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BACKGROUND: Oxidation taking place during the use of oil leads to the deterioration of both nutritional and sensorial qualities. Natural antioxidants from herbs and plants are rich in phenolic compounds and could therefore be more efficient than synthetic ones in preventing lipid oxidation reactions. This study was aimed at the valorization of Tunisian aromatic plants and their active compounds as new sources of natural antioxidant preventing oil oxidation. RESULTS: Carnosol, rosmarinic acid and thymol were isolated from Rosmarinus officinalis and Thymus capitatus by column chromatography and were analyzed by nuclear magnetic resonance. Their antioxidant activities were measured by DPPH, ABTS and FRAP assays. These active compounds were added to soybean oil in different proportions using a simplex–centroid mixture design. Antioxidant activity and oxidative stability of oils were determined before and after 20 days of accelerated oxidation at 60 °C. CONCLUSION: Results showed that bioactive compounds are effective in maintaining oxidative stability of soybean oil. However, the binary interaction of rosmarinic acid and thymol caused a reduction in antioxidant activity and oxidative stability of soybean oil. Optimum conditions for maximum antioxidant activity and oxidative stability were found to be an equal ternary mixture of carnosol, rosmarinic acid and thymol. © 2016 Society of Chemical Industry

The genus Naegleria, of free-living amoeba (FLA) group, has been investigated mainly due to its human health impact resulting in deadly infections and their worldwide distribution on freshwater systems. Naegleria fowleri, colloquially known as the “brain-eating amoeba”, is the most studied Naegleria species because it causes Primary Amoebic Meningoencephalitis (PAM) of high lethality. The assessment of FLA biodiversity is fundamental to evaluate the presence of pathogenic species and the possibility of human contamination. However, the knowledge of FLA distribution in Brazil is unknown, and to rectify this situation we present a research on identifying Naegleria spp. in the Monjolinho River, as a model study. The river is a public Brazilian freshwater source that crosses the city of São Carlos. Five distinct sampling sites were examined through limnological features, trophozoites culturing and PCR against internal transcribed spacers (ITS) regions and 5.8S rRNA sequence. The results identified N. philippinensis, N. canariensisi, N. australiensis, N.gruberi, N. dobsoni sequences, as well as a Vahlkampfia sequence. The methodology delineated here represents the first Brazilian Naegleria spp. study on a freshwater system. Our result stresses the urgency of a large scale evaluation of the presence of free-living amoebas in Brazil.

Indolocarbazoles are a family of natural alkaloids characterized by their potent protein kinase and topoisomerase I inhibitory activity. Among them, staurosporine (1) has exhibited promising inhibitory activity against parasites. Based on new insights on the activity and mechanism of action of STS in Acanthamoeba parasites, this work reports the isolation, identification, and the anti-Acanthamoeba activity of the minor metabolites 7-oxostaurosporine (2), 4&prime, demethylamino-4&prime, oxostaurosporine (3), and streptocarbazole B (4), isolated from cultures of the mangrove strain Streptomyces sanyensis. A clear correlation between the antiparasitic activities and the structural elements and conformations of the indolocarbazoles 1&ndash, 4 was observed. Also, the study reveals that 7-oxostaurosporine (2) affects membrane permeability and causes mitochondrial damages on trophozoites of A. castellanii Neff.

Leishmaniasis and American trypanosomiasis are parasitic diseases that cause significant clinical, social and economic impact on the population of tropical and subtropical countries. Their current treatment is limited and presents multiple drawbacks, including high toxicity, high cost, lengthy treatment plans, as well as the emergence of resistant species. Therefore, there is a need to find new lead compounds with high potency against parasites and low toxicity in patients. In the present work, the bioguided fractionation of an endemic plant from the Canary Islands, Withania aristata, led to the identification of withanolide-type metabolites (1–3) with leishmanicidal and trypanocidal activities. Compounds 1 and 3 showed a significant dose-dependent inhibition effect on the proliferation of L. amazonensis promastigotes and T. cruzi epimastigotes, higher than the reference drugs, miltefosine and benznidazole, respectively. Moreover, compounds 1–3 were more potent (IC50 0.055–0.663 µM) than the reference drug against the intracellular amastigote stage of L. amazonensis, with a high selectivity index on murine macrophage cells (SI 58.66–216.73). Studies on the mechanism of death showed that the compounds induced programmed cell death or that which was apoptosis-like. The present findings underline the potential of withanolides as novel therapeutic antikinetoplastid agents.

Chagas disease and leishmaniasis are tropical neglected diseases caused by kinetoplastids protozoan parasites of Trypanosoma and Leishmania genera, and a public health burden with high morbidity and mortality rates in developing countries. Among difficulties with their epidemiological control, a major problem is their limited and toxic treatments to attend the affected populations; therefore, new therapies are needed in order to find new active molecules. In this work, sixteen Laurencia oxasqualenoid metabolites, natural compounds 1–11 and semisynthetic derivatives 12–16, were tested against Leishmania amazonensis, Leishmania donovani and Trypanosoma cruzi. The results obtained point out that eight substances possess potent activities, with IC values in the range of 5.40–46.45 µM. The antikinetoplastid action mode of the main metabolite dehydrothyrsiferol (1) was developed, also supported by AFM images. The semi-synthetic active compound 28-iodosaiyacenol B (15) showed an IC 5.40 µM against Leishmania amazonensis, turned to be non-toxic against the murine macrophage cell line J774A.1 (CC > 100). These values are comparable with the reference compound miltefosine IC 6.48 ± 0.24 and CC 72.19 ± 3.06 μM, suggesting that this substance could be scaffold for development of new antikinetoplastid drugs., This work was funded by INTERREG-MAC/1.1b/042 (BIOTRANSFER2), PI18/01380 from Instituto de Salud Carlos III, Spain and RICET [RD16/0027/0001 project, from Programa Redes Temáticas de Investigación Cooperativa, FIS (Ministerio Español de Salud, Madrid, Spain), FEDER. IS, ALA and ARDM were funded by the Agustín de Betancourt Programme (Cabildo de Tenerife – ULL). Authors acknowledge the use AFM Service of General Research Support Services of University of La Laguna (SEGAI-ULL).

The current chemotherapy of Acanthamoeba keratitis relies on few drugs with low potential and limited efficacy, for all this there is an urgent need to identify new classes of anti-Acanthamoeba agents. In this regard, natural products play an important role in overcoming the current need and medicinal chemistry of natural products represents an attractive approach for the discovery and development of new agents. Ursolic acid, a natural pentacyclic triterpenoid compound, possesses a broad spectrum of activities including anti-Acanthamoeba. Herein, we report on the development by chemical transformation of an ursolic acid-based series of seven compounds (2&ndash, 8), one of them reported for the first time. The structure-activity relationship (SAR) analysis of their anti-Acanthamoeba activity revealed that acylation/ether formation or oxidation enhances their biological profile, suggesting that the hydrophobic moiety contributes to activity, presumably by increasing the affinity and/or cell membrane permeability. These ursolic acid derivatives highlight the potential of this source as a good base for the development of novel therapeutic agents against Acanthamoeba infections.

Naegleria fowleri causes a deadly disease called primary amoebic meningoencephalitis (PAM). Even though PAM is still considered a rare disease, the number of reported cases worldwide has been increasing each year. Among the factors to be considered for this, awareness about this disease, and also global warming, as these amoebae thrive in warm water bodies, seem to be the key factors. Until present, no fully effective drugs have been developed to treat PAM, and the current options are amphotericin B and miltefosine, which present side effects such as liver and kidney toxicity. Statins are able to inhibit the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which is a key enzyme for the synthesis of ergosterol of the cell membrane of these amoebae. Therefore, the in vitro activity of a group of statins was tested in this study against two types of strains of Naegleria fowleri. The obtained results showed that fluvastatin was the most effective statin tested in this study and was able to eliminate these amoebae at concentrations of 0.179 &plusmn, 0.078 to 1.682 &plusmn, 0.775 &micro, M depending on the tested strain of N. fowleri. Therefore, fluvastatin could be a potential novel therapeutic agent against this emerging pathogen.

Acanthamoeba genus is a widely distributed and opportunistic parasite with increasing importance worldwide as an emerging pathogen in the past decades. This protozoan has an active trophozoite stage, a cyst stage, and is dormant and very resistant. It can cause Acanthamoeba keratitis, an ocular sight-threatening disease, and granulomatous amoebic encephalitis, a chronic, very fatal brain pathology. In this study, the amoebicidal activity of sixteen Laurencia oxasqualenoid metabolites and semisynthetic derivatives were tested against Acanthamoeba castellanii Neff. The results obtained point out that iubol (3) and dehydrothyrsiferol (1) possess potent activities, with IC50 values of 5.30 and 12.83 &micro, M, respectively. The hydroxylated congeners thyrsiferol (2) and 22-hydroxydehydrothyrsiferol (4), active in the same value range at IC50 13.97 and 17.00 &micro, M, are not toxic against murine macrophages, thus, they are solid candidates for the development of new amoebicidal therapies.

Protozoan pathogens that cause neglected tropical diseases are a major public health concern in tropical and developing countries. In the course of our ongoing search for new lead compounds as potential antiprotozoal agents, this study aims to perform a bio-guided fractionation of Pituranthos battandieri, using an in vitro assay against Leishmania amazonensis and Trypanosoma cruzi. Two known polyacetylenes, (−)-panaxydiol (1) and (−)-falcarindiol (2) were identified from the ethanolic extract of the aerial parts of P. battandieri as the main bioactive constituents. Compounds 1 and 2 showed similar potency (IC50 values of 5.76 and 5.68 μM, respectively) against L. amazonensis to miltefosine (IC50 value of 6.48 μM), the reference drug, and low toxicity on macrophage cell lines J774. Moreover, compound 1 exhibited moderate activity (IC50 23.24 μM) against T. cruzi. In addition, three known furanocoumarins, 8-geranyloxypsoralen (3), 8-geranyloxy-5-methoxypsoralen (4), and phellopterin (5) were isolated. Their structures were elucidated by NMR and MS analysis. Compounds 1 and 2 are described for the first time in the Pituranthos genus, and this is the first report on their antiprotozoal activity. These results highlight this type of polyacetylenes as an interesting scaffold for the development of novel antiparasitic drugs.

Acanthamoeba are free living amoeba that have been isolated from different environments like soil, water, air dust. Moreover, they are also able to act as opportunist pathogens, mainly causing a fatal encephalitis and also keratitis in both human and animals. This study was aimed to evaluate the activity of the Medicines for Malaria Venture (MMV) compounds against the trophozoite stage of Acanthamoeba castellanii Neff. Sixteen compounds showed ≥90% inhibition of parasite growth in the initial screen (10 μM). Those set were further evaluated to determine the inhibitor concentration that inhibit the 50% of the initial population and cytotoxicity against murine macrophages. Among the compounds included in the pathogen box, pentamidine and posaconazole were the most effective against this parasite with an of IC50 of 0.567 ± 0.04 and 0.630 ± 0.11, respectively.

Acanthamoeba keratitis (AK), a severe sight-threatening corneal infection, has become a significant medical problem, especially among contact lens wearers. The disease manifests as eye pain, congestion, blurred vision, lachrymation, and ring-shaped infiltrates of the cornea, and can lead to permanent blindness. Inappropriate habits of contact lens users may result in an increased risk of AK infection. The anti-amoebic efficiency of popular multipurpose contact lens solutions is insufficient to reduce this risk. An effective and non-toxic therapy against AK has not yet been developed. The prevention of AK is crucial to reduce the number of AK infections. Nanoparticles are known to be active agents against bacteria, viruses, and fungi and were also recently tested against protozoa, including Acanthamoeba spp. In our previous studies, we proved the anti-amoebic and anti-adhesive activity of silver nanoparticles against Acanthamoeba castellanii. The aim of this study is to evaluate the activity, cytotoxicity, and anti-adhesive properties of silver nanoparticles conjugated with five commonly used multipurpose contact lens solutions against the Acanthamoeba castellanii NEFF strain. The obtained results show a significant increase in anti-amoebic activity, without increasing the overall cytotoxicity, of Solo Care Aqua and Opti Free conjugated with nanoparticles. The adhesion of Acanthamoeba trophozoites to the contact lens surface is also significantly reduced. We conclude that low concentrations of silver nanoparticles can be used as an ingredient in contact lens solutions to decrease the risk of Acanthamoeba keratitis infection.

Primary Amoebic Encephalitis due to Naegleria fowleri species is a fatal infection of the Central Nervous System mostly affecting children and young adults. Infections often occur after performance of risk activities in aquatic habitats such as swimming and splashing. PAḾs therapy remain a key issue to be solved which needs an urgent development. Recently, statins have been highlighted as possible novel compounds to treat PAM. Furthermore, type 2 statins due to improved pharmacological properties and lower toxicity could be use in the future. In the present work, three type 2 statins were checked for their activity against two type strains of N. fowleri. In addition, the effects at the cellular level triggered in treated amoebae were checked in order to evaluate if programmed cell death was induced. The obtained results showed that the tested statins, rosuvastatin, pitavastatin and cerivastatin were able to eliminate N. fowleri trophozoites and also induced PCD. Therefore, type 2 statins could be used in the near future for the treatment of PAM.

The use of freshwater in agricultural systems represents a high percentage of total water consumption worldwide. Therefore, alternative sources of water for irrigation will need to be developed, particularly in arid and semi-arid areas, in order to meet the growing demand for food in the future. The use of recycled wastewater (RWW), brackish water (BW) or desalinated brackish water (DBW) are among the different non-conventional water resources proposed. However, it is necessary to evaluate the health risks for humans and animals associated with the microbiological load of these waters. Protozoa such as free-living amoebae (FLA) are considered an emerging group of opportunistic pathogens capable to cause several diseases in humans (e.g. cutaneous and ocular infections, lung, bone or adrenal gland conditions or fatal encephalitis). In the present study we evaluate FLA presence in three different irrigation water qualities (RWW, BW and DBW) and its survival in irrigated agricultural soils of an extremely arid insular ecosystem (Fuerteventura, Canary Islands, Spain). Samples were cultured on 2% Non-Nutrient Agar (NNA) plates covered with a thin layer of heat killed E. coli and checked daily for the presence of FLA. According to the prevalence of FLA, Vermamoeba vermiformis (53,8%), Acanthamoeba spp. (30,8%), Vahlkampfia avara (7,7%) and Naegleria australiensis (7,7%) were detected in the analysed water samples, while Acanthamoeba (83,3%), Cercozoa spp. (8,3%) and Vahlkampfia orchilla (8,3%) were isolated in irrigated soils. Only Acanthamoeba strains were isolated in no irrigated soils used as control, evidencing the capability of these protozoa to resist environmental harsh conditions. Additionally, all analysed water sources and the irrigated soils presented growth of several pathogenic bacteria. Therefore, the coexistence in water and soils of pathogenic bacteria and FLA, can mean an increased risk of infection in agroecosystems.

Introduction: Free Living Amoebae (FLA) are unicellular protozoa common to most soil and aquatic environments. Only members of the genus Acanthamoeba and the species Naegleria fowleri, Balamuthia mandrillaris and Sappinia diploidea are known to infect humans and other animals, often with fatal consequences. Acanthamoeba species are the causative agents of a sight threating infection of the cornea known as Acanthamoeba keratitis (AK) that mainly affects immunocompetent contact lens wearers and has shown an increased incidence in the recent years worldwide. To date, most of the available treatments and contact lens maintenance solutions are not fully effective against these pathogens.Areas covered: There is a clear need for novel fully effective therapeutic agents against AK. In the recent years the application of ‘phylopharmacology’ and RNAi has shown to be a reliable tool for the elucidation of novel drug targets and therapeutic agents. Current therapeutic options for AK as well as ongoing researc...

The present research, included in the project “ A history of shepherding on the Island of La Gomera ”, investigates the historical development of shepherding on this island from prehistoric to modern times, and from a social, economic, territorial, ecological, and cultural perspective. Within the framework of the project and its multidisciplinary approach, we present the initial phase of the archaeological study, which aims to delve into the management and consumption of livestock during the island's prehistory. In order to track this “culture” from an archeological standpoint, residential contexts have been proposed as a first stage in the survey, as they have not so far been addressed as an important issue for the archeology of La Gomera. To this end, thirteen archaeological sondages have been carried out in five separate points of the island. Exhumed archaeological materials relating them to the stratigraphic context of each domestic space are analyzed. Data from the consumption of food, especially those from the livestock, has been studied. The text provides preliminary information on shepherding and management of the island territory by ancient Gomerans.

The genus Acanthamoeba includes pathogenic strains which are causative agents of keratitis and encephalitis that often may end fatal in humans and other animals. In the present study, forty soil samples were collected in the island of El Hierro, Canary Islands, Spain, and checked for the presence of Acanthamoeba. Samples were cultivated onto 2 % non-nutrient agar plates seeded with a layer of heat killed Escherichia coli. Amplification by PCR and sequencing of the DF3 region of the 18S rDNA of Acanthamoeba was carried out in order to confirm morphological identification of the amoebae. Furthermore, Acanthamoeba spp. was isolated from 47.5 % of soil samples. Moreover, genotypes T2, T4, and T11 were identified in these samples. To the best of our knowledge, this is the first study to establish genotypes T2, T4, and T11 in soil sources from El Hierro island.

Naegleria fowleri is the causative agent of a type of encephalitis called Primary Amoebic Encephalitis (PAM). Almost 98 % of PAM cases reported worldwide are fatal and affect mostly immunocompetent children and young adults. The current therapeutic option against PAM cases includes a combination of miltefosine, amphotericin B and other drugs which are unfortunately associated with severe side effects. In a recent study in our group, statins were tested in vitro against Naegleria fowleri trophozoites showing activity against these pathogens at low concentrations causing low toxicity. Consequently, there is an urgent need to develop novel PAM therapeutic options. Therefore, this study was undertaken to evaluate the pathway of cell death induced by two of the previously tested molecules, fluvastatin and atorvastatin. Moreover, these statins were compared to miltefosine and amphotericin B. Furthermore, the induction of Programmed Cell Death (PCD) instead of necrosis in treated amoebae would be the ideal situation since necrosis could lead to non-desired inflammation processes in the infected individual. The obtained results revealed that both statins induced PCD in the treated amoebae after the observation of condensed chromatin, cell membrane damages, mitochondrial membrane potential and ATP levels collapse and ROS generation. In conclusion, both fluvastatin and atorvastatin could be potential new candidates for PAM therapy since they are active at low concentrations, induce low toxicity and cause PCD in the treated amoebae, hence avoiding the activation of inflammation pathways.

Photodynamic therapy (PDT) is a potentially non-invasive therapeutic strategy that involves the use of a photosensitizing agent molecule capable to absorb light at a specific wavelength and to transfer the absorbed energy to generate singlet oxygen. This oxidizing agent reacts with double bonds of membrane phospholipids and sterols present in biological membranes causing damages and/or cell permeability. With the aim to analyze and to determine the effectiveness of possible photosensitizing agents, in this work we evaluate the in vitro amoebicidal effect of nine cosmetic dyes, 1–9, belonging to the xanthene, chlorophyll, aminoanthraquinone, and pyrene group of chromophores, against Acanthamoeba castellanii Neff. Red 22 (2, eosin Y) and red 28 (4) were the most effective photosensitizers to inhibit cell growth, with 2 showing the highest growth inhibition when it is activated by white light. The study of their mechanisms of action reveals that red 22 (2) increases membrane permeability and induces chromatin condensation of parasites, and could be used in combined therapies with currently used drugs.

A collection of N-substituted quinolin-2(1H)-ones were screened against a panel of clinically relevant protozoa (Leishmania, Trypanosoma and Acanthamoeba). Three quinolin-2(1H)-one compounds were identified as selective anti-Acanthamoeba agents. Further assessment revealed that these compounds were active against both trophozoite and cyst forms of A. castellanii Neff, and caused protozoa death via apoptosis. The data presented herein identify N-acyl quinolin-2(1H)-ones as a promising new class of selective anti-Acanthamoeba agents.

Acanthamoeba is a genus of free-living amoebae widely distributed in nature, associated with the development of encephalitis and keratitis. Despite the fact that it is common to find genotype T5 in environmental samples, only a few cases have been associated with clinical cases in humans. The wide distribution of Acanthamoeba, the characteristic of being amphizoic and the severity of the disease motivate researchers to focus on the isolation of these organisms, but also in demonstrating direct and indirect factors that could indicate a possible pathogenic potential. Here, we performed the characterization of the pathogenic potential of an Acanthamoeba T5 isolate collected from a water source in a hospital. Osmo- and thermotolerance, the secretion of proteases and the effect of trophozoites over cell monolayers were analyzed by different methodologies. Additionally, we confirm the secretion of extracellular vesicles (EVs) of this isolate incubated at two different temperatures, and the presence of serine and cysteine proteases in these vesicles. Finally, using atomic force microscopy, we determined some nanomechanical properties of the secreted vesicles and found a higher value of adhesion in the EVs obtained at 37 &deg, C, which could have implications in the parasite&acute, s survival and damaging potential in two different biological environments.

The look for benefits antioxidant agents has attracted much attention from consumers, health and food industries. Certainly, the interactions occurring between different compounds affect the antioxidant efficiency. The current study aims the investigation of the antioxidant combined effect of Rhamnus alaternus fractions using DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) assay. The three most active fractions were subject for binary and ternary combinations using the augmented simplex-centroid design. Two known compounds emodin (1) and kaempferol (2) were identified from the ethanolic extract of the leaves of Rhamnus alaternus. Their structures were elucidated using spectroscopy data. The antioxidant potent of the crude extract and the mixture was compared with the antioxidant activity of the fractions separately. Important synergetic effects were observed in binary mixture between the most active fractions, besides the response optimizer allowed us to optimize the mixture presenting the highest antioxidant activity at 82.54 %. The results from the present work could have interesting applications in both medical formulations and functional food design.

Focused on our interest to develop novel antiparasistic agents, the present study was aimed to evaluate the biological activity of an extract of Laurencia johnstonii collected in Baja California Sur, Mexico, against an Acantamoeba castellanii Neff strain. Bioassay-guided fractionation allowed us to identify the amoebicidal diastereoisomers &alpha, bromocuparane (4) and &alpha, isobromocuparane (5). Furthermore, bromination of the inactive laurinterol (1) and isolaurinterol (2) yielded four halogenated derivatives, (6)&ndash, (9), which improved the activity of the natural sesquiterpenes. Among them, the most active compound was 3&alpha, bromojohnstane (7), a sesquiterpene derivative which possesses a novel carbon skeleton johnstane.

Focused on our interest to develop novel antiparasistic agents, the present study was aimed to evaluate the biological activity of an extract of Laurencia johnstonii collected in Baja California Sur, Mexico, against an Acantamoeba castellanii Neff strain. Bioassay-guided fractionation allowed us to identify the amoebicidal diastereoisomers α-bromocuparane (4) and α-isobromocuparane (5). Furthermore, bromination of the inactive laurinterol (1) and isolaurinterol (2) yielded four halogenated derivatives, (6)–(9), which improved the activity of the natural sesquiterpenes. Among them, the most active compound was 3α-bromojohnstane (7), a sesquiterpene derivative which possesses a novel carbon skeleton johnstane.

Saline groundwater desalination has recently emerged as an alternative source of irrigation water in arid and semiarid regions due to the gradual reduction in the quantity and quality of conventional water resources for agricultural use. In Fuerteventura Island (Spain), an extremely arid territory in the European Union, brackish water desalination is one of the few available water sources for agricultural production. Very little research has been conducted on the microbiological quality of this water mainly used for irrigation of vegetable crops. Free-living amoebae (FLA) are widely distributed protozoa in the environment and have been isolated from many environmental sources such as dust, soil and water. Among the pathogenic genera included in this group, Acanthamoeba spp., Naegleria fowleri and Balamuthia mandrillaris have been reported to be causative agents of lethal encephalitis, disseminated infections and keratitis. Particularly, Naegleria fowleri is a pathogenic FLA species which causes primary amoebic meningoencephalitis (PAM). In the present study, the presence of pathogenic FLA strains on desalinated brackish water samples for irrigation has been evaluated during 7 months. From the analysed samples, only one was positive for Naegleria australiensis. This is the first report of Naegleria spp. in desalinated brackish water for irrigation in Spain.

Free-living amoebae of the genus Acanthamoeba are the etiological agents of cutaneous lesions, granulomatous amoebic encephalitis (GAE) and amoebic keratitis (AK), which are chronic infections with poor prognosis if not diagnosed promptly. Currently, there is no optimal therapeutic scheme to eradicate the pathologies these protozoa cause. In this study we report the morphological and molecular identification of three species of the genus Acanthamoeba, belonging to T4 group; A. polyphaga isolated from the corneal ulcer of a patient sample of AK case; A. castellanii isolated from the contact lens of an AK patient and A. palestinensis obtained from a soil sample. The in vitro activity of chlorhexidine, itraconazole and voriconazole drugs against trophic stage was also evaluated through a colorimetric assay based on the oxidation-reduction of alamar blue. The strains in the study were sensitive to the evaluated drugs; although when determining the 50% inhibitory concentration (IC50) statistically significant differences were observed. A. castellanii showed to be highly sensitive to voriconazole (0.66 ± 0.13 μM) but the least sensitive to chlorhexidine and itraconazole (8.61 ± 1.63 and 20.14 ± 4.93 μM, respectively), A. palestinensis showed the highest sensitivity to itraconazole (0.502 ± 0.11 μM) and A. polyphaga expressed moderate sensitivity to chlorhexidine and itraconazole and lower sensitivity to voriconazole (10.10 ± 2.21 μM). These results showed that species of the genus Acanthamoeba express different sensitivity to the tested drugs, which could explain the problems surrounding the establishment of a treatment of choice in the infections caused by these amoebae. We consider that although chlorhexidine and itraconazole show good activity on these amoebae and have been used in cases of AK in Mexico with acceptable results, voriconazole should be considered as the first therapeutic option of future Acanthamoeba infections that will be diagnosed in our country.

Acanthamoeba keratitis (AK) is a sight-threatening corneal infection. The early symptoms include redness, pain, photophobia and intense tearing. Chronic infection usually progresses to stromal inflammation, ring ulcers, corneal opacification and hypopyon. Here we document an AK case in a high myopic 38-year-old woman from Mexico City, with a history of wearing contact lenses while swimming. Corneal scrapes cultures were positive only for amoebae, consequently a treatment including netilmicin 0.3% and oral itraconazole 100 mg/12 h was prescribed. The infection was resolved after 8 months, leaving a slight leucoma outside the visual axis, with a visual acuity of 20/150. In the laboratory, the amoebic isolate was axenized in PYG medium, with an optimal growth at 30 °C, and was identified morphologically as Acanthamoeba polyphaga according to the taxonomic criteria of Page (1988) and placed in the T4 group by genotyping. The virulence of this strain (40%) was determined by intranasal inoculation of 1 × 106/20 μl trophozoites in BALB/c mice recovering from brain, proving their invasion ability and by the interaction with monolayers of epithelial cells of the established MDCK line of canine kidney origin (1:2 ratio of interaction), at 1, 3, 6, 8 and 24 h; trophozoites migrated to cell junctions inducing few lytic zones. In addition to the biological characterization, in vitro drug sensitivity tests were performed using chlorhexidine, itraconazole, netilmicin and voriconazole. Results revealed that voriconazole was the most effective compound. A. polyphaga remains as one of the most frequently isolated species producing AK. The treatment of AK case using netilmicin and oral itraconazole solved the disease, but the healing process was wide-ranging (8 months). The use of voriconazole and chlorhexidine may be an alternative treatment of future AK cases in Mexico.

Amoebae of the genus Acanthamoeba are ubiquitous protists that have been isolated from many sources such as soils, water and the air. They are responsible for infections including fatal encephalitis and a severe keratitis in humans. To date, there is no satisfactorily effective therapeutic agent against this pathogen and the infections it causes are exacerbated by the existence of a resistant cyst stage produced by this amoeba. As dry eye syndrome is a risk factor for Acanthamoeba keratitis, we aimed to evaluate the anti-Acanthamoeba activity of a variety of proprietary eye drops intended to treat dry eye syndrome. From the nine eye drop formulations tested, “Systane Ultra” was determined to be the most active against all tested Acanthamoeba strains. During our investigations into the mode of action of Systane Ultra, we discovered that it decreases mitochondrial membrane potential and ATP levels, induces chromatin condensation, and increases the permeability of the plasma-membrane.

Free-living amoebae belonging to Acanthamoeba genus are widely distributed protozoans which are able to cause infection in humans and other animals such as keratitis and encephalitis. Acanthamoeba keratitis is a vision-threatening corneal infection with currently no available fully effective treatment. Moreover, the available therapeutic options are insufficient and are very toxic to the eye. Therefore, there is an urgent need for the development of more effective anti-amoebic agents. Nanotechnology approaches have been recently reported to be useful for the elucidation antimicrobial, antiviral, antifungal and antiprotozoal activities and thus, they could be a good approach for the development of anti-Acanthamoeba agents. Therefore, this study was aimed to explore the activity and cytotoxicity of tannic acid-modified silver nanoparticles, pure silver nanoparticles and pure gold nanoparticles against clinical strains of Acanthamoeba spp. The obtained results showed a significant anti-amoebic effect of the tannic acid-modified silver nanoparticles which also presented low cytotoxicity. Moreover, tannic acid-modified silver nanoparticles were well absorbed by the trophozoites and did not induce encystation. On the other hand, pure silver nanoparticles were only slightly active against the trophozoite stage and pure gold nanoparticles did not show any activity. In conclusion and based on the observed results, silver nanoparticle conjugation with tannic acid may be considered as potential agent against Acanthamoeba spp.

Balamuthia mandrillaris is an opportunistic free-living amoebae (FLA) which has been reported as the causative agent of the fatal Balamuthia amoebic encephalitis (BAE). Currently, the transmission dynamics of this pathogen remain poorly understood although the organism has been associated with soils, and thus, soil exposure has been identified as a risk factor for this pathogenic amoeba. Nevertheless, environmental isolation of B. mandrillaris is a rare event and strains of this amoebic species have been isolated from soil and dust sources only in seven previous reports (Iran, Mexico, USA, Peru, Costa Rica, Guinea Bissau, and Jamaica). In Iran, a previous study reported the isolation of B. mandrillaris from dust collected in the city of Tehran and free of known infectious involvement. Therefore, in this work, 55 soil samples collected from public and recreational areas of East Azerbaijan, North-Western Iran, were investigated for the presence of this pathogen. Samples were cultured in 2% non-nutrient agar plates and were monitored for the presence of B. mandrillaris-like trophozoites and/or cysts. Those samples that were positive for these amoebae by microscopic criteria were then confirmed by PCR amplification and DNA sequencing of the mitochondrial 16S ribosomal DNA (rDNA) of B. mandrillaris. The obtained results revealed the presence of this emerging pathogen in 5 of 55 samples included in the study. Homology analysis of the obtained DNA sequences revealed high similarity with previously isolated strains from clinical and environmental sources. To the best of our knowledge, this is the first report on the isolation of B. mandrillaris from soil sources in Iran related to human activity and the second time that this pathogen is isolated from the environment in this country.

Pathogenic strains of Acanthamoeba are causative agents of a sight-threatening infection of the cornea known as Acanthamoeba keratitis, which is often associated with the misuse of contact lenses. However, there is still a question remaining to be answered, which is whether these micro-organisms are present on the ocular surface of healthy individuals. Therefore, the aim of this study was to determine the presence of Acanthamoeba on the ocular surface in healthy patients and also in those with other ocular surface infections. Sterile Schirmer test strips were used to collect samples from a group of patients who attended an ophthalmology consultation at the Hospital del Norte, Icod de los Vinos, Tenerife, Canary Islands. Most of the patients (46 individuals, 79.31 %) presented ocular surface pathologies such as blepharitis or conjunctivitis; the rest did not present any pathology. None of the patients included in the study wore contact lenses. The collected samples were cultured in 2 % non-nutrient agar plates and positive plates were then cultured in axenic conditions for further analyses. Molecular analysis classified all isolated strains as belonging to Acanthamoeba genotype tbl4, and osmotolerance and thermotolerance assays revealed that all strains were potentially pathogenic. Furthermore, all strains were assayed for sensitivity against voriconazole and chlorhexidine. Assays showed that both drugs were active against the tested strains. In conclusion, the Schirmer strip test is proposed as an effective tool for the detection of Acanthamoeba on the ocular surface.

Free-living amoebae are protozoa widely distributed in nature, which can be found in a variety of environments. Four genera are recognized as causal agents of infections in humans and animals: Acanthamoeba, Naegleria, Balamuthia, and Sappinia. In this study, the presence of Acanthamoeba in dental units was determined and the isolates obtained were molecularly characterized; osmotolerance and thermotolerance assays were also performed to evaluate multiplication under these conditions, frequently associated with pathogenicity. The morphological analysis and partial sequencing of the 18S rDNA gene revealed the presence of Acanthamoeba genotype T4 in 14% of the units sampled. Osmotolerance and thermotolerance tests were positive for more than 80% of the isolates. Up to date, this is the first study that reports the detection, identification, and genotyping of Acanthamoeba isolated from dental units in Costa Rica and even in Latin-America. Further assays to determine the potential pathogenicity of these Acanthamoeba isolates are underway.

The 13th International Workshops on Opportunistic Protists (IWOP-13) was held November 13 to 15, 2014 in Seville, Spain. The objectives of the IWOP meetings are to: (1) Serve as a forum for exchange of new information among active researchers concerning the basic biology, molecular genetics, immunology, biochemistry, pathogenesis, drug development, therapy, and epidemiology of these immunodeficiency associated pathogenic eukaryotic microorganisms that are seen in patients with AIDS; and (2) to foster the entry of new and young investigators into these underserved research areas. The IWOP meeting focuses on opportunistic protists; e.g. the free-living amoebae, Pneumocystis, Cryptosporidium, Toxoplasma, the Microsporidia, and kinetoplastid flagellates. This conference represents the major conference which brings together research groups working on these opportunistic pathogens. Progress has been achieved on understanding the biology of these pathogenic organisms, their involvement in disease causation in both immune deficient and immune competent hosts and is providing important insights into these emerging and reemerging pathogens. A continuing concern of the participants is the ongoing loss of scientific expertise and diversity in this research community. This decline is due to the small size of these research communities and an ongoing lack of understanding by the broader scientific community of the challenges and limitations faced by researchers working on these organisms, which makes these research communities very sensitive to declines in research funding.

The in vitro activity of a novel group of compounds, hexaazatrinaphthylene derivatives, against two species of Leishmania is described in this study. These compounds showed a significant dose-dependent inhibition effect on the proliferation of the parasites, with 50% inhibitory concentrations (IC 50 s) ranging from 1.23 to 25.05 μM against the promastigote stage and 0.5 to 0.7 μM against intracellular amastigotes. Also, a cytotoxicity assay was carried out to in order to evaluate the possible toxic effects of these compounds. Moreover, different assays were performed to determine the type of cell death induced after incubation with these compounds. The obtained results highlight the potential use of hexaazatrinaphthylene derivatives against Leishmania species, and further studies should be undertaken to establish them as novel leishmanicidal therapeutic agents.

Members of the genus Acanthamoeba are facultative pathogens of humans, causing a sight-threatening keratitis and a life-threatening encephalitis. In order to treat those infections properly, it is necessary to target the treatment not only to the trophozoite but also to the cyst. Furthermore, it may be advantageous to avoid parasite killing by necrosis, which may induce local inflammation. We must also avoid toxicity of host tissue. Many drugs which target eukaryotes are known to induce programmed cell death (PCD), but this process is poorly characterized in Acanthamoeba . Here, we study the processes of programmed cell death in Acanthamoeba , induced by several drugs, such as statins and voriconazole. We tested atorvastatin, fluvastatin, simvastatin, and voriconazole at the 50% inhibitory concentrations (IC 50 s) and IC 90 s that we have previously established. In order to evaluate this phenomenon, we investigated the DNA fragmentation, one of the main characteristics of PCD, with quantitative and qualitative techniques. Also, the changes related to phosphatidylserine exposure on the external cell membrane and cell permeability were studied. Finally, because caspases are key to PCD pathways, caspase activity was evaluated in Acanthamoeba . All the drugs assayed in this study induced PCD in Acanthamoeba . To the best of our knowledge, this is the first study where PCD induced by drugs is described quantitatively and qualitatively in Acanthamoeba .

Free living amoebae (FLA) are amphizoic protozoa that are ubiquitous in nature. Infection with FLA may result in neurological, ocular and skin infections. Exposure to Acanthamoeba occurs frequently through water contact and knowledge of the presence of the organisms in water sources is important in understanding transmission dynamics. The distribution of Acanthamoeba was studied in recreational and domestic water samples collected from across Jamaica. Morphological assessment and polymerase chain reaction revealed Acanthamoeba spp. isolates in 50.6% (42/83) and 17.3% (14/81) of recreational and domestic water, respectively. Sequencing of the DF3 region of the 18S rDNA resulted in the identification of genotypes T3, T4, T5, T10 and T11 corresponding to Acanthamoeba spp: A. griffini, A. triangularis, A. lenticulata, A. culbertsoni and A. hatchetti. Moreover, T4 was the most frequently isolated genotype in both recreational and domestic water. Thermotolerance and osmotolerance assays indicated that most isolates were potentially pathogenic. This is the first report of T3 and T10 genotypes in the Caribbean and the first report of these Acanthamoeba spp. in Jamaican waters. The study shows that there is potential risk of infection to contact wearers who practise poor lens care. Further, Acanthamoeba should be considered as a cause of neurological infections in Jamaica.

Amoebic keratitis, a sight-threatening corneal infection, mainly occurs in contact lens wearers who wash their eyes with tap water. The present research was conducted to identify the occurrence of potentially pathogenic free-living amoebae (FLA) in tap water sources on Kish Island, a tourist region in Iran. Amoebae were detected using a culture-enriched method and by polymerase chain reaction (PCR)/sequencing of the diagnostic fragment 3 region of the 18S rRNA gene of Acanthamoeba. In the case of other free-living amoebae species, PCR/sequencing analysis of the 18S rDNA was conducted. Results of this study showed the presence of Acanthamoeba belonging to T3, T4, T5, and T11 genotypes in tap water sources. Additionally, Vermamoebae vermiformis was detected in three water samples. This is the first report of the Acanthamoeba genotypes T3, T4, T5, and T11 and V. vermiformis species in tap water sources in a tourist region in Iran.

Helicobacter pylori is considered to be responsible for the most common gastric infections in humans worldwide. In animals, other Helicobacter species are linked to gastritis with and without the presence of ulcers in their respective hosts. Moreover, gastric ulcers have been reported for decades in wild and captive dolphins. Clinical signs include lack of appetite, anorexia, abdominal tenderness, depression, and occasional unresponsiveness. In this study, serum and stool of nine bottlenose dolphins from Loro Parque collection Tenerife, Spain were examined for the presence of Helicobacter spp. The aim of our study was to evaluate the use of two commercially available kits for the detection of H. pylori in humans: a stool antigen immunoassay (Letitest H. pylori CARD) and a Western blot assay (EUROLINE-WB H. pylori) that were adapted to identify specific Helicobacter spp. antibodies in the tested Loro Parque bottlenose dolphin collection. The utility of these diagnostic kits for their application in dolphins is demonstrated, and their use in the future for the diagnosis of Helicobacter spp. in both wild and captive dolphins is proposed in this study.

Leishmaniasis represents a serious threat to the health as one of the most important neglected tropical diseases as designated by the World Health Organization. The disease is endemic in 82 countries, among them Tunisia is an indigenous area for cutaneous Leishmaniasis. In a previous work, two tritepenic acids namely oleanolic and maslinic acids have been isolated from olive leaf extract. In the present paper, the in vitro activity against amastigotes stage of Leishmania (L.) infantum and Leishmania (L.) amazonensis was investigated. Maslinic acid showed the highest activity, against L. amazonensis, with an IC50 of 1.417 ± 0.401 µg/mL and a selectivity index of 9.405. Although, the oleanolic acid exhibit a better activity against L. infantum with an IC50 of 0.999 ± 0.089 µg/mL and selectivity index of 8.111.

The lack of an effective chemotherapy for treatment of protozoan disease urges a wide investigation for active compounds, and plant-derived compounds continue to provide key leads for therapeutic agents. The current study reports the in vitro antiprotozoal evaluation of the Algerian medicinal plant Pulicaria inuloides against Leishmania amazonensis, Trypanosoma cruzi, and Acanthamoeba castellanii str. Neff. All the extracts from the aerial part showed to be present a higher leishmanicidal activity than anti-Acanthamoeba or Trypanosoma. Therefore, bioguided fractionation of the active CHCl3 extract led to the isolation and characterization of the flavonol, quercetagetin-3,5,7,3′-tetramethyl ether (1) as the main component. The structure of compound 1 was established by extensive 1D and 2D NMR spectroscopic analysis (COSY, HSQC, HMBC, and ROESY experiments), chemical transformation (derivatives 2 and 3), and comparison with data in the literature. Compound 1 and derivatives 2 and 3 were further evaluated against the promastigote and amastigote stage of L. amazonensis. Compounds 1–3 exhibited moderate leishmanicidal activity with IC50 values ranging from 0.234 to 0.484 mM and from 0.006 to 0.017 mM for the promastigote and amastigote forms, respectively, as well as low toxicity levels on macrophages (CC50 ranging from 0.365 to 0.664 mM). This study represents the first report of the antiprotozoal evaluation of Pulicaria inuloides, and the results highlight this species as a promising source of leishmanicidal agents.

Acanthamoeba is a free-living amoeba genus that causes several diseases namely, amoebic keratitis which is a painful sight threatening eyes disease. Its treatment is difficult and the exploration for new drugs is very important. The main objective of the present study was to evaluate the chemical composition of the Essential Oils (EO) obtained from leaves and flowers and aerial parts of Ammoides pusilla by an alternative method “Hydrodistillation’’. Identification and quantification were realized by Gas Chromatography-Mass Spectrometry (GC-MS) and Gas Chromatography with Flame Ionization Detection (GC-FID). The main components of leaves and flowers and aerials parts were thymol (39.6% and 33.05%), γ-terpinene (28.97% and 28.19%), p-cymene (13.69% and 15.31%) and thymol methyl ether (7.33% and 8.91%), respectively. The antiparasitic activity of the EO was evaluated against Acanthamoeba castellanii Neff by the Alamar Blue® assay. Results showed that Ammoides pusilla amoebicidal activity from leaves and flowers essential oil (IC50 = 65.32 ± 5.43 μg/mL) was more important than those of aerial parts EO (IC50 = 97.18 ± 1.43 μg/ml).

In order to promote a local Tunisian product, this study was designed to examine, for the first time, the anti-Acanthamoeba activity (Acanthamoeba castellanii Neff) of the essential oils of Tunisian Citrus sinensis peels (Maltese half-blood) and the effect of viroid plant infection on this activity. To do so, three samples of peels’ essential oils were studied: from a healthy plant (Control), a plant inoculated with Citrus exocortis viroid (CEVd) and one inoculated with hot stunt cachexia viroid (HSVd). The samples were extracted by hydrodistillation from dried peels and characterized by GC–MS. Limonene was the major component with a percentage ranging from 90.76 to 93.34% for (CEVd) sample and (Control), respectively. Anti-Acanthamoeba activity of the tested oils was determined by the Alamar Blue® assay. Primary results showed a strong potential anti-Acanthamoeba activity with an IC50 ranging from 36.6 to 54.58 μg/ml for (HSVd) and (CEVd) samples, respectively. In terms of the effect of viroid infection, a strong positive correlation was observed between different chemical classes and anti-Acanthamoeba activity.

The present study aimed to evaluate the activity of methanolic extract of Rubus ulmifolius Schott against the Acanthamoeba castellani Neff Strain as well as its antioxidant and antimicrobial effects. The tested extract has a good amoebicidal activity with low IC 50 (61.785 ± 1.322 μg/ml) and also has significant activity against both Gram-positive ( S. aureus, S. agalactiae ) and Gram-negative bacteria ( E. coli, S. typhimurium ) and against C. albicans . The inhibition zones diameters (IZD) and minimal inhibitory concentration (MIC) values were in the range of 22.5–50 mm and 02.29–4.76 mg ml −1 , respectively. In the other hand, the in vitro ROS scavenging activity was evaluated, the tested extract exhibited a good effect on the ·OH radical (89.99% at a concentration of 100 μg/ml) when compared to the ascorbic acid (68.81%). Moreover, the inhibition percentage of superoxide generation by R. ulmifolius extract at 100 μg/ml was greater than ascorbic acid (79.55; 64.79%, respectively). Also, the tested extract showed a high percentage of H 2 O 2 scavenging activity (99.95% at 100 μg/ml). Our findings suggest that R. ulmifolius could be a potential source of natural antioxidant in preventing many diseases associated with oxidative stress, amoebic and bacterial infections.

Acanthamoeba species are free-living amoebae widely distributed in the environment and which cause serious human infections. The treatment of Acanthamoeba infections is always very difficult and not constantly effective. More efficient drugs against Acanthamoeba must be developed and medicinal plants can be useful in this case. Our research focused on the examination of the anti-Acanthamoeba activity of the essential oil and the ethanolic-aqueous extract from Thymus capitatus L. The essential oil showed best activity with an IC50 of 2.73 μg/ml. The conducted Bio-guided fractionation of thyme extract result to the identification of two active compounds against the trophozoite stage of Acanthamoeba: thymol and 2,3-dihydroxy-p-cymene. The results have clearly shown that the investigated products may be successfully used against Acanthamoeba infections. These molecules that are found in plants may be an alternative for the development of new drugs.

Granulomatous amoebic encephalitis (GAE) is a chronic, difficult to resolve infection caused by amphizoic amoebae of the genus Acanthamoeba , which in most cases occurs in immunosuppressed persons or with chronic diseases such as diabetes. In this study, we describe the early events of A. culbertsoni infection of GAE in diabetic mice model. Diabetes was induced in male BALB/c mice, with a dose of streptozotocin (130 mg/kg). Healthy and diabetic mice were inoculated via intranasal with 1 × 10 6 trophozoites of A. culbertsoni . Then were sacrificed and fixed by perfusion at 24, 48, 72 and 96 h post-inoculation, the brains and nasopharyngeal meatus were processed to immunohistochemical analysis. Invasion of trophozoites in diabetic mice was significantly greater with respect to inoculated healthy mice. Trophozoites and scarce cysts were immunolocalized in respiratory epithelial adjacent bone tissue, olfactory nerve packets, Schwann cells and the epineurium base since early 24 h post-inoculation. After 48 h, trophozoites were observed in the respiratory epithelium, white matter of the brain, subcortical central cortex and nasopharyngeal associated lymphoid tissue (NALT). At 72 h, cysts and trophozoites were immunolocalized in the olfactory bulb with the presence of a low inflammatory infiltrate characterized by polymorphonuclear cells. Scarce amoebae were observed in the granular layer of the cerebellum without evidence of inflammation or tissue damage. No amoebas were observed at 96 h after inoculation, suggesting penetration to other tissues at this time. In line with this, no inflammatory infiltrate was observed in the surrounding tissues where the amoebae were immunolocalized, which could contribute to the rapid spread of infection, particularly in diabetic mice. All data suggest that trophozoites invade the tissues by separating the superficial cells, penetrating between the junctions without causing cytolytic effect in the adjacent cells and subsequently reaching the CNS, importantly, diabetes increases the susceptibility to amoebae infection, which could favor the GAE development.