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Balamuthia mandrillaris is an emerging cause of encephalitis in humans. The transmission dynamics are poorly understood due to the high fatality rate and the sporadic nature of cases. Seventy-two soil samples were collected from beaches and the banks of lagoons, rivers, ponds, mineral springs and streams from across Jamaica and assayed for the presence of B. mandrillaris. Seventy-nine sites were sampled and the mitochondrial 16S rDNA gene of B. mandrillaris was amplified and sequenced to confirm the presence of the amoeba. One isolate of B. mandrillaris was recovered from soil from mineral spring which hosts an informal therapeutic mud bath business. Although B. mandrillaris is less frequently isolated from soil than other free-living amoebae, rubbing mud containing the organism onto the skin increases the likelihood of exposure and infection. This first report on the isolation of B. mandrillaris in the Caribbean and its presence in soil where human contact is likely warrants further investigation using serological methods to elucidate exposure patterns. [ABSTRACT FROM AUTHOR]

Free-living amoebae (FLA) are widely distributed in the environment. Among these, Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris, and Vermamoeba vermiformis have been reported as human pathogens with health effects ranging from lethal encephalitis to different epithelial disorders. Despite this, FLA still present many diagnostic challenges. The aim of this study was to develop a rapid and efficient multiplex real-time quantitative polymerase chain reaction (qPCR) to simultaneously detect Acanthamoeba spp., N. fowleri, B. mandrillaris, and V. vermiformis in different water sources. For the validation of the qPCR assay, 38 samples (19 tap water and 19 stagnant water sources) were analyzed. The qPCR assay accurately identified the four types of FLA with no cross-reactivity. Considering water samples with results subsequently confirmed by conventional PCR, the multiplex qPCR assay detected 18/38 (47.4%) positive samples (Acanthamoeba spp. in 44.7% and V. vermiformis in 31.6%) and growth in nonnutritive agar (NNA) cultures identified 7/38 (18.4%) positive samples. Of the tap water samples analyzed, 26.3% of samples positive for FLA were detected by growth in NNA culture whereas 31.6% were identified by qPCR. In addition, FLA were detected in 2/19 stagnant water samples (10.5%) by growth in NNA culture and in 12/19 stagnant water samples (63.2%) by qPCR. Neither N. fowleri nor B. mandrillaris was detected in the water samples analyzed. In conclusion, the qPCR developed showed its potential as a rapid tool for detection of Acanthamoeba spp., N. fowleri, B. mandrillaris, and V. vermiformis. Moreover, FLA species were detected in half of the water sources evaluated, suggesting the importance of the surveillance of these potential infectious agents.

The Tajogaite Volcano erupted on the western slope of the Cumbre Vieja mountain range on La Palma Island in the Canary Islands, Spain, in 2021. As one of the multiple consequences of this eruption, a layer of tephra was deposited, to a variable extent, over a large part of the island. Tephra deposits affect all aspects of vegetation recovery, the water cycle, and the long-term availability of volcanic nutrients. Protozoa, including free-living amoeba (FLA), are known to be among the first microorganisms capable of colonizing harsh environments. In the present study, the presence of FLA has been evaluated in the Tajogaite Volcano deposits. Samples of the tephra were collected and incubated at 26 °C on 2% non-nutrient agar plates with a layer of heat-killed E. coli . Morphological features, as well as the DF3 region sequence of the 18S rDNA, confirmed the presence of a T4 genotype strain of Acanthamoeba. Thermotolerance and osmotolerance assays were used to evaluate the strain's pathogenic potential. This strain was considered thermotolerant but poorly osmotolerant. To the best of our knowledge, this is the first report of Acanthamoeba being isolated from a recently erupted volcano.

Acanthamoeba spp. can cause a sight threatening disease. At present, the current treatments used to treat Acanthamoeba spp. Infections, such as biguanide-based antimicrobials, remain inefficacious, with the appearance of resistant forms and high cytotoxicity to host cells. In this study, an initial screening was conducted against Acanthamoeba castellanii Neff and murine macrophages J774A.1 using alamarBlue™. Among the 160 compounds included in the cited box, 90% exhibited an inhibition of the parasite above 80%, while only 18.75% of the compounds inhibited the parasite with a lethality towards murine macrophage lower than 20%. Based on the amoebicidal activity, the cytotoxicity assay, and availability, Terconazole was chosen for the elucidation of the action mode in two clinical strains, Acanthamoeba culbertsoni and Acanthamoeba castellanii L10. A fluorescence image-based system and proteomic techniques were used to investigate the effect of the present azole on the cytoskeleton network and various programmed cell death features, including chromatin condensation and mitochondria dysfunction. Taking all the results together, we can suggest that Terconazole can induce programmed cell death (PCD) via the inhibition of sterol biosynthesis inhibition.

Organic and synthetic chemistry plays a crucial role in drug discovery fields. Moreover, chemical modifications of available molecules to enhance their efficacy, selectivity and safety have been considered as an attractive approach for the development of new bioactive agents. Indoles, a versatile group of natural heterocyclic compounds, have been widely used in pharmaceutical industry due to their broad spectrum of activities including antimicrobial, antitumoral and anti-inflammatory among others. Herein, we report the amoebicidal activity of different indole analogs on Acanthamoeba castellanii Neff. Among the 40 tested derivatives, eight molecules were able to inhibit this protistan parasite. The structure-activity relationship (SAR) analysis of their anti- Acanthamoeba activity would suggest that a carboxylation of C-3 position and the incorporation of halogen as chlorine/fluorine would enhance their biological profile, presumably by increasing their lipophilicity and therefore their ability to cross the cell membrane. Fluorescence image base system was used to investigate the effect of indole 6o c-6 on the cytoskeleton network and various programmed cell death features. We were able to highlight that the methyl 6-chloro-1H-indole-3-carboxylate could induce program cell death by the mitochondrial dysfunction., Competing Interests: The authors declare no conflict of interest.

Acanthamoeba is a ubiquitous genus of amoebae that can act as opportunistic parasites in both humans and animals, causing a variety of ocular, nervous and dermal pathologies. Despite advances in Acanthamoeba therapy, the management of patients with Acanthamoeba infections remains a challenge for health services. Therefore, there is a need to search for new active substances against Acanthamoebae. In the present study, we evaluated the amoebicidal activity of nitroxoline against the trophozoite and cyst stages of six different strains of Acanthamoeba . The strain A. griffini showed the lowest IC 50 value in the trophozoite stage (0.69 ± 0.01 µM), while the strain A. castellanii L-10 showed the lowest IC 50 value in the cyst stage (0.11 ± 0.03 µM). In addition, nitroxoline induced in treated trophozoites of A. culbertsoni features compatibles with apoptosis and autophagy pathways, including chromatin condensation, mitochondrial malfunction, oxidative stress, changes in cell permeability and the formation of autophagic vacuoles. Furthermore, proteomic analysis of the effect of nitroxoline on trophozoites revealed that this antibiotic induced the overexpression and the downregulation of proteins involved in the apoptotic process and in metabolic and biosynthesis pathways.

Few studies have been conducted in the cooling systems of power plants; they have focused on Naegleria fowleri , leaving a gap in the knowledge of other pathogenic free-living amoebae in this environment. The objective of this study was to determine the occurrence of saline-tolerant pathogenic Acanthamoeba in a geothermal power plant. The identification of isolated amoebae at genus level was carried out, observing their morphological characteristics; the determination of genotype and species of Acanthamoeba was performed via molecular biology (PCR). Water temperature ranged from 18 to 43 °C and conductivity from 4.0 × 10 4 to 8.7 × 10 4 μS/cm; this last value was greater than the seawater value. Only five amoeba genera were found. Acanthamoeba was in all the sampling sites, showing high saline tolerance. The high temperature, but mainly high conductivity, were the environmental conditions that determined the presence of pathogenic free-living amoebae in the hot water. All the strains of Acanthamoeba culbertsoni killed the mice, having a mortality of 40 to 100%. Acanthamoeba genotypes T10 and T5 were identified, T10 is rarely isolated from the environment, while T5 is more frequent. This is the first time that genotypes T5 and T10 have been reported in the environment in Mexico.

Despite the Naegleria genus being isolated from different natural environments such as water, soil, and air, not all Naegleria species are capable of causing infections in humans, and they are capable of completing their life cycle in environmental niches. However, the presence of this genus may suggest the existence of one of the highly pathogenic free-living amoeba (FLA) species: Naegleria fowleri or the brain-eating amoeba. This facultative parasitic protozoon represents a risk to public health, mainly related to domestic and agricultural waters. In this research, our main objective was to determine the existence of pathogenic protozoa in the Santa Cruz wastewater treatment plant, Santiago Island. Using 5 L of water we confirmed the presence of potentially pathogenic Naegleria australiensis, being the first report on Naegleria species in Cape Verde. This fact demonstrates the low efficiency in the treatment of wastewater and, consequently, a potential threat to public health. Nevertheless, more studies will be needed for the prevention and control of possible infections in this Macaronesian country.

Free Living Amoeba (FLA) infections caused by Acanthamoeba genus include chronic nervous system diseases such as Granulomatous Amoebic Encephalitis (GAE), or a severe eye infection known as Acanthamoeba keratitis (AK). Current studies focused on therapy against these diseases are aiming to find novel compounds with amoebicidal activity and low toxicity to human tissues. Brown algae, such as Gongolaria abies-marina (previously known as Cystoseira abies-marina, S.G. Gmelin), presents bioactive molecules of interest, including some with antiprotozoal activity. In this study, six meroterpenoids were isolated and purified from the species Gongolaria abies-marina. Gongolarones A (1), B (2) and C (3) were identified as new compounds. Additionally, cystomexicone B (4), 1'-methoxyamentadione (5) and 6Z-1'-methoxyamentadione (6) were isolated. All compounds exhibited amoebicidal activity against Acanthamoeba castellanii Neff, A. polyphaga and A. griffini strains. Gongolarones A (1) and C (3) showed the lowest IC 50 values against the two stages of these amoebae (trophozoite and cyst). Structure-activity relationship revealed that the cyclization by ether formation from C-12 to C-15 of 1, and the isomerization Δ 2 t to Δ 3 t of 3, increased the antiamoeboid activity of both compounds. Furthermore, gongolarones A (1) and C (3) triggered chromatin condensation, mitochondrial malfunction, oxidative stress, and disorganization of the tubulin-actin cytoskeleton in treated trophozoites. Moreover, transmission electron microscopy (TEM) images analysis revealed that compounds 1 and 3 induced autophagy process and inhibited the encystation process. All those results suggest that both compounds could induce programmed cell death (PCD) in Acanthamoeba., Competing Interests: Conflict of interest The authors declare that there is not conflict of interest with the submission., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Naegleria . fowleri, a protozoa belonging to the free-living amoeba group, is the causative agent of a central nervous system affecting disease that is fatal in more than the 95% of the reported cases. This parasite can be found in warm water bodies such as lakes, rivers or inadequately disinfected swimming pools. On the other hand, chlorination and UV light treatment are two of the most extensively used disinfection methods in recreational water facilities. In this study the effect of chlorination and UV light on N. fowleri trophozoites was studied in a close water circuit with the aim to assess the efficacy of this disinfection methods in large pools. The obtained results showed that the chlorination was able to decrease the number of viable cells despite the elimination was not totally achieved. Nonetheless, the combination of the UV light with the chlorination allowed the complete removal of the N. fowleri trophozoites from the water in experimental testing conditions., Competing Interests: The authors declare no conflict of interest., (© 2022 The Author(s).)

Statins are effective sterol lowering agents with high amoebicidal activity. Nevertheless, due to their poor aqueous solubility, they remain underused especially in eye drop formulation. The aim of the present study is to develop Pitavastatin loaded nanoparticles suitable for ophthalmic administration and designed for the management of Acanthamoeba Keratitis. These nanocarriers are aimed to solve both the ophthalmic route-associated problems and the limited aqueous drug solubility issues of Pitavastatin. Nanoparticles were obtained by a nanoprecipitation-solvent displacement method and their amoebicidal activity was evaluated against four strains of Acanthamoeba: A. castellanii Neff, A. polyphaga, A. griffini and A. quina. In Acanthamoeba polyphaga, the effect of the present nanoparticles was investigated with respect to the microtubule distribution and several programmed cell death features. Nanoparticles were able to eliminate all the tested strains and Acanthamoeba polyphaga was determined to be the most resistance strain. Nanoparticles induced chromatin condensation, autophagic vacuoles and mitochondria dysfunction., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: Acanthamoeba keratitis is a severe corneal infection caused by a ubiquitous opportunistic protozoan pathogen known as acanthamoeba. For the last decade, the approach to treating this infection typically includes the use of polyhexamethylene biguanide (0.02%) and/or chlorhexidine (Chx) (0.02%). Although chlorhexidine is reportedly effective, its mode of action towards this type of cell is not clear. The aim of this work was to study the effect of chlorhexidine on the oxidative status of Acanthamoeba polyphaga., Methods: The effect of chlorhexidine (Chx) on the oxidative state of Acanthamoeba polyphaga was studied using different antiradical methods including ABTS, DPPH and FRAP and measuring the activity of a couple of antioxidant enzyme namely SOD, NADH-FRD and SDH., Results: The chlorhexidine was able to induce oxidative imbalance in cells by over expression of reactive oxygen species and/or inhibiting the antioxidant enzymes. In addition to enhancing the antiradical activity in response to oxidative stress, the present drug was able to reduce the activity of two antioxidant enzymes, superoxide dismutase (SOD) and reduced flavin adenine dinucleotide-fumarate reductase (NADH-FRD), to 30% and 40%, respectively., Conclusions: We could observe an increase of the antiradical capacity of cell's lysate supernatant, to cope with the overproduction of ROS. The imbalance state The inhibition of both SOD and NADH-FRD activities could have a major role in cell oxidative imbalance., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Free-living amoebae (FLA) are protozoa which have been reported in different countries worldwide from diverse sources (water, soil, dust, air), contributing to the environmental microbiological contamination. Most of the FLA species present a life cycle with two different phases: an active vegetative and physiologically form named trophozoite, and an extremely resistant phase called cyst. Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris, Sapinia pedata, Vahlkampfia spp., Paravahlkampfia spp. and Vermamoeba vermiformis have been reported not only as causal agents of several opportunistic diseases including fatal encephalitis or epithelial disorders, but also as capable to favour the intracellular survival of common pathogenic bacteria, which could avoid the typical water disinfection systems, non-effective against FLAs cysts. Even though Santiago Island possesses high levels of humidity compared to the rest of the archipelago of Cape Verde, the water resources are scarce. Therefore, it is important to carry out proper microbiological quality controls, which currently do not contemplate the FLA presence in most of the countries. In the present work, we have reported the presence of Acanthamoeba spp. (69.2%); Vannella spp. (15.4%); Vermamoeba vermiformis (7.7%) and the recently discovered Stenamoeba dejonckheerei (7.7%) in different water sources of Santiago Island., (© 2022. The Author(s).)

Free-living amoeba (FLA) group includes the potentially pathogenic genera Acanthamoeba, Naegleria, Balamuthia, Sappinia, and Vermamoeba, causative agents of human infections (encephalitis, keratitis, and disseminated diseases). In Brazil, the first report on pathogenic FLA was published in the 70s and showed meningoencephalitis caused by Naegleria spp. FLA studies are emerging, but no literature review is available to investigate this trend in Brazil critically. Thus, the present work aims to integrate and discuss these data. Scopus, PubMed, and Web of Science were searched, retrieving studies from 1974 to 2020. The screening process resulted in 178 papers, which were clustered into core and auxiliary classes and sorted into five categories: wet-bench studies, dry-bench studies, clinical reports, environmental identifications, and literature reviews. The papers dating from the last ten years account for 75% (134/178) of the total publications, indicating the FLA topic has gained Brazilian interest. Moreover, 81% (144/178) address Acanthamoeba-related matter, revealing this genus as the most prevalent in all categories. Brazil's Southeast, South, and Midwest geographic regions accounted for 96% (171/178) of the publications studied in the present work. To the best of our knowledge, this review is the pioneer in summarising the FLA research history in Brazil.

The genus Acanthamoeba is characterized by being a group of ubiquitous and free-living amoebae that inhabit a variety of environments. Generally, human infections by this parasite are associated with Acanthamoeba keratitis, especially in contact lens wearers, and with chronic but fatal granulomatous amoebic meningoencephalitis. Current treatments used for eradication of amoeba from infection sites represent a challenge for pharmacotherapy, due to the lack of effective treatment and the amoebae highly resistant to anti-amoebic drugs. In this study, we describe the results of the assessment of the IC 50 of 10 isobenzofuran-1(3H)-one derivatives (QOET) against four Acanthamoeba strains. The compounds QOET-3 and QOET-9 were the selected derivatives with the lowest IC 50 in A. castellanii Neff trophozoites (73.71 ± 0.25 and 69.99 ± 15.32 µM, respectively). Interestingly, analysis of the compound effects on the cell apoptosis-like features showed that both active molecules triggered programmed cell death (PCD) in A. castellanii Neff. The results obtained in this study highlights that isobenzofuranone derivatives could represent an interesting source for developing novel antiamoebic drugs., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Acanthamoeba is a ubiquitous opportunistic protozoan pathogen that is known to cause blinding keratitis and rare, but usually fatal, granulomatous encephalitis. The difficulty in treating infections and the toxicity issues of the current treatments emphasize the need to use alternative agents with amoebicidal activity. The aim of this study was to evaluate the in vitro antiamoebic activity of three third-generation statins-cerivastatin, pitavastatin and rosuvastatin-against both cysts and trophozoites of the following four strains of Acanthamoeba: A. castellanii Neff, A. polyphaga, A. griffini and A. quina . Furthermore, programmed cell death (PCD) induction traits were evaluated by measuring chromatin condensation, damages at the mitochondrial level, production of reactive oxygen species (ROS) and the distribution of actin cytoskeleton fibers. Acanthamoeba castellanii Neff was the strain most sensitive to all the statins, where cerivastatin showed the lowest amoebicidal activity for both trophozoite and cyst forms (0.114 ± 0.050 and 0.704 ± 0.129 µM, respectively). All the statins were able to cause DNA condensation, collapse in the mitochondrial membrane potential and a reduction in ATP level production, and disorganization of the total actin fibers in the cytoskeleton of all the evaluated Acanthamoeba strains. Our results showed that the tested statins were able to induce PCD compatible events in the treated amoebae, including chromatin condensation, collapse in the mitochondrial potential and ATP levels, cytoskeleton disassembly and ROS generation.

Free-living amoebae of Acanthamoeba spp. are causative agents of human infections such as granulomatous amoebic encephalitis (GAE) and Acanthamoeba keratitis (AK). The exploration of innovative chemical entities from natural sources that induce intrinsic apoptotic pathway or a Programmed Cell Death (PCD) in Acanthamoeba protozoa is essential to develop new therapeutic strategies. In this work, the antiamoeboid activity of squamins C-F (1-4), four cyclooctapeptides isolated from Annona globiflora was tested in vitro against Acanthamoeba castellanii Neff, A. polyphaga, A. quina, and A. griffini, and a structure-activity relationship was also established. The most sensitive strain against all tested cyclooctapeptides was A. castellanii Neff being the R conformers of the S-oxo-methionine residue, squamins D (2) and F (4), the most active against the trophozoite stage. It is remarkable that all four peptides showed no cytotoxic effects against murine macrophages cell line J774A.1. The analysis of the mode of action of squamins C-F against A. castellanii indicate that these cyclopeptides induced the mechanisms of programmed cell death (PCD). All peptides trigger mitochondrial damages, significant inhibition of ATP production compared to the negative control, chromatin condensation and slight damages in membrane that affects its permeability despite it conserves integrity at the IC 90 for 24 h. An increase in reactive oxygen species (ROS) was observed in all cases., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The protozoan parasite Leishmania causes a spectrum of diseases and there are over 1 million infections each year. Current treatments are toxic, expensive, and difficult to administer, and resistance to them is emerging. In this study, we screened the antileishmanial activity of the Pathogen Box compounds from the Medicine for Malaria Venture against Leishmania amazonensis , and compared their structures and cytotoxicity. The compounds MMV676388 (3), MMV690103 (5), MMV022029 (7), MMV022478 (9) and MMV021013 (10) exerted a significant dose-dependent inhibition effect on the proliferation of L. amazonensis promastigotes and intracellular amastigotes. Moreover, studies on the mechanism of cell death showed that compounds 3 and 5 induced an apoptotic process while the compounds 7, 9 and 10 seem to induce an autophagic mechanism. The present findings underline the potential of these five molecules as novel therapeutic leishmanicidal agents.

Primary amoebic encephalitis (PAM) caused by the opportunistic pathogen Naegleria fowleri is characterized as a rapid and lethal infection of the brain which ends in the death of the patient in more than 90% of the reported cases. This amoeba thrives in warm water bodies and causes infection after individuals perform risky activities such as splashing or diving, mostly in non-treated water bodies such as lakes and ponds. Moreover, the infection progresses very fast and no fully effective molecules have currently been found to treat PAM. In this study, naphthyridines fused with chromenes or chromenones previously synthetized by the group were tested in vitro against the trophozoite stage of two strains of N. fowleri . In addition, the most active molecule was evaluated in order to check the induction of programmed cell death (PCD) in the treated amoebae. Compound 3 showed good anti- Naegleria activity (61.45 ± 5.27 and 76.61 ± 10.84 µM, respectively) against the two different strains (ATCC ® 30808 and ATCC ® 30215) and a good selectivity compared to the cytotoxicity values (>300 µM). In addition, it was able to induce PCD, causing DNA condensation, damage at the cellular membrane, reduction in mitochondrial membrane potential and ATP levels, and ROS generation. Hence, naphthyridines fused with chromenes or chromenones could be potential therapeutic agents against PAM in the near future.

Efficacious treatments against Acanthamoeba Keratitis (AK) is challenging, often ineffective and linked to the intragenotype variation in the drug efficacy. Increased oxygen can facilitate host response and can inhibit some organisms. Herein, we report the effect of increased oxygen concentrations on Acanthamoeba spp. growth and its effect on ROS (reactive oxygen species) production. The exposition to pure oxygen could reduce cell growth by at least 60% for Acanthamoeba castellanii Neff, Acanthamoeba polyphaga, and Acanthamoeba griffini. The increase in ROS production confirming that oxygen cell's growth inhibition was due to oxidative stress. Further studies are needed to determine oxygen saturation level, time of oxygen exposition, and number of sessions needed to eliminate the parasite., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Free-Living Amoebae (FLA) are widely distributed protozoa, which contain some groups considered as pathogenic microorganisms. These members are able to produce several opportunistic diseases including epithelial disorders, such as keratitis and fatal encephalitis. Even though they have been reported in numerous sources, such as soils, dust and water, there is no legislation related to the presence of these protozoa in soil-related environments worldwide. Therefore, there are no established prevention or disinfection protocols to advise the population regarding FLA infections or eliminate these microorganisms from human-related environments to date. Acanthamoeba spp. are the most common FLA isolated in soil samples, which is also the most common genera found in clinical cases. Thus, the aim of the present study was to evaluate the presence of potentially pathogenic FLA in human-related soil samples of Santiago Island, Cabo Verde. A total of 26 soil samples were seeded in non-nutrient agar plates (2%), incubated at 26 °C, and monitored daily to evaluate the presence of FLA. DNA was extracted from those plates on which there was suspected FLA growth, and PCR amplification of the 18S rRNA gene was carried out. A total of 17 from the 26 analysed samples were positive for FLA, where Acanthamoeba is the most abundant isolated genus (14/17; 82.4%), with the T4 genotype being the most common (13/14; 92.9%), followed by the T5 genotype, A. lenticulata (1/14; 7.1%). Moreover, Vermamoeba vermiformis , Stenamoeba dejonckheerei and Vannella pentlandi were isolated in three other samples. To the best of our knowledge, this is the first report of FLA presence in Cape Verde and the first report of V. vermiformis in beach sand worldwide.

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 (IC 50 values of 5.76 and 5.68 μM, respectively) against L. amazonensis to miltefosine (IC 50 value of 6.48 μM), the reference drug, and low toxicity on macrophage cell lines J774. Moreover, compound 1 exhibited moderate activity (IC 50 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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The validation of anti-Acanthamoeba activity of commercial eye drops has gained a great interest recently and a growing number of commercials eye drop were evaluated for their aptitude to inhibit Acanthamoeba as a second pharmacological effect. In the present study, three different eye drops, commercializing in Spain, including TobraDex, Cusimolol and Colircusi antiedema have been tested in vitro against trophozoites and cysts stage of the facultative pathogen Acanthamoeba. The alamarBlue™ method was used to evaluate both trophocidal and cysticidal properties. The most active eye drops were tested for their impact on some programmed cell death features. We found out that the cells inhibition was strain and eye drop dependent, and 5% eye drop was able to eliminate both trophozoite and cyst stage of Acanthamoeba spp. A treatment of 24 h with 5% of TobraDex or Cusimolol was able to show DNA condensation, collapse in the mitochondrial membrane potential and reduction of the ATP level production in Acanthamoeba. We could assume that the present eye drops could induce programed cell death like process in Acanthamoeba via mitochondrial pathway., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Opportunistic parasitic protozoa of genus Acanthamoeba are responsible to cause severe infections in humans such as Acanthamoeba Keratitis or Amoebic Granulomatous Encephalitis. Current treatments are usually toxic and inefficient and there is a need to access new therapeutic agents. The antiamoebic effects of nephthediol (1) and fourteen germacranolide and eudesmanolide sesquiterpene lactones (2-5, 7-12) isolated from the indigenous zoanthid Palythoa aff. clavata collected at the coast of Lanzarote, Canary Islands were studied against Acanthamoeba castellanii Neff, and the clinical strains A. polyphaga and A. griffini. 4-epi-arbusculin A (11) presented the lowest IC 50 value (26,47 ± 1,69 µM) against A. castellanii Neff and low cytotoxicity against murine macrophages, followed by isobadgerin (2), which also showed to be active against A. castellanii Neff cysts. The studies on the mode of action of compounds 2 and 11 revealed these sesquiterpene lactones induce mechanisms of PDC on A. castellanii Neff., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Pathogenic and opportunistic free-living amoebae such as Acanthamoeba spp. can cause keratitis ( Acanthamoeba keratitis [AK]), which may ultimately lead to permanent visual impairment or blindness. Acanthamoeba can also cause rare but usually fatal granulomatous amoebic encephalitis (GAE). Current therapeutic options for AK require a lengthy treatment with nonspecific drugs that are often associated with adverse effects. Recent developments in the field led us to target cAMP pathways, specifically phosphodiesterase. Guided by computational tools, we targeted the Acanthamoeba phosphodiesterase RegA. Computational studies led to the construction and validation of a homology model followed by a virtual screening protocol guided by induced-fit docking and chemical scaffold analysis using our medicinal and biological chemistry (MBC) chemical library. Subsequently, 18 virtual screening hits were prioritized for further testing in vitro against Acanthamoeba castellanii , identifying amoebicidal hits containing piperidine and urea imidazole cores. Promising activities were confirmed in the resistant cyst form of the amoeba and in additional clinical Acanthamoeba strains, increasing their therapeutic potential. Mechanism-of-action studies revealed that these compounds produce apoptosis through reactive oxygen species (ROS)-mediated mitochondrial damage. These chemical families show promise for further optimization to produce effective antiacanthamoebal drugs., (Copyright © 2021 American Society for Microbiology.)

Primary amoebic meningoencephalitis (PAM), caused by the pathogenic free-living amoeba Naegleria fowleri , is a rare but fatal disease. Nowadays, no fully effective therapy is available to erradicate or prevent this disease. Natural products could constitute a promising source of useful bioactive compounds in drug discovery. The present study is a characterization of main active compounds from the ethanolic extract of Inula viscosa (Asteraceae) leaves against N. fowleri trophozoites. Four compounds ( 1 - 4 ) were successfully identified by spectroscopic techniques, but only inuloxin A displayed a potential antiamoebic activity with an IC 50 of 21.27 μM. The specificity of this compound toward the studied strain leads us to analyze the insight into its mechanism of action by performing in vitro assays of programmed cell death markers and to discuss the structure-activity relationship (SAR). The obtained results demonstrated that inuloxin A interferes with various processes leading to membrane damage, mitochondria alteration, chromatin condensation, and ROS accumulation, which highlight features specific to apoptosis. The current findings could be a promising step for developing new effective drugs against PAM.

Recently, indoor swimming pool activities have increased to promote health-enhancing physical activities, which require establishing suitable protocols for disinfection and water quality control. Normally, the assessment of the microbial quality of the water in the pools only considers the presence of different bacteria. However, other less frequent but more resistant pathogens, such as free-living amoebas (FLA), are not contemplated in both existing recommendation and research activities. FLA represent a relevant human health risk, not only due to their pathogenicity but also due to the ability to act as vehicles of other pathogens, such as bacteria. Therefore, this work aimed to study the physicochemical characteristics and the occurrence of potentially pathogenic FLA and bacteria in water samples from 20 public indoor swimming facilities in Northern Portugal. Our results showed that some swimming pools presented levels of pH, free chlorine, and conductivity out of the recommended limits. Pathogenic FLA species were detected in two of the facilities under study, where we also report the presence of both, FLA and pathogenic bacteria. Our findings evidence the need to assess the occurrence of FLA and their existence in the same environmental niche as pathogenic bacteria in swimming pool facilities worldwide and to establish recommendations to safeguard the health of the users., (© 2021 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

The in vitro activity against Leishmania spp. of five novel designed compounds, phenalenone derivatives, is described in this study. Previous works have shown that some phenalenones present leishmanicidal activity, some of which could induce programmed cell death events in L. amazonensis parasites. In this research, we focused on the determination of the programmed cell death evidence by detecting the characteristic features of the apoptosis-like process, such as phosphatidylserine exposure and mitochondrial membrane potential, among others. The results showed that the new derivatives have comparable or better activity and selectivity than the commonly prescribed anti-leishmanial drug. This result was obtained by inducing stronger mitochondrial depolarization or more intense phosphatidylserine exposure than miltefosine, highlighting compound 8 with moreover 9-times better selectivity index. In addition, the new five molecules activated the apoptosis-like process in the parasite. All the signals observed were indicative of the death process that the parasites were undergoing., (Copyright © 2020 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Interest in periocular (eyelid and eyelashes margins) hygiene has attracted attention recently and a growing number of commercials eye cleanser and shampoos have been marketed. In the present study, a particular eye cleanser foam, Belcils® has been tested against trophozoites and cysts on the facultative pathogen Acanthamoeba. Viability was tested by the alamarBlue™ method and the foam was tested for the induction of programmed cell death in order to explore its mode of action. We found that a 1% solution of the foam eliminated both trophozoite and cyst stage of Acanthamoeba spp. After 90 min of incubation, Belcils® induced, DNA condensation, collapse in the mitochondrial membrane potential and reduction of the ATP level production in Acanthamoeba. We conclude that the foam destroys the cells by the induction of an apoptosis-like process. The current eye cleanser could be used as part of AK therapy protocol and as prevention from AK infections for contact lens users and post-ocular trauma patients., (Copyright © 2020. Published by Elsevier Ltd.)

Primary amoebic encephalitis (PAM) is a lethal disease caused by the opportunistic pathogen, Naegleria fowleri. This amoebic species is able to live freely in warm aquatic habitats and to infect children and young adults when they perform risk activities in these water bodies such as swimming or splashing. Besides the need to increase awareness of PAM which will allow an early diagnosis, the development of fully effective therapeutic agents is needed. Current treatment options are amphotericin B and miltefosine which are not fully effective and also present toxicity issues. In this study, the in vitro activity of various sesquiterpenes isolated from the red alga Laurencia johnstonii were tested against the trophozoite stage of a strain of Naegleria fowleri. Moreover, the induced effects (apoptotic cell death) of the most active compound, laurinterol (1), was evaluated by measuring DNA condensation, damages at the mitochondrial level, cell membrane disruption and production of reactive oxygen species (ROS). The obtained results demonstrated that laurinterol was able to eliminate the amoebae at concentrations of 13.42 ± 2.57 µM and also to induced programmed cell death (PCD) in the treated amoebae. Moreover, since ATP levels were highly affected and laurinterol has been previously reported as an inhibitor of the Na + /K + -ATPase sodium-potassium ion pump, comparison with known inhibitors of ATPases were carried out. Our results points out that laurinterol was able to inhibit ENA ATPase pump at concentrations 100 times lower than furosemide.

Members of the genus Naegleria are free-living amoebae that are widely distributed in water and soil environments. Moreover, Naegleria fowleri is a pathogenic amoeba species that causes a fatal disease in the central nervous system known as primary amoebic meningoencephalitis (PAM) in humans. Since most reported infections due to N. fowleri are reported in recreational waters worldwide, this study was aimed to describe the presence of these amoebic genus in Mexicali Valley irrigation channels of recreational use. A total of nine water samples were collected and processed by triplicate, in nine different sites of the Valley. After filtering and culturing the samples, plates were examined, and the observed amoebae were morphologically identified at the genus level. In addition, the pathogenicity of these amoebic isolates was checked, and molecular characterization was performed by PCR/sequencing. The results revealed the presence of Naegleria spp. in all the channels sampled. Finally, molecular identification confirmed the presence of five different species of Naegleria: N. fowleri, N. australiensis, N. gruberi, N. clarki and N. pagei . The presence of these protists, particularly N. fowleri , should be considered as a potential human health risk in the region.

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., (Copyright © 2020 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Neglected tropical diseases such as leishmaniasis and American trypanosomiasis represent an increasing health problem. Current treatments are not satisfactory which remains an urgent need for novel, cheap and safe chemotherapies. In the course of our ongoing search for new potential anti-protozoal agents, this study aimed to perform a bio-guided fractionation of Inula viscosa (Asteraceae) using in vitro assays against three strains of Leishmania and Trypanosma genus. Eight known compounds were identified from the ethanolic extract of leaves, sesquiterpenoids (3 and 4) and flavonoids (5 and 6) were characterized as the main bioactive constituents. Sesquiterpene lactones 3 and 4 (IC 50 values between 4.99 and 14.26 μM) showed promising antiparasitic activity against promastigotes of L. donovani, L. amazonensis and epimastigotes of T. cruzi. Their structures were successfully characterized by spectroscopic techniques including 1D and 2D NMR experiments. Furthermore, the main bioactive compounds 4, 5 and 6 displayed higher potency (IC 50 values between 0.64 and 2.13 μM) against amastigotes of L. amazonensis than miltefosine (IC 50 3.11 μM), and a low toxicity on macrophages cell line (SI > 45). The analysis of structure-activity relationship (SAR) of the anti-protozoal activity revealed that lactonization or oxidation enhanced the biological profile, suggesting that the hydrophobic moiety was presumably involved in the activity by increasing the affinity and/or cell membrane permeability. In order to get an insight into the mechanism of action of these compounds, programmed cell death (PCD) experiments were performed, and the obtained results suggest that the reported compounds induced PCD in the treated parasites. These results highlight that sesquiterpenoids and flavonoids from I. viscosa could constitute an interesting scaffold for the development of novel antikinetoplastid agents., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Naegleria fowleri is an opportunistic pathogenic free-living amoeba which is able to rapidly colonize the central nervous system (CNS) and causes a lethal infection known as primary amoebic meningoencephalitis (PAM). Furthermore, more than 98% of the known cases of PAM are fatal and affect mainly children under 12 and young adults. Until now, no fully effective therapeutic agents against N. fowleri are available and hence the urgent need to find novel agents to treat PAM. At present, PAM therapy is based on the combination of amphotericin B, miltefosine, among others, with unwanted toxic effects. Recently, our team isolated various indolocarbazoles (ICZs) from the culture of a mangrove strain of Streptomyces sanyensis which showed activity against kinetoplastids and the Acanthamoeba genus. Hence, in this study, the activity of the previously isolated ICZs, staurosporine (STS), 7-oxostaurosporine (7OSTS), 4'-demethylamino-4'-oxostaurosporine, and streptocarbazole B, was evaluated against two type strains of N. fowleri . Furthermore, the performed activity assays revealed that STS was the most active ICZ presenting an inhibitory concentration 50 (IC 50 ) of 0.08 ± 0.02 µM (SI 109.3). Moreover, STS induced programmed cell death (PCD) in the treated amoebae by triggering DNA condensation, mitochondrial disfunction, cell membrane disruption, and reactive oxygen species (ROS) generation. Therefore, STS could be a promising therapeutic agent against PAM., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Free living, cosmopolitan amoebae from Acanthamoeba genus present a serious risk to human health. As facultative human parasites, these amoebae may cause Acanthamoeba keratitis (AK). Acanthamoeba keratitis is a severe, vision-threatening corneal infection with non-specific symptoms. The number of reported AK cases worldwide has been increasing every year. Moreover, 90% of Acanthamoeba keratitis cases are related to contact lens use. Wearing and storage contact lenses not in accordance with the physicians and manufacturers recommendations are the primary key risk factors of this disease. Amoebae can easily adhere to the contact lens surface and transmit to the corneal epithelium. Preventing amoebae adhesion to the contact lens surface could significantly decrease the number of AK infections. Until now, the effective therapy against AK is still under development. Currently proposed therapies are mainly limited to the chlorhexidine digluconate combined with propamidine isethionate or hexamidine applications, which are insufficient and very toxic to the eye. Due to lack of effective treatment, looking for new potential preventive agents is crucial to decrease the number of Acanthamoeba keratitis infections, especially among contact lens users. Nanoparticles have been already included in several novel therapies against bacteria, viruses, fungi, and protist. However, their anti-amoebic potential has not been fully tested yet. The aim of this study was to assess silver nanoparticles (AgNPs) and platinum nanoparticles (PtNPs) anti-amoebic activity and influence on the amoebae adhesion to the surface of four different groups of contact lenses-classified according to the Food and Drugs Administration (FDA) guidelines. The obtained results show that both tested nanoparticles were effective against Acanthamoeba trophozoites and decreased the amoebae adhesion to the contact lens surface. AgNPs showed better anti-amoebic activity to cytotoxicity dependence and reduced amoebae adhesion in a wider spectrum of the tested contact lenses. Our studies also confirmed that ionization next to hydration of the contact lens material is a crucial parameter influencing the Acanthamoeba adhesion to the contact lens surface. In conclusion, silver nanoparticles might be considered as a novel preventive agent against Acanthamoeba keratitis infection., Competing Interests: The authors declare no conflict of interest.

The establishment of an effective therapeutic agent against Acanthamoeba keratitis (AK), remains until present, an issue to be solved due to the existence of a cyst stage in the life cycle of Acanthamoeba . Moreover, the effectiveness of the current standard therapeutic agents varies depending on the tested Acanthamoeba strains and its resistance pattern. In the present study, two 10-point augmented simplex-centroid designs were used to formulate a three-component mixture system using water, atorvastatin, and Diclofenaco-lepori or Optiben. The amoebicidal effects and in vitro -induced toxicity in a eukaryotic cell line were determined for all experiments. The optimal mixture to inhibit the parasite without inducing toxicity was established in the first plan as 30% Optiben, 63.5% atorvastatin, and 3.1% water. As for the second experimental design, the optimal mixture to inhibit Acanthamoeba with lower toxicity effect was composed of 17.6% Diclofenaco-lepori and 82.4% atorvastatin.

The genus Naegleria , of the 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 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, in São Paulo state, Brazil. Five distinct sampling sites were examined through limnological features, trophozoites culturing, and PCR against internal transcribed spacer (ITS) regions and 5.8S rRNA sequences. The results identified N. philippinensis , N. canariensisi , N. australiensis , N. gruberi , N. dobsoni sequences, as well as a Hartmannella sequence. The methodology delineated here represents the first Brazilian Naegleria spp . study on a freshwater system. Our results stress the urgency of a large scale evaluation of the presence of free-living amoebas in Brazil., Competing Interests: The authors declare no conflict of interest.