Multiplex qPCR assay to determine Leishmania infantum load in Lutzomyia longipalpis sandfly samples.

The study aimed to develop a multiplex qPCR to detect Leishmania infantum load in different sandfly sample settings using Leishmania kDNA and sandfly vacuolar ATPase (VATP) subunit C as internal control gene. The amplification of Lutzomyia longipalpis VATP gene was evaluated together with Leishmania infantum kDNA in a multiplex reaction. The concentration of VATP gene oligonucleotides was adjusted until no statistically significant difference was observed between all multiplex standard curves and singleplex curves, that is, only kDNA amplification. Limit of detection (LoD) was measured using a probit model and a cut-off defined by receiver operating characteristic analysis. Limit of quantification (LoQ) was assessed by a linear model using the coefficient of variation threshold of 25%. After assuring VATP gene amplification, its primer-probe concentrations were best at 100 nM/10 nM, respectively. The cut-off Cq value for L. infantum kDNA was defined as 35.46 with 100% of sensitivity and specificity. A total of 95% LoD was determined to be of 0.162 parasites while LoQ was 5.858. Our VATP/kDNA multiplex qPCR assay shows that it can be used to evaluate both DNA integrity and determine L. infantum load in L. longipalpis even for low yielded samples, that is, individual midguts.

Plant-feeding phlebotomine sand flies, vectors of leishmaniasis, prefer Cannabis sativa.

Blood-sucking phlebotomine sand flies (Diptera: Psychodidae) transmit leishmaniasis as well as arboviral diseases and bartonellosis. Sand fly females become infected with Leishmania parasites and transmit them while imbibing vertebrates' blood, required as a source of protein for maturation of eggs. In addition, both females and males consume plant-derived sugar meals as a source of energy. Plant meals may comprise sugary solutions such as nectar or honeydew (secreted by plant-sucking homopteran insects), as well as phloem sap that sand flies obtain by piercing leaves and stems with their needle-like mouthparts. Hence, the structure of plant communities can influence the distribution and epidemiology of leishmaniasis. We designed a next-generation sequencing (NGS)-based assay for determining the source of sand fly plant meals, based upon the chloroplast DNA gene ribulose bisphosphate carboxylase large chain (rbcL). Here, we report on the predilection of several sand fly species, vectors of leishmaniasis in different parts of the world, for feeding on Cannabis sativa We infer this preference based on the substantial percentage of sand flies that had fed on C. sativa plants despite the apparent "absence" of these plants from most of the field sites. We discuss the conceivable implications of the affinity of sand flies for C. sativa on their vectorial capacity for Leishmania and the putative exploitation of their attraction to C. sativa for the control of sand fly-borne diseases.

Identification of Bioactive Compounds against Aedes aegypti (Diptera: Culicidae) by Bioassays and in Silico Assays.

Most of the hematophagous insects act as disease vectors, including Aedes aegypti, responsible for transmitting some of the most critical arboviruses globally, such as Dengue. The use of repellents based on natural products is a promising alternative for personal protection compared to industrial chemical repellents. In this study, the repellent effect of essential oils extracted from Lippia thymoides, Lippia alba, Cymbopogon winterianus, and Eucalyptus globulus leaves was evaluated. Essential oils used showed repellent activity against Ae. aegypti in laboratory bioassays, obtaining protection rates above 70 % from 3.75 mg/mL and higher concentration for all analyzed oils. GC/MS identified 57 constituents, which were used in the ligand-based pharmacophore model to expose compounds with requirements for repellents that modulate mosquitoes behavior through odorant-binding protein 1 Ae. aegypti. Ligand-based pharmacophore model approach results suggested that repellent activity from C. winterianus, L. alba, and L. thymoides essential oils' metabolites is related to Citronelal (QFIT=26.77), Citronelol (QFIT=11.29), Citronelol acetate (QFIT=52.22) and Geranil acetate (QFIT=10.28) with synergistic or individual activity. E. globulus essential oil's repellent activity is associated with Ledol (0.94 %; QFIT=41.95). Molecular docking was applied to understand the binding mode and affinity of the essential oils' data set at the protein binding site. According to molecular docking, Citronelol (ChemPLP=60.98) and geranyl acetate (ChemPLP=60.55) were the best-classified compounds compared to the others and they can be explored to develop new repellents.

Identification of biological factors of Lutzomyia longipalpis using MALDI-TOF peptide/protein profiles.

Sand flies are vectors of great public health importance, since they constitute a group of hematophagous insects responsible for etiological agents transmission of zoonotic diseases such a visceral leishmaniasis. In face of the expansion of these diseases, efficient control strategies are needed which depend on comprehending the sand fly eco-epidemiology. In this regard, MALDI-TOF mass spectrometry has been used for bacteria, fungi and yeast detection studies through peptide/protein profiles. However, little is known about interference of biological factors associated with vector ecology, such as blood meal preferences and even sand fly age on the peptide/protein profiles. Thus, the present study aimed to evaluate the differences in peptide/protein profiles of the sand fly Lutzomyia longipalpis, by means of MALDI-TOF, due to the sand fly's age, sex, blood meal source and Leishmania infantum infection. Sample preparation was made removing both head and last abdomen segments keeping the thorax, its appendices and the rest of the abdomen. Five specimens per pool were used to obtain peptide/protein extract of which 1 µL solution was deposited over 1 µL MALDI matrix dried. Characteristic spectra were analyzed using principal coordinate analysis as well as indicator species analysis to discriminate differences in sand flies's peptide/protein profile by sex, age, blood meal source and L. infantum infection. The results show that the evaluated variables produced distinct peptide/protein profiles, demonstrated by the identification of specific diagnostic ions. It was found that the interference of biological factors should be taken into account when using the MALDI-TOF analysis of sand fly species identification and eco-epidemiological applications in field studies. Based on our results, we believe that it is possible to identify infected specimens and the source of blood meal in a collection of wild sand flies, serving to measure infectivity and understand the dynamics of the vector's transmission chain. Our results may be useful for epidemiological studies that look at the ecology of sand flies and leishmaniasis, as well as for raising awareness of biological characteristics' impact on peptide/protein profiles in sand fly species identification.

The sound of silent RNA in tuberculosis and the lncRNA role on infection.

Tuberculosis (TB) is one of the leading causes of death worldwide, and Diabetes Mellitus is one of the major comorbidities (TB/DM) associated with the disease. A total of 103 differentially expressed ncRNAs have been identified in the TB and TB/DM comparisons. A machine learning algorithm was employed to identify the most informative lncRNAs: ADM-DT, LINC02009, LINC02471, SOX2-OT, and GK-AS1. These lncRNAs presented substantial accuracy in classifying TB from HC (AUCs >0.85) and TB/DM from HC (AUCs >0.90) in the other three countries. Genes with significant correlations with the five lncRNAs enriched common pathways in Brazil and India for both TB and TB/DM. This suggests that lncRNAs play an important role in the regulation of genes related to the TB immune response.

Effects of larval rearing substrates on some life-table parameters of Lutzomyia longipalpis sand flies.

Sand flies are the insects responsible for transmitting Leishmania parasites, the causative agents of leishmaniasis in humans. However, the effects of sand fly breeding sites on their biology and ecology remain poorly understood. Herein, we studied how larval nutrition associated with putative breeding sites of the sand fly Lutzomyia longipalpis affects their oviposition, development, microbiome, and susceptibility to Leishmania by rearing L. longipalpis on substrates collected from an endemic area for leishmaniasis in Brazil. The results showed that female L. longipalpis select the oviposition site based on its potential to promote larval maturation and while composting cashew leaf litter hindered the development, larvae reared on chicken feces developed rapidly. Typical gut microbial profiles were found in larvae reared upon cashew leaf litter. Adult females from larvae reared on substrate collected in chicken coops were infected with Leishmania infantum, indicating that they were highly susceptible to the parasite. In conclusion, the larval breeding sites can exert an important role in the epidemiology of leishmaniasis.

Immune response dynamics and Lutzomyia longipalpis exposure characterize a biosignature of visceral leishmaniasis susceptibility in a canine cohort.

BACKGROUND: Reports have shown correlations between the immune response to vector saliva and Leishmaniasis outcome. We followed dogs in an endemic area for two years characterizing resistance or susceptibility to canine visceral leishmaniasis (CVL) according to Leishmania infantum diagnosis and clinical development criteria. Then, we aimed to identify a biosignature based on parasite load, serum biological mediators' interactions, and vector exposure intensity associated with CVL resistance and susceptibility. METHODOLOGY/PRINCIPAL FINDINGS: A prospective two-year study was conducted in an area endemic for CVL. Dogs were evaluated at 6-month intervals to determine infection, clinical manifestations, immune profile, and sandfly exposure. CVL resistance or susceptibility was determined upon the conclusion of the study. After two years, 78% of the dogs were infected with L. infantum (53% susceptible and 47% resistant to CVL). Susceptible dogs presented higher splenic parasite load as well as persistence of the parasite during the follow-up, compared to resistant ones. Susceptible dogs also displayed a higher number of correlations among the investigated biological mediators, before and after infection diagnosis. At baseline, anti-saliva antibodies, indicative of exposure to the vector, were detected in 62% of the dogs, reaching 100% in one year. Higher sandfly exposure increased the risk of susceptibility to CVL by 1.6 times (CI: 1.11-2.41). We identified a discriminatory biosignature between the resistant and susceptible dogs assessing splenic parasite load, interaction of biological mediators, PGE2 serum levels and intensity of exposure to sandfly. All these parameters were elevated in susceptible dogs compared to resistant animals. CONCLUSIONS/SIGNIFICANCE: The biosignature identified in our study reinforces the idea that CVL is a complex multifactorial disease that is affected by a set of factors which are correlated and, for a better understanding of CVL, should not be evaluated in an isolated way.

Natural infection by Leishmania infantum in the Lutzomyia longipalpis population of an endemic coastal area to visceral leishmaniasis in Brazil is not associated with bioclimatic factors.

Visceral leishmaniasis (VL) is a zoonosis caused by the protozoan Leishmania infantum and in Brazil is transmitted mainly by the bite of Lutzomuyia longipalpis sand flies. Data about the presence, distribution, natural infection rate, seasonal and monthly dynamics of the vector population are important for optimizing the measures to control VL in endemic areas. This study aimed to identify sand fly fauna in an endemic area for VL to detect the prevalence of L. infantum infection in the Lu. longipalpis population and to elucidate the influence of bioclimatic factors on the monthly fluctuations of this vector. HP light traps were monthly set in the intradomicile and peridomicile of residences located in the central and beachfront areas of Camaçari, a VL endemic area. The sand fly collection was conducted in two periods: i) period 1-between December 2011 and November 2012 and ii) period 2-August 2014 and July 2015. Sand fly species were identified and detection of L. infantum infection by qPCR was performed in pools of female Lu. longipalpis. For the first time, the parasite load of positive pools was correlated with the number of Lu. longipalpis captured per month in both periods. Correlation analyses between the monthly fluctuation of the sand fly population and bioclimatic indices of the municipality in both collection periods were also performed. In both evaluated periods, more than 98% of the collected sand flies were Lu. longipalpis, confirming the predominance of this species in the region. It was captured mostly in the beachfront area in all months evaluated (99%). For the period 1, Leishmania DNA was detected in 81% of tested pools representing a minimal infection rate of 9.6%. In the period 2, 40% of the pools were positive with a minimal infection rate of 10.2%. Infected sand flies were only detected in the beachfront area in both periods. The parasite load was low and did not vary in the evaluated months despite the number of collected sand flies. No correlation was observed for climatic factors in both areas of Camaçari. These findings emphasize the high risk of Leishmania transmission in Camaçari regardless of the season and that other factors, aside from bioclimatic elements, are influencing the sand fly population monthly fluctuation.

Xenodiagnosis on dogs with visceral leishmaniasis: Canine and sand fly aspects related to the parasite transmission.

One of the main limitations for the effective control of canine leishmaniasis in endemic areas is the difficulty in identifying infectious dogs. The objective of this study was to determine factors, related to dogs and to parasite detection in sand flies, which are associated with the positive xenodiagnosis of Leishmania infantum using the vector Lutzomyia longipalpis. The xenodiagnosis was performed in 50 owned dogs residing in endemic areas, which were divided into three different groups: G1-26 dogs proved to be infected and classified by severity of VL clinical signs on physical examination; G2-15 dogs proved to be infected and classified by severity of clinical signs and intensity of laboratory abnormalities; G3-nine dogs that were seropositive for anti-Leishmania IgG in ELISA tests. Parasite search in the sand flies after having fed on dogs in the xenodiagnosis was performed by both methodologies, PCR and dissection followed by microscopy. In G1, 58% (15/26) of dogs were able to transmit Leishmania to the vector, when parasite detection in sand flies were performed by PCR technique, 5 days after blood meal, whereas in G2, 53% (8/15) transmitted the parasite to the vector, however, confirmation was performed by direct observation of parasite through optical miscroscopy held 10 days after blood meal. Rate of infectiousness of dogs to sand flies was positively associated to severity of disease (p=0.042 and p=0.040), regardless the method used for clinical classification or for parasite detection in sand flies after xenodiagnosis. In G1 30% (3/10) of dogs with subclinical infection were infectious to the vector, while 80% (12/16) of dogs with clinical disease were also infectious. Even more, 17% (1/6) of dogs that had moderate disease were infectious to the sand flies, while 78% (7/9) of dogs with severe disease were infectious in G2. Still in G2, the proportion of sand flies infected (grade of infectiousness) was significantly lower (p=0.0098) when they fed on dogs with moderate disease (1%) in comparison with dogs with severe disease (38%). The dogs from G3 presented a rate of infectiousness of 11% (1/9), demonstrating that the indirect ELISA is not a good indicator of infectiousness and, therefore, should not be used as a confirmatory test for the euthanasia of dogs, as it is currently done in Brazil.

Seroepidemiological survey of canine visceral leishmaniasis in a continental island of northeast Brazil / Investigação soroepidemiológica da leishmaniose visceral canina em uma ilha continental do Nordeste do Brasil

Introduction: Visceral leishmaniasis (VL) is endemic disease in the neighboring municipalities of the continental island Ilha de Maré, Salvador, Bahia, Brazil. Nevertheless, VL has not been reported in the island itself. Objective: the present study aimed to investigate the seroprevalence and clinical signs of Canine Visceral Leishmaniasis (CVL) and to identify the sand fly population present in the village of Botelho, Ilha de Maré. Methodology: sera of local dogs were tested for anti-Leishmania IgG by immunoassays (screening with TR DPP ™ rapid test and confirmatory with indirect ELISA, Bio-Manguinhos/Fiocruz) and an entomological survey was conducted to estimate and identify the phlebotomine fauna of the region. Results: seven out of 106 samples (6.6%) were positive using rapid test. These positive samples were sent to the Central Laboratory of Bahia for confirmation by indirect ELISA. However, all samples presentednegative results. Nine specimens of Pressatia choti, subfamily Phlebotominae were identified, being this species frequently found in areas with cutaneous leishmaniasis transmission in Brazil. Conclusion: although this work did not confirm the presence of CVL in Ilha de Maré, new serological and entomological studies in a larger area are required for the maintenance of the epidemiological surveillance in the emphasized insular area.

Introdução: Leishmaniose Visceral (LV) é uma doença endêmica em municípios vizinhos à Ilha de Maré, situada na plataforma continental do município de Salvador, Bahia, Brasil. Entretanto, casos de LV não tem sido notificados nesta Ilha. Objetivo: O presente trabalho objetivou investigar a soroprevalência e sinais clínicos de Leishmaniose Visceral Canina (LVC) e identificar a população de flebótomos presentes no povoado de Botelho, Ilha de Maré. Metodologia: soro de cães locais foram testados para IgG anti-Leishmania por imunoensaios (triagem com teste rápido TR DPP™ e confirmatório com Elisa Indireto, Bio-Manguinhos/Fiocruz) e uma investigação entomológica foi conduzida para estimar e identificar a fauna flebotomínea da região. Resultados: sete de 106 amostras (6,6%) foram positivas usando o teste rápido. As amostras positivas foram encaminhadas ao Laboratório Central da Bahia para a confirmação por ELISA indireto. Entretanto, essas amostras apresentaram resultado negativo. Foram encontrados nove exemplares da espécie Pressatia choti, subfamília Phlebotominae, espécie frequentemente encontrada em áreas de transmissão de leishmaniose cutânea no Brasil. Conclusão: apesar deste trabalho não ter confirmado a presença de LVC na Ilha de Maré, novos inquéritos sorológicos e analises entomológicas em uma maior área são necessários para a manutenção de uma vigilância epidemiológica na região insular em destaque.

Primeiro relato da ocorrência de evandromyia sallesi e evandromyia cortelezzii (diptera: psychodidae) na região metropolitana de Salvador, Bahia, Brasil / First report of the occurrence of evandromyia sallesi e evandromyia cortelezzii (diptera: psychodidae) at the metropolitan area of the city of Salvador, Bahia state, Brazil / Primer reporte de ocurrencia de evandromyia sallesi y evandromyia cortelezzii (díptera Psychodidae) en la región metropolitana de la ciudad de Salvador, estado de Bahia, Brasil

Este é o primeiro relato da ocorrência de Evandromyia sallesi (Galvão e Coutinho) e Evandromyia cortelezzii (Brèthes) na região metropolitana de Salvador, Bahia. Os flebotomíneos foram capturados com armadilhas luminosas CDC, durante um estudo investigativo em 24 localidades. A vigilância entomológica foi realizada entre setembro/2009 e março/2010, sendo coletados 13 espécimes de cinco bairros diferentes e classificados dentro do gênero Evandromyia.

This is the first report of Evandromyia sallesi (Galvão e Coutinho) and Evandromyia cortelezzii (Brèthes) occurrence at the metropolitan area of Salvador, Bahia, Brazil. The sand flies were captured with CDC luminous traps, during an investigative study in 24 localities. The entomological survey was performed between September/2009 and March/2010, being thirteen specimens collected and classified into the genus Evandromyia, from five different neighborhoods.

Este es el primer relato de ocurrencia de Evandromyia sallesi (Galvão y Coutinho) y Evandromyia cortelezzii (Brèthes) en la región metropolitana de Salvador, Bahia. Los flebotominos fueron capturados con trampas de luz CDC durante un estudio de investigación en 24 localidades. El muestreo entomológico se realizó entre Septiembre/2009 y Marzo/2010, recolectando 13 ejemplares, de cinco barrios diferentes, clasificados en el género Evandromyia.