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Abstract Giant viruses (Nucleocytoviricota) are significant lethality agents of various eukaryotic hosts. Although metagenomics indicates their ubiquitous distribution, available giant virus isolates are restricted to a very small number of protist and algal hosts. Here we report on the first viral isolate that replicates in the amoeboflagellate Naegleria. This genus comprises the notorious human pathogen Naegleria fowleri, the causative agent of the rare but fatal primary amoebic meningoencephalitis. We have elucidated the structure and infection cycle of this giant virus, Catovirus naegleriensis (a.k.a. Naegleriavirus, NiV), and show its unique adaptations to its Naegleria host using fluorescence in situ hybridization, electron microscopy, genomics, and proteomics. Naegleriavirus is only the fourth isolate of the highly diverse subfamily Klosneuvirinae, and like its relatives the NiV genome contains a large number of translation genes, but lacks transfer RNAs (tRNAs). NiV has acquired genes from its Naegleria host, which code for heat shock proteins and apoptosis inhibiting factors, presumably for host interactions. Notably, NiV infection was lethal to all Naegleria species tested, including the human pathogen N. fowleri. This study expands our experimental framework for investigating giant viruses and may help to better understand the basic biology of the human pathogen N. fowleri.

Naegleria fowleri, known as the brain-eating amoeba, is the pathogen that causes the primary amoebic meningoencephalitis (PAM), a severe neurodegenerative disease with a fatality rate exceeding 95%. Moreover, PAM cases commonly involved previous activities in warm freshwater bodies that allow amoebae-containing water through the nasal passages. Hence, awareness among healthcare professionals and the general public are the key to contribute to a higher and faster number of diagnoses worldwide. Current treatment options for PAM, such as amphotericin B and miltefosine, are limited by potential cytotoxic effects. In this context, the repurposing of existing compounds has emerged as a promising strategy. In this study, the evaluation of the COVID Box which contains 160 compounds demonstrated significant in vitro amoebicidal activity against two type strains of N. fowleri. From these compounds, terconazole, clemastine, ABT-239 and PD-144418 showed a higher selectivity against the parasite compared to the remaining products. In addition, programmed cell death assays were conducted with these four compounds, unveiling compatible metabolic events in treated amoebae. These compounds exhibited chromatin condensation and alterations in cell membrane permeability, indicating their potential to induce programmed cell death. Assessment of mitochondrial membrane potential disruption and a significant reduction in ATP production emphasized the impact of these compounds on the mitochondria, with the identification of increased ROS production underscoring their potential as effective treatment options. This study emphasizes the potential of the mentioned COVID Box compounds against N. fowleri, providing a path for enhanced PAM therapies.

Acanthamoeba is a ubiquitous genus of amoebae that can trigger a severe and progressive ocular disease known as Acanthamoeba Keratitis (AK). Furthermore, current treatment protocols are based on the combination of different compounds that are not fully effective. Therefore, an urgent need to find new compounds to treat Acanthamoeba infections is clear. In the present study, we evaluated staurosporine as a potential treatment for Acanthamoeba keratitis using mouse cornea as an ex vivo model, and a comparative proteomic analysis was conducted to elucidate a mechanism of action. The obtained results indicate that staurosporine altered the conformation of actin and tubulin in treated trophozoites of A. castellanii. In addition, proteomic analysis of treated trophozoites revealed that this molecule induced overexpression and a downregulation of proteins related to key functions for Acanthamoeba infection pathways. Additionally, the ex vivo assay used validated this model for the study of the pathogenesis and therapies of AK. Finally, staurosporine eliminated the entire amoebic population and prevented the adhesion and infection of amoebae to the epithelium of treated mouse corneas.

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.

Naegleria fowleri is an opportunistic protozoan, belonging to the free-living amoeba group, that can be found in warm water bodies. It is causative agent the primary amoebic meningoencephalitis, a fulminant disease with a rapid progression that affects the central nervous system. However, no 100% effective treatments are available and those that are currently used involve the appearance of severe side effects, therefore, there is an urgent need to find novel antiamoebic compounds with low toxicity. In this study, the in vitro activity of six oxasqualenoids obtained from the red algae Laurencia viridis was evaluated against two different strains of N. fowleri (ATCC® 30808 and ATCC® 30215) as well as their cytotoxicity against murine macrophages. Yucatecone was the molecule with the highest selectivity index (>2.98 and 5.23 respectively) and it was selected to continue with the cell death type determination assays. Results showed that yucatone induced programmed cell death like responses in treated amoebae causing DNA condensation and cellular membrane damage among others. In this family of oxasqualenoids, it seems that the most significative structural feature to induce activity against N. fowleri is the presence of a ketone at C-18. This punctual oxidation transforms an inactive compound into a lead compound as the yucatecone and 18-ketodehydrotyrsiferol with IC50 values of 16.25 and 12.70 μM, respectively. The assessment of in silico ADME/Tox analysis revealed that the active compounds showed good Human Oral Absorption and demonstrate that are found to be within the limit of approved drug parameter range. Hence, the study highlights promising potential of yucatone to be tested for therapeutic use against primary amoebic meningoencephalitis.

IntroductionFree-living amoebae are an extensive group of protistans that can be found in a wide variety of environments. Among them, the Acanthamoeba genus and Naegleria fowleri stand out as two of the most pathogenic amoebae and with a higher number of reported cases. N. fowleri is mainly found in warm freshwater water bodies whereas amoebae of the Acanthamoeba genus are broadly distributed through natural and anthropogenic environments. In this regard, the management and the control of the amoebic populations in swimming pools has become a major public health challenge for institutions.MethodsThe aim of this work was to evaluate the growth pattern of trophozoites of A. griffini and N. fowleri at different temperatures and salt concentrations.Results and discussionOur results showed that A. griffini resisted a higher concentration of salt than N. fowleri. Moreover, no trophozoites could withstand the salt levels of the sea in in vitro conditions. This work supports the contention that salinity could represent an important and useful tool for the control of the most pathogenic amoebic populations in recreational water bodies.

ABSTRACT: 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.

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 IC50 value in the trophozoite stage (0.69 ± 0.01 µM), while the strain A. castellanii L-10 showed the lowest IC50 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.

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 IC90 for 24 h. An increase in reactive oxygen species (ROS) was observed in all cases.

The Free-Living Amoeba species, Naegleria fowleri is the causative agent of a lethal encephalitis known as Primary Amoebic Encephalitis (PAM). Moreover, most of the reported cases are often related to swimming and/or diving in aquatic environments. In addition, the current therapeutic options against PAM are not fully effective and hence, there is an urgent need to develop novel therapeutic agents against this disease. Previously isobenzofuranones compounds have been reported to present antiprotozoal and antifungal activity among others. However, to the best of our knowledge, these molecules have not been previously tested against N. fowleri. Therefore, the aim of this study was to evaluate the activity of 14 novel isobenzofuranones against this pathogenic amoeba. The most active and less toxic molecules, were assayed in order to check induction of Programmed Cell Death (PCD) in the treated amoebae. The obtained results showed that these molecules were able to eliminate N. fowleri trophozoites and also induced PCD. Therefore, the tested isobenzofuranones could be potential therapeutic candidates for the treatment of PAM.

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.

Among the pathogenic free-living amoebae (FLA), Naegleria fowleri is the etiological agent of a fatal disease known as primary amoebic meningoencephalitis (PAM). Once infection begins, the lesions generated in the central nervous system (CNS) result in the onset of symptoms leading to death in a short period of time. Currently, there is no standardized treatment against the infection, which, due to the high virulence of the parasite, results in a high case fatality rate (>97%). Therefore, it is essential to search for new therapeutic sources that can generate a rapid elimination of the parasite. In recent years, there have already been several successful examples of drug repurposing, such as Nitroxoline, for which, in addition to its known bioactive properties, anti-Balamuthia activity has recently been described. Following this approach, the anti-Naegleria activity of Nitroxoline was tested. Nitroxoline displayed low micromolar activity against two different strains of N. fowleri trophozoites (IC50 values of 1.63 ± 0.37 µM and 1.17 ± 0.21 µM) and against cyst stages (IC50 of 1.26 ± 0.42 μM). The potent anti-parasitic activity compared to the toxicity produced (selectivity index of 3.78 and 5.25, respectively) in murine macrophages and human cell lines (reported in previous studies), together with the induction of programmed cell death (PCD)-related events in N. fowleri make Nitroxoline a great candidate for an alternative PAM treatment.

Naegleria fowleri is the causative agent of a central nervous system affecting disease called primary amoebic meningoencephalitis. It is a fulminant disease with a rapid progression that affects mainly children and young adults who report previous water exposure. Current treatment options are not totally effective and involve several side effects. In this work, six meroterpenoids isolated from the brown algae Gongolaria abies-marina were evaluated against N. fowleri. Gongolarone B (1), 6Z-1′-methoxyamentadione (2), and 1′-methoxyamentadione (3) were the most active molecules against N. fowleri with IC50 values between 13.27 ± 0.96 µM and 21.92 ± 1.60 µM. However, cystomexicone B (6) was the molecule with the highest selectivity index (>8.5). Moreover, all these compounds induced different cellular events compatible with the apoptosis-like PCD process, such as chromatin condensation, damages at the mitochondrial level, cell membrane disruption, and production of reactive oxygen species (ROS). Therefore, G. abies-marina could be considered as a promising source of active molecules to treat the N. fowleri infections.

ABSTRACT Primary amoebic meningoencephalitis (PAM) is a lethal and rapid infection that affects the central nervous system and is caused by the free-living amoeba Naegleria fowleri. The life cycle of this protozoa consists of three different stages: The trophozoite, flagellate and cyst stages. Currently, no fully effective molecules have been found to treat PAM. In the search of new antiamoebic molecules, most of the efforts have focused on the trophozoidal activity of the compounds. However, there are no reports on the effect of the compounds on the N. fowleri cyst viability. In the present study, the cysticidal activity of four different molecules was evaluated using an alamarBlue based fluorometric assay. All the tested compounds were active against the cyst stage of N. fowleri. In fact, all the molecules except the amphotericin B, showed highest activity toward the cyst stage than the trophozoite stage. This work could be an effective protocol to select molecules with cysticidal and trophozoidal activity that can be considered a future PAM treatment. IMPORTANCE In the search of new anti-Naegleria fowleri compounds, most of the works focus on the activity of different molecules against the trophozoite stage; however, none of them include the effect of those compounds on the cyst viability. This manuscript presents a solid and reliable assay to evaluate the activity of compounds against the cyst stage of N. fowleri.

Abstract Background The in vitro activity of the brown seaweed Dictyota spiralis against both Leishmania amazonensis and Trypanosoma cruzi was evaluated in a previous study. Processing by bio-guided fractionation resulted in the isolation of three active compounds, classified as diterpenes. In the present study, we performed several assays to detect clinical features associated to cell death in L. amazonensis and T. cruzi with the aim to elucidate the mechanism of action of these compounds on parasitic cells. Methods The aims of the experiments were to detect and evaluate specific events involved in apoptosis-like cell death in the kinetoplastid, including DNA condensation, accumulation of reactive oxygen species and changes in ATP concentration, cell permeability and mitochondrial membrane potential, respectively, in treated cells. Results The results demonstrated that the three isolated diterpenes could inhibit the tested parasites by inducing an apoptosis-like cell death. Conclusions These results encourage further investigation on the isolated compounds as potential drug candidates against both L. amazonensis and T. cruzi. Graphic abstract

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.

Naegleria fowleri is an opportunistic protozoon that can be found in warm water bodies. It is the causative agent of the primary amoebic meningoencephalitis. Focused on our interest to develop promising lead structures for the development of antiparasitic agents, this study was aimed at identifying new anti-Naegleria marine natural products from a collection of chamigrane-type sesquiterpenes with structural variety in the levels of saturation, halogenation and oxygenation isolated from Laurencia dendroidea. (+)-Elatol (1) was the most active compound against Naegleria fowleri trophozoites with IC50 values of 1.08 μM against the ATCC 30808™ strain and 1.14 μM against the ATCC 30215™ strain. Furthermore, the activity of (+)-elatol (1) against the resistant stage of N. fowleri was also assessed, showing great cysticidal properties with a very similar IC50 value (1.14 µM) to the one obtained for the trophozoite stage. Moreover, at low concentrations (+)-elatol (1) showed no toxic effect towards murine macrophages and could induce the appearance of different cellular events related to the programmed cell death, such as an increase of the plasma membrane permeability, reactive oxygen species overproduction, mitochondrial malfunction or chromatin condensation. Its enantiomer (−)-elatol (2) was shown to be 34-fold less potent with an IC50 of 36.77 μM and 38.03 μM. An analysis of the structure–activity relationship suggests that dehalogenation leads to a significant decrease of activity. The lipophilic character of these compounds is an essential property to cross the blood-brain barrier, therefore they represent interesting chemical scaffolds to develop new drugs.

Leishmaniasis and Chagas disease affect millions of people worldwide. The available treatments against these parasitic diseases are limited and display multiple undesired effects. The brown alga belonging to the genus Gongolaria has been previously reported as a source of compounds with different biological activities. In a recent study from our group, Gongolaria abies-marine was proven to present antiamebic activity. Hence, this brown alga could be a promising source of interesting molecules for the development of new antiprotozoal drugs. In this study, four meroterpenoids were isolated and purified from a dichloromethane/ethyl acetate crude extract through a bioguided fractionation process targeting kinetoplastids. Moreover, the in vitro activity and toxicity were evaluated, and the induction of programmed cell death was checked in the most active and less toxic compounds, namely gongolarone B (2), 6Z-1′-methoxyamentadione (3) and 1′-methoxyamentadione (4). These meroterpenoids triggered mitochondrial malfunction, oxidative stress, chromatin condensation and alterations of the tubulin network. Furthermore, a transmission electron microscopy (TEM) image analysis showed that meroterpenoids (2–4) induced the formation of autophagy vacuoles and ER and Golgi complex disorganization. The obtained results demonstrated that the mechanisms of action at the cellular level of these compounds were able to induce autophagy as well as an apoptosis-like process in the treated parasites.

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.

Abstract Background The in vitro activity against Leishmania spp. of a novel group of compounds, phenalenone derivatives, is described in this study. Previous studies have shown that some phenalenones present leishmanicidal activity, and induce a decrease in the mitochondrial membrane potential in L. amazonensis parasites, so in order to elucidate the evidence of programmed cell death occurring inside the promastigote stage, different assays were performed in two different species of Leishmania. Methods 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, mitochondrial membrane potential, and chromatin condensation among others. Results The results showed that four molecules activated the apoptosis-like process in the parasite. All the signals observed were indicative of the death process that the parasites were undergoing. Conclusions The present results highlight the potential use of phenalenone derivatives against Leishmania species and further studies should be undertaken to establish them as novel leishmanicidal therapeutic agents.

Objective: To evaluate the combination of several statins (atorvastatin, fluvastatin and simvastatin) and azoles (voriconazole, posaconazole and itraconazole) against Acanthamoeba spp. Methods: The efficiency of the different drug combinations against the trophozoite stage of different Acanthamoeba strains were evaluated by Alamar Blue assay. Effect on the cyst stage was observed by inverted microscope. Cytotoxicity of combinations of azoles and statins was evaluated by measuring the release of lactate dehydrogenase from a murine macrophage cell line. Results: Combinations of any of the tested statins and voriconazole or posaconazole were more efficient in inhibiting Acanthamoeba compared to statins or azoles individually. The drug combinations at the combined inhibitory concentrations 50% showed lower toxicity compared to that of the compounds alone. Conclusions: The combinations of statins together with voriconazole and posaconazole are more efficient than these drugs alone, and these combinations have lower cytotoxicity in mammalian cell lines.

In this study, the application of amphipods in vivo assays was evaluated. The main aim of this work was to check the potential use of this model in biocompatibility assessments of metal-organic frameworks (MOFs). Hence, six different MOFs were synthesized and the in vitro and ex vivo cytotoxicity was first assessed using a colorimetric assay and a macrophage cell line. Obtained results were compared to validate the in vivo toxicity tests carried out using amphipods and increasing concentrations of the different MOFs. Amphipods do not require the need of ethics approval and also are less expensive to keep than conventional in vivo models, showing its potential as a fast and reliable platform in toxicity studies. The obtained results showed that the amphipods based-assay was simple, easy to replicate and yielded toxicity data corresponding to the type of MOFs tested. In addition, it was observed that only CIM-80(Al) and CIM-84(Zr) did not show any toxicity to the animals at the different tested concentrations. Therefore, the developed in vivo model could be applied as a high-throughput toxicity screening method to evaluate the toxicity of numerous materials, chemicals and therapeutic agents among others.

Abstract 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.

Acanthamoeba is a free-living amoeba genus able to cause severe infections, such as Granulomatous amoebic encephalitis (GAE), epithelial disorders and a sight-threatening disease called Acanthamoeba keratitis (AK) [...]

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.

Abstract 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.

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.

Despite intensified efforts to develop an effective antibiotic, S. aureus is still a major cause of mortality and morbidity worldwide. The multidrug resistance of bacteria has considerably increased the difficulties of scientific research and the concomitant emergence of resistance is to be expected. In this study we have investigated the in vitro activity of 15 ethanol extracts prepared from Moroccan medicinal plants traditionally used for treatment of skin infections. Among the tested species I. viscosa, C. oxyacantha, R. tinctorum, A. herba alba, and B. hispanica showed moderate anti-staphylococcal activity. However, R. alaternus showed promising growth-inhibitory effects against specific pathogenic bacteria especially methicillin-susceptible Staphylococcus aureus Panton-Valentine leucocidin positive (MSSA-PVL) and methicillin-resistant S. aureus (MRSA). The bioguided fractionation of this plant using successive chromatographic separations followed by nuclear magnetic resonance (NMR) and mass spectrometry (MS) including EIMS and HREIMS analysis yielded the emodin (1) and kaempferol (2). Emodin being the most active with MICs ranging between 15.62 and 1.95 µg/mL and showing higher activity against the tested strains in comparison with the crude extract, its mechanism of action and the structure-activity relationship were interestingly discussed. The active compound has not displayed toxicity toward murine macrophage cells. The results obtained in the current study support the traditional uses of R. alaternus and suggest that this species could be a good source for the development of new anti-staphylococcal agents.

The neglected infection known as Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, results in more than 7000 deaths per year, with an increasing number of cases in non-endemic areas such as Europe or the United States. Moreover, with the current available therapy, only two compounds which are active against the acute phase of the disease are readily available. In addition, these therapeutic agents display multiple undesired side effects such as high toxicity, they are expensive, the treatment is lengthy and the resistant strain has emerged. Therefore, there is a need to find new compounds against Chagas disease which should be active against the parasite but also cause low toxicity to the patients. In the present work, the activity of novel acrylonitriles against Trypanosoma cruzi was evaluated as well as the analysis of the physiological events induced in the treated parasites related to the cell death process. Hence, the characteristic features of an apoptosis-like process such as chromatin condensation and mitochondrial membrane potential, among others, were studied. From the 32 compounds tested against the epimastigote stage of T. cruzi, 11 were selected based on their selectivity index to determine if these compounds were able to induce programmed cell death (PCD) in the treated parasites. Furthermore, acrylonitriles Q5, Q7, Q19, Q27 and Q29 were shown to trigger physiological events related in the PCD. Therefore, this study highlights the therapeutic potential of acrylonitriles as novel trypanocidal agents.

Background: Free-living amoebae (FLA) are group of protozoa distributed worldwide in many habitats mainly water and soil related sources. Some members of FLA are able to act as opportunistic pathogens and are environmental carriers of other pathogenic agents such as bacteria and viruses. Vermamoeba vermiformis is a highly abundant FLA species in water bodies and has recently gained environmental importance as it acts as a vehicle of many pathogenic bacteria such as Legionella pneumophila.Cases Report: In this study, water samples were collected from the island of El Hierro, Canary Islands, Spain during 2015. El Hierro island was designated by UNESCO as a biosphere reserve and it is currently the less populated of the Canary Islands. The water samples were culture 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. After a week, V. vermiformis amoebae were observed in the plates incubated at room temperature and 37 ºC. Molecular characterization was carried out by amplifying the 18S rDNA gene and DNA sequencing, confirming that the isolated strain belonged to Vermamoeba vermiformis species.Conclusion: To the best of our knowledge, this is the first report of Vermamoeba vermiformis isolation in the island of El Hierro and the second report of this species in the Canary Islands.

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 (IC50) 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.

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.

Chagas disease and leishmaniasis are neglected tropical diseases caused by kinetoplastid parasites of Trypanosoma and Leishmania genera that affect poor and remote populations in developing countries. These parasites share similar complex life cycles and modes of infection. It has been demonstrated that the particular group of phosphorylating enzymes, protein kinases (PKs), are essential for the infective mechanisms and for parasite survival. The natural indolocarbazole staurosporine (STS, 1) has been extensively used as a PKC inhibitor and its antiparasitic effects described. In this research, we analyze the antikinetoplastid activities of three indolocarbazole (ICZs) alkaloids of the family of staurosporine STS, 2–4, and the commercial ICZs rebeccamycin (5), K252a (6), K252b (7), K252c (8), and arcyriaflavin A (9) in order to establish a plausive approach to the mode of action and to provide a preliminary qualitative structure–activity analysis. The most active compound was 7-oxostaurosporine (7OSTS, 2) that showed IC50 values of 3.58 ± 1.10; 0.56 ± 0.06 and 1.58 ± 0.52 µM against L. amazonensis; L. donovani and T. cruzi, and a Selectivity Index (CC50/IC50) of 52 against amastigotes of L. amazonensis compared to the J774A.1 cell line of mouse macrophages.

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.

Therapy against Acanthamoeba infections such as Granulomatous Amoebic Encephalitis (GAE) and Acanthamoeba Keratitis (AK), remains as an issue to be solved due to the existence of a cyst stage which is highly resistant to most chemical and physical agents. Recently, the activity of Olive Leaf Extracts (OLE) was demonstrated against Acanthamoeba species. However, the molecules involved in this activity were not identified and/or evaluated. Therefore, the aim of this study was to evaluate the activity of the main molecules which are present in OLE and secondly to study their mechanism of action in Acanthamoeba. Among the tested molecules, the observed activities ranged from an IC50 of 6.59 in the case of apigenine to an IC50 > 100 μg/ml for other molecules. After that, elucidation of the mechanism of action of these molecules was evaluated by the detection of changes in the phosphatidylserine (PS) exposure, the permeability of the plasma membrane, the mitochondrial membrane potential and the ATP levels in the treated cells. Vanillic, syringic and ursolic acids induced the higher permeabilization of the plasma membrane. Nevertheless, the mitochondrial membrane was altered by all tested molecules which were also able to decrease the ATP levels to less than 50% in IC90 treated cells after 24 h. Therefore, all the molecules tested in this study could be considered as a future therapeutic alternative against Acanthamoeba spp. Further studies are needed in order to establish the true potential of these molecules against these emerging opportunistic pathogenic protozoa.

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.

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′-demethylamino-4′-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 1−4 was observed. Also, the study reveals that 7-oxostaurosporine (2) affects membrane permeability and causes mitochondrial damages on trophozoites of A. castellanii Neff.

Leishmaniasis remains a major world health problem, and in particular, Algeria ranks second for the incidence of cutaneous leishmaniasis. Pulicaria inuloides is a well-known Arabian Peninsula medicinal plant. In the present study, the chloroform, ethyl acetate and n-butanol extracts from the roots of Pulicaria inuloides were analyzed for antioxidant activity and its correlation with the total phenolic and flavonoid contents. The highest antioxidant activity using a DPPH assay was showed by the ethyl acetate extract (IC50 4.08 µg/mL), which also had the highest total phenolic content (307.12 µgAGE). Furthermore, P. inuloides root extracts were evaluated against Leishmania amazonensis and Leishmania donovani. The results highlighted the chloroform extract as the most active one against both tested Leishmania strains. A bioguided fractionation of the chloroform extract led to the isolation of (8R:8S)-(75:25 er)-10-isobutyryloxy-8,9-epoxy-thymol isobutyrate as the main bioactive component, showing a potent leishmanicidal activity on L. amazonensis promatigote and amastigote stages (IC50 5.03 and 2.87 µM, respectively) and a good selectivity index on murine macrophages (CC50 19.37 µM). This study provides the first report of the antioxidant and leishmanicidal activities of P. inuloides root extracts and the results point to this species as a source of potential bioactive agents.

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 ± 0.078 to 1.682 ± 0.775 µM depending on the tested strain of N. fowleri. Therefore, fluvastatin could be a potential novel therapeutic agent against this emerging pathogen.

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 (2−8), 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.

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 µM, respectively. The hydroxylated congeners thyrsiferol (2) and 22-hydroxydehydrothyrsiferol (4), active in the same value range at IC50 13.97 and 17.00 µM, are not toxic against murine macrophages; thus, they are solid candidates for the development of new amoebicidal therapies.

Acanthamoeba is a free-living amoebae genus which is present worldwide in natural and artificial environments. These amoebae are clinically important as causative agents of diseases in humans and other animals such as a fatal encephalitis or a sight threatening Acanthamoeba keratitis (AK). Lately; studies have focused on the search of novel therapeutic options for AK but also to prevent infections. Furthermore; the evaluation of commercialized products seems to be an option for this case since not clinical assays would be required. Thus; we aimed to test the amoebicidal activity of different mixtures of two commercial ophthalmic solutions: Systane® Ultra; which has already shown anti-Acanthamoeba properties; and Naviblef® Daily Care. In addition, we tested their cytotoxic effect against murine macrophages. At the individual level; Naviblef® Daily Care showed to be the most active product against Acanthamoeba spp. Nevertheless; the combinations of Systane® Ultra and Naviblef® Daily Care; showed an improvement in the activity against trophozoites and cysts of Acanthamoeba castellanii Neff. Moreover; the concentration necessary to generate cytotoxic effect against murine macrophages (J774.1) was much higher than the required for the amoebicidal and cysticidal effect achieved in the most effective mixtures.

Bioassay-guided fractionation of the antikinetoplastid extract of the brown alga Dictyota spiralis has led to the isolation of spiralyde A (1), a new dolabellane aldehyde, along with other five known related diterpenes (2–6). Their structures were determined by HRESIMS, 1D and 2D NMR spectroscopy, and comparison with data reported in the literature. The antiparasitic activity of all compounds was evaluated. Spiralyde A (1) and the known compound 3,4-epoxy-7,18-dolabelladiene (2) were the most active compounds against Leishmania amazonensis and Trypanosoma cruzi. Spiralyde A (1) was the most potent compound, comparable to benznidazole, the reference drug for trypanocidal activity.

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.