities where VL transmission is observed. The purpose of this study was to determine conclusively if L. migonei can be found naturally infected with L. infantum in key focus in Fortaleza. Using a CDC traps we performed phlebotomine capture during one year. External morphological features and qPCR targeting species-specific gene sequences of Lutzomyia species were used to identify the female phlebotomine sand flies. The molecular identification of the Leishmania species was performed using qPCR targeting species-specific gene sequences of L. infantum and Leishmania braziliensis. The males L. migonei abundance was higher in the rainy season. Humidity and rainfall positively correlated with males L. migonei abundance, while temperature showed a negative correlation. The correlation between the density of L. migonei female with rainfall, relative air humidity, and temperature were not statistically significant. According to the molecular data produced by qPCR amplifications, three positive sand flies were identified as L. longipalpis, and one was identified as L. migonei. The infection rate was 0.35% and 0.18%, respectively. The parasite load was 32,492±2572 L. infantum in L. migonei while the L. longipalpis had parasite loads between 2,444,964.6±116,000 and 6,287,130±124,277. Our findings confirm L. migonei as a potential vector of VL in Fortaleza at a molecular level.

Epidemiological survey of Lutzomyia longipalpis infected by Leishmania infantum in an endemic area of Brazil.

The objective of this study was to perform an epidemiological survey to determine the areas at risk of visceral leishmaniasis through the detection and quantification of natural infection by Leishmania infantum in Lutzomyia longipalpis. The sandflies were captured between February 2009 and January 2010, at 21 sites in four regions of the Fortaleza municipality. Samples were screened for the presence of Leishmania DNA by Real Time PCR (qPCR), amplification of kDNA minicircle sequence. Out of the 123 pools of analyzed sandflies, 45 were positive for L.infantum, and the minimum infection rate was 3.7%. In the north, south, east and west regions, the pool screen assay predicted sand-fly infection prevalence of 3.4%, 4.7%, 4.9% and 8.4%, respectively. The parasite load ranged from 2.45 ± 0.96 to 2,820,246 ± 106,072. No statistical differences were found with respect to the frequency of sand-fly infection between the regions (P=0.3014), seasons (P = 0.3906) or trap locations (P = 0.8486). Statistical differences were found with respect to the frequency of sand-fly infection between the two seasons only in the west region (P=0.0152). The qPCR was able to detect and quantify L. infantum in L. longipalpis, therefore succeeding in identifying the areas of greatest risk of VL transmission.

Epidemiological survey of Lutzomyia longipalpis infected by Leishmania infantum in an endemic area of Brazil / Levantamento epidemiológico de Lutzomyia longipalpis infectada por Leishmania infantum em área endêmica do Brasil

The objective of this study was to perform an epidemiological survey to determine the areas at risk of visceral leishmaniasis through the detection and quantification of natural infection by Leishmania infantum in Lutzomyia longipalpis. The sandflies were captured between February 2009 and January 2010, at 21 sites in four regions of the Fortaleza municipality. Samples were screened for the presence of Leishmania DNA by Real Time PCR (qPCR), amplification of kDNA minicircle sequence. Out of the 123 pools of analyzed sandflies, 45 were positive for L.infantum, and the minimum infection rate was 3.7%. In the north, south, east and west regions, the pool screen assay predicted sand-fly infection prevalence of 3.4%, 4.7%, 4.9% and 8.4%, respectively. The parasite load ranged from 2.45 ± 0.96 to 2,820,246 ± 106,072. No statistical differences were found with respect to the frequency of sand-fly infection between the regions (P=0.3014), seasons (P = 0.3906) or trap locations (P = 0.8486). Statistical differences were found with respect to the frequency of sand-fly infection between the two seasons only in the west region (P=0.0152). The qPCR was able to detect and quantify L. infantum in L. longipalpis, therefore succeeding in identifying the areas of greatest risk of VL transmission.

O objetivo foi realizar um estudo epidemiológico para determinar as áreas de risco de transmissão de leishmaniose visceral pela detecção e quantificação de infecções naturais por Leishmania infantum em Lutzomyia longipalpis. As coletas foram realizadas entre fevereiro de 2009 e janeiro de 2010 em 21 locais, distribuídos em quatro regiões do município de Fortaleza. As amostras foram testadas quanto à presença de DNA de Leishmania por PCR em tempo real (qPCR). Dos 123 pools de flebotomíneos investigados, 45 foram positivos para L. infantum, e a taxa de infecção mínima foi de 3,7%. Nas regiões Norte, Sul, Leste e Oeste, a prevalência de flebotomíneos infectados foi de 3,4%, 4,7%, 4,9% e 8,4%, respectivamente. A carga de parasitas nos pools variou de 2,45 ± 0,96 a 2.820.246 ± 106.072. Não foram observadas diferenças significativas na frequência de flebotomíneos infectados entre as regiões (P = 0,3014), estação do ano (P = 0,3906) ou localização da armadilha (P = 0,8486). Foram observadas diferenças significativas na frequência de flebotomíneos somente na região oeste durante a estação chuvosa (P = 0,0152). A qPCR foi capaz de detectar e quantificar L. infantum em L. longipalpis, identificando as áreas de maior risco de transmissão de leishmaniose visceral.

In vitro effect of Aloe vera, Coriandrum sativum and Ricinus communis fractions on Leishmania infantum and on murine monocytic cells.

In South America, visceral leishmaniasis is a zoonosis caused by the protozoan species Leishmania infantum (syn. L. chagasi) and is primarily transmitted through the bite of the female Lutzomyia longipalpis. Its main reservoir in urban areas is the dog. The application of control measures recommended by health agencies have not achieved significant results in reducing the incidence of human cases, and the lack of effective drugs to treat dogs resulted in the prohibition of this course of action in Brazil. Therefore, it is necessary to search new alternatives for the treatment of canine and human visceral leishmaniasis. The objectives of this study were to evaluate the in vitro effect of fractions from Aloe vera (aloe), Coriandrum sativum (coriander), and Ricinus communis (castor) on promastigotes and amastigotes of L. infantum and to analyze the toxicity against the murine monocytic cells RAW 264.7. To determine the viability of these substances on 50% parasites (IC50), we used a tetrazolium dye (MTT) colorimetric assay (bromide 3-4.5-dimethylthiazol-2-yl-2,5-dephenyltetrazolium), and on amastigotes we performed an in situ ELISA. All fractions were effective against L. infantum promastigotes and did not differ from the positive control pentamidine (p>0.05). However, the R. communis ethyl acetate and chloroform fractions, as well as the C. sativum methanol fraction, were the most effective against amastigotes and did not differ from the positive control amphotericin B (p>0.05). The R. communis ethyl acetate fraction was the least toxic, presenting 83.5% viability of RAW 264.7 cells, which was similar to the results obtained with amphotericin B (p>0.05). Based on these results, we intend to undertake in vivo studies with R. communis ethyl acetate fractions due the high effectiveness against amastigotes and promastigotes of L. infantum and the low cytotoxicity towards murine monocytic cells.