The anti-amoebic activity of Pinus densiflora leaf extract against the brain-eating amoeba Naegleria fowleri.

Naegleria fowleri invades the brain and causes a fatal primary amoebic meningoencephalitis (PAM). Despite its high mortality rate of approximately 97%, an effective therapeutic drug for PAM has not been developed. Approaches with miltefosine, amphotericin B, and other antimicrobials have been clinically attempted to treat PAM, but their therapeutic efficacy remains unclear. The development of an effective and safe therapeutic drug for PAM is urgently needed. In this study, we investigated the anti-amoebic activity of Pinus densiflora leaf extract (PLE) against N. fowleri. PLE induced significant morphological changes in N. fowleri trophozoites, resulting in the death of the amoeba. The IC50 of PLE on N. fowleri was 62.3±0.95 µg/ml. Alternatively, PLE did not significantly affect the viability of the rat glial cell line C6. Transcriptome analysis revealed differentially expressed genes (DEGs) between PLE-treated and non-treated amoebae. A total of 5,846 DEGs were identified, of which 2,189 were upregulated, and 3,657 were downregulated in the PLE-treated amoebae. The DEGs were categorized into biological process (1,742 genes), cellular component (1,237 genes), and molecular function (846 genes) based on the gene ontology analysis, indicating that PLE may have dramatically altered the biological and cellular functions of the amoeba and contributed to their death. These results suggest that PLE has anti-N. fowleri activity and may be considered as a potential candidate for the development of therapeutic drugs for PAM. It may also be used as a supplement compound to enhance the therapeutic efficacy of drugs currently used to treat PAM.

Flucofuron as a Promising Therapeutic Agent against Brain-Eating Amoeba.

Primary amoebic meningoencephalitis (PAM) is a rare and fulminant neurodegenerative disease caused by the free-living amoeba Naegleria fowleri. Currently, there is a lack of standardized protocols for therapeutic action. In response to the critical need for effective therapeutic agents, we explored the Global Health Priority Box, a collection of 240 compounds provided by the Medicines for Malaria Venture (MMV). From this pool, flucofuron emerged as a promising candidate, exhibiting high efficacy against trophozoites of both N. fowleri strains (ATCC 30808 IC50 : 2.58 ± 0.64 µM and ATCC 30215 IC50: 2.47 ± 0.38 µM), being even active against the resistant cyst stage (IC50: 0.88 ± 0.07 µM). Moreover, flucofuron induced diverse metabolic events that suggest the triggering of apoptotic cell death. This study highlights the potential of repurposing medications for treating challenging diseases, such as PAM.

Structural informatics approach for designing an epitope-based vaccine against the brain-eating Naegleria fowleri.

Primary Amoebic Meningoencephalitis (PAM), a severe lethal brain disease, is caused by a parasite, Naegleria fowleri, also known as the "brain-eating amoeba". The chances of a patient's recovery after being affected by this parasite are very low. Only 5% of people are known to survive this life-threatening infection. Despite the fact that N. fowleri causes a severe, fatal infection, there is no proper treatment available to prevent or cure it. In this context, it is necessary to formulate a potential vaccine that could be able to combat N. fowleri infection. The current study aimed at developing a multi-epitope subunit vaccine against N. fowleri by utilizing immunoinformatics techniques and reverse vaccinology approaches. The T- and B-cell epitopes were predicted by various tools. In order to choose epitopes with the ability to trigger both T- and B-cell-mediated immune responses, the epitopes were put through a screening pipeline including toxicity, antigenicity, cytokine-inductivity, and allergenicity analysis. Three vaccine constructs were designed from the generated epitopes linked with linkers and adjuvants. The modeled vaccines were docked with the immune receptors, where vaccine-1 showed the highest binding affinity. Binding affinity and stability of the docked complex were confirmed through normal mode analysis and molecular dynamic simulations. Immune simulations developed the immune profile, and in silico cloning affirmed the expression probability of the vaccine construct in Escherichia coli (E. coli) strain K12. This study demonstrates an innovative preventative strategy for the brain-eating amoeba by developing a potential vaccine through immunoinformatics and reverse vaccinology approaches. This study has great preventive potential for Primary Amoebic Meningoencephalitis, and further research is required to assess the efficacy of the designed vaccine.

Cyanomethyl Vinyl Ethers Against Naegleria fowleri.

Naegleria fowleri is a pathogenic amoeba that causes a fulminant and rapidly progressive disease affecting the central nervous system called primary amoebic meningoencephalitis (PAM). Moreover, the disease is fatal in more than 97% of the reported cases, mostly affecting children and young people after practicing aquatic activities in nontreated fresh and warm water bodies contaminated with these amoebae. Currently, the treatment of primary amoebic meningoencephalitis is based on a combination of different antibiotics and antifungals, which are not entirely effective and lead to numerous side effects. In the recent years, research against PAM is focused on the search of novel, less toxic, and fully effective antiamoebic agents. Previous studies have reported the activity of cyano-substituted molecules in different protozoa. Therefore, the activity of 46 novel synthetic cyanomethyl vinyl ethers (QOET-51 to QOET-96) against two type strains of N. fowleri (ATCC 30808 and ATCC 30215) was determined. The data showed that QOET-51, QOET-59, QOET-64, QOET-67, QOET-72, QOET-77, and QOET-79 were the most active molecules. In fact, the selectivity index (CC50/IC50) was sixfold higher when compared to the activities of the drugs of reference. In addition, the mechanism of action of these compounds was studied, with the aim to demonstrate the induction of a programmed cell death process in N. fowleri.

A clinical case report of Balamuthia granulomatous amoebic encephalitis in a non-immunocompromised patient and literature review.

BACKGROUND: Balamuthia granulomatous amoebic encephalitis (GAE) is a peculiar parasitic infectious disease of the central nervous system, about 39% of the infected Balamuthia GAE patients were found to be immunocompromised and is extremely rare clinically. The presence of trophozoites in diseased tissue is an important basis for pathological diagnosis of GAE. Balamuthia GAE is a rare and highly fatal infection for which there is no effective treatment plan in clinical practice. CASE PRESENTATION: This paper reports clinical data from a patient with Balamuthia GAE to improve physician understanding of the disease and diagnostic accuracy of imaging and reduce misdiagnosis. A 61-year-old male poultry farmer presented with moderate swelling pain in the right frontoparietal region without obvious inducement three weeks ago. Head computed tomography(CT) and magnetic resonance imaging(MRI) revealed a space-occupying lesion in the right frontal lobe. Intially clinical imaging diagnosed it as a high-grade astrocytoma. The pathological diagnosis of the lesion was inflammatory granulomatous lesions with extensive necrosis, suggesting amoeba infection. The pathogen detected by metagenomic next-generation sequencing (mNGS) is Balamuthia mandrillaris, the final pathological diagnosis was Balamuthia GAE. CONCLUSION: When a head MRI shows irregular or annular enhancement, clinicians should not blindly diagnose common diseases such as brain tumors. Although Balamuthia GAE accounts for only a small proportion of intracranial infections, it should be considered in the differential diagnosis.

Role of cathepsin B of Naegleria fowleri during primary amebic meningoencephalitis.

Naegleria fowleri causes primary amoebic meningoencephalitis in humans and experimental animals. It has been suggested that cysteine proteases of parasites play key roles in metabolism, nutrient uptake, host tissue invasion, and immune evasion. The aim of this work was to evaluate the presence, expression, and role of cathepsin B from N. fowleri in vitro and during PAM. Rabbit-specific polyclonal antibodies against cathepsin B were obtained from rabbit immunization with a synthetic peptide obtained by bioinformatic design. In addition, a probe was designed from mRNA for N. fowleri cathepsin B. Both protein and messenger were detected in fixed trophozoites, trophozoites interacted with polymorphonuclear and histological sections of infected mice. The main cathepsin B distribution was observed in cytoplasm or membrane mainly pseudopods and food-cups while messenger was in nucleus and cytoplasm. Surprisingly, both the messenger and enzyme were observed in extracellular medium. To determine cathepsin B release, we used trophozoites supernatant recovered from nasal passages or brain of infected mice. We observed the highest release in supernatant from recovered brain amoebae, and when we analyzed molecular weight of secreted proteins by immunoblot, we found 30 and 37 kDa bands which were highly immunogenic. Finally, role of cathepsin B during N. fowleri infection was determined; we preincubated trophozoites with E-64, pHMB or antibodies with which we obtained 60%, 100%, and 60% of survival, respectively, in infected mice. These results suggest that cathepsin B plays a role during pathogenesis caused by N. fowleri mainly in adhesion and contributes to nervous tissue damage.

A Fluorometric Assay for the In Vitro Evaluation of Activity against Naegleria fowleri Cysts.

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.

Cyclolauranes as plausible chemical scaffold against Naegleria fowleri.

Primary amoebic meningoencephalitis (PAM) is a central nervous system (CNS) disease caused by Naegleria fowleri that mainly affects children and young adults with fatal consequences in most of the cases. Treatment protocols are based on the combination of different antimicrobial agents, nonetheless there is the need to develop new anti-Naegleria compounds with low toxicity and full effects compared to the currently used drug combination. The marine environment is a well-established source of bioactive natural products. In this work, we have focused on the structure of Laurencia cyclolaurane-type sesquiterpenes as potential chemical model against Naegleria species. The effects of debromolaurinterol (1) to induce PCD/apoptosis-like events in Naegleria fowleri have been evaluated, revealing that this compound induced reduction of ATP production showing a decrease of 99.98% in treated parasite cells. A SAR analysis have been supported with molecular modeling and analysis of the in silico ADME/Tox properties of the Laurencia sesquiterpenes debromolaurinterol (1), laurinterol (2) and allolaurinterol (3), which reinforce cyclolaurane metabolites as plausible molecular models to develop PAM treatments.

Differential Growth Rates and In Vitro Drug Susceptibility to Currently Used Drugs for Multiple Isolates of Naegleria fowleri.

The free-living amoeba Naegleria fowleri, which typically dwells within warm, freshwater environments, can opportunistically cause primary amoebic meningoencephalitis (PAM), a disease with a mortality rate of >97%. The lack of positive treatment outcomes for PAM has prompted the discovery and development of more effective therapeutics, yet most studies utilize only one or two clinical isolates. The inability to assess possible heterogenic responses to drugs among isolates from various geographical regions hinders progress in the discovery of more effective drugs. Here, we conducted drug efficacy and growth rate determinations for 11 different clinical isolates by applying a previously developed CellTiter-Glo 2.0 screening technique and flow cytometry. We found significant differences in the susceptibilities of these isolates to 7 of 8 drugs tested, all of which make up the cocktail that is recommended to physicians by the U.S. Centers for Disease Control and Prevention. We also discovered significant variances in growth rates among isolates, which draws attention to the differences among the amoeba isolates collected from different patients. Our results demonstrate the need for additional clinical isolates of various genotypes in drug assays and highlight the necessity for more targeted therapeutics with universal efficacy across N. fowleri isolates. Our data establish a needed baseline for drug susceptibility among clinical isolates and provide a segue for future combination therapy studies as well as research related to phenotypic or genetic differences that could shed light on mechanisms of action or predispositions to specific drugs. IMPORTANCE Naegleria fowleri, also known as the brain-eating amoeba, is ubiquitous in warm freshwater and is an opportunistic pathogen that causes primary amoebic meningoencephalitis. Although few cases are described each year, the disease has a case fatality rate of >97%. In most laboratory studies of this organism, only one or two well-adapted lab strains are used; therefore, there is a lack of data to discern if there are major differences in potency of currently used drugs for multiple strains and genotypes of the amoeba. In this study, we found significant differences in the susceptibilities of 11 N. fowleri isolates to 7 of the 8 drugs currently used to treat the disease. The data from this study provide a baseline of drug susceptibility among clinical isolates and suggest that new drugs should be tested on a larger number of isolates in the future.

A case of fatal amoebic encephalitis caused by Balamuthia mandrillaris, China.

We reported a case of B.mandrillaris amoebic encephalitis in mainland China. Metagenomics next-generation sequencing helped initial diagnosis and then polymerase chain reaction of the B.mandrillaris in the infected nasal skin tissues reported positive and amoeba cysts were found in the tissue under microscopic observation.

Primary Amoebic Meningoencephalitis by Naegleria fowleri: Pathogenesis and Treatments.

Naegleria fowleri is a free-living amoeba (FLA) that is commonly known as the "brain-eating amoeba." This parasite can invade the central nervous system (CNS), causing an acute and fulminating infection known as primary amoebic meningoencephalitis (PAM). Even though PAM is characterized by low morbidity, it has shown a mortality rate of 98%, usually causing death in less than two weeks after the initial exposure. This review summarizes the most recent information about N. fowleri, its pathogenic molecular mechanisms, and the neuropathological processes implicated. Additionally, this review includes the main therapeutic strategies described in case reports and preclinical studies, including the possible use of immunomodulatory agents to decrease neurological damage.

Comparative Genomic and Transcriptomic Analysis of Naegleria fowleri Clinical and Environmental Isolates.

Out of over 40 species of Naegleria, which are free-living thermophilic amebae found in freshwater and soil worldwide, only Naegleria fowleri infects humans, causing primary amebic meningoencephalitis (PAM), a typically fatal brain disease. To understand the population structure of Naegleria species and the genetic relationships between N. fowleri isolates and to detect pathogenic factors, we characterized 52 novel clinical and environmental N. fowleri genomes and a single Naegleria lovaniensis strain, along with transcriptomic data for a subset of 37 N. fowleri isolates. Whole-genome analysis of 56 isolates from three Naegleria species (N. fowleri, N. lovaniensis, and Naegleria gruberi) identified several genes unique to N. fowleri that have previously been linked to the pathogenicity of N. fowleri, while other unique genes could be associated with novel pathogenicity factors in this highly fatal pathogen. Population structure analysis estimated the presence of 10 populations within the three Naegleria species, of which 7 populations were within N. fowleri. The whole-nuclear-genome (WNG) phylogenetic analysis showed an overall geographical clustering of N. fowleri isolates, with few exceptions, and provided higher resolution in identifying potential clusters of isolates beyond that of the traditional locus typing. There were only 34 genes that showed significant differences in gene expression between the clinical and environmental isolates. Genomic data generated in this study can be used for developing rapid molecular assays and to conduct future population-based global genomic analysis and will also be a valuable addition to genomic reference databases, where shotgun metagenomics data from routine water samples could be searched for the presence of N. fowleri strains. IMPORTANCE N. fowleri, the only known Naegleria species to infect humans, causes fatal brain disease. PAM cases from 1965 to 2016 showed <20 cases per year globally. Out of approximately 150 cases in North America since 1962, only four PAM survivors are known, yielding a >97% case fatality rate, which is critically high. Although the pathogenesis of N. fowleri has been studied for the last 50 years, pathogenetic factors that lead to human infection and breaching the blood-brain barrier remain unknown. In addition, little is known regarding the genomic diversity both within N. fowleri isolates and among Naegleria species. In this study, we generated novel genome sequences and performed comparative genomic and transcriptomic analysis of a set of 52 N. fowleri draft genome sequences from clinical and environmental isolates derived from all over the world in the last 53 years, which will help shape future genome-wide studies and develop sensitive assays for routine surveillance.

On the diversity and clinical importance of Acanthamoeba spp. from Group 1.

Group 1 acanthamoebae are morphologically and phylogenetically distinct from all other Acanthamoeba species. They include five species, each labelled by its genotype: A. astronyxis (T7), A. tubiashi (T8), A. comandoni (T9), unnamed Acanthamoeba sp. (T17), and A. byersi (T18). Thought only environmental, they have recently attracted attention due to their recovery in cases of human keratitis and encephalitis, the main diseases caused by Acanthamoeba, where the usual causative agents are mainly species of Groups 2 and 3. Analysis of the available data confirms the pathogenic importance of these species, although it is probably minor compared to that of the species in Groups 2 and 3. In addition, it should be noted that there are difficulties in identifying genotypes by widely used molecular methods, and some misidentifications are revealed.

In silico analysis of Naegleria fowleri cathepsin B paralogs: important drug targets.

Naegleria fowleri is a deadly human pathogen that causes primary amoebic meningoencephalitis (PAM). In this study, in silico investigations of two important N. fowleri cathepsin B paralogs, i.e., copies of genes resulting from a gene duplication event, were carried out using comparative modeling and molecular dynamics (MD) simulations. Comparative models of both paralogs showed significant architectural similarity with their template, i.e., rat cathepsin B. However, in N. fowleri cathepsin B (UniProt ID: X5D761) and putative cathepsin B (UniProt ID: M1HE19) enzymes, eleven and fifteen residues in the occluding loop regions were deleted, respectively, suggesting that these enzymes have a short occluding loop. Thus, it is concluded that N. fowleri cathepsin B and putative cathepsin B enzymes lack exopeptidase activity but possess enhanced endopeptidase activity and an affinity for macromolecular inhibitors. MD simulations further confirmed that prosegments (macromolecular inhibitors) bond more tightly with both enzymes than with wild-type cathepsin B. Additionally, a mutation was identified at an important N-glycosylation site; this mutation is believed to affect cathepsin B targeting inside the cell and make cathepsin B available in the extracellular environment. Due to this important N-glycosylation site mutation, these enzymes are secreted in the extracellular environment via an alternative, still unknown, posttranslational processing strategy. The present study is the first to predict the three-dimensional folds of N. fowleri cathepsin B paralogous enzymes, including a detailed description of the active site architecture and information about propeptide binding mode. This information can contribute to the discovery of novel and selective treatments that are effective against N. fowleri.

Case Report: Encephalitis Caused by Balamuthia mandrillaris in a 3-Year-Old Iranian Girl.

It is about half a century since free-living amoebae were recognized as pathogenic organisms, but there is still much we should learn about these rare fatal human infectious agents. A recently introduced causative agent of granulomatous amoebic encephalitis, Balamuthia mandrillaris, has been reported in a limited number of countries around the world. A 3-year-old girl was referred to our tertiary hospital because of inability to establish a proper diagnosis. She had been experiencing neurologic complaints including ataxia, altered level of consciousness, dizziness, seizure, and left-sided hemiparesis. The patient's history, physical examination results, and laboratory investigations had led to a wide differential diagnosis. Computed tomography (CT) scan and magnetic resonance imaging analyses revealed multiple mass lesions. As a result, the patient underwent an intraoperative frozen section biopsy of the brain lesion. The frozen section study showed numerous cells with amoeba-like appearances in the background of mixed inflammatory cells. Medications for free-living amoebic meningoencephalitis were administered. PCR assay demonstrated B. mandrillaris as the pathogenic amoeba. Unfortunately, the patient died 14 days after her admission. To our knowledge, this is the first report of B. mandrillaris meningoencephalitis in the Middle East and the first time we have captured the organism during a frozen-section study.

Drugs used for the treatment of cerebral and disseminated infections caused by free-living amoebae.

Free-living amoebae (FLAs) are protozoa developing autonomously in diverse natural or artificial environments. The FLAs Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria fowleri represent a risk for human health as they can become pathogenic and cause severe cerebral infections, named granulomatous amoebic encephalitis (GAE), Balamuthia amoebic encephalitis (BAE), and primary amoebic meningoencephalitis (PAM), respectively. Additionally, Acanthamoeba sp. can also rarely disseminate to diverse organs, such as the skin, sinuses, or bones, and cause extracerebral disseminated acanthamebiasis (EDA). No consensus treatment has been established for cerebral FLA infections or EDA. The therapy of cerebral and disseminated FLA infections often empirically associates a large diversity of drugs, all exhibiting a high toxicity. Nevertheless, these pathologies lead to a high mortality, above 90% of the cases, even in the presence of a treatment. In the present work, a total of 474 clinical cases of FLA infections gathered from the literature allowed to determine the frequency of usage, as well as the efficacy of the main drugs and drug combinations used in the treatment of these pathologies. The efficacy of drug usage was determined based on the survival rate after drug administration. The most efficient drugs, drug combinations, and their mechanism of action were discussed in regard to the present recommendations for the treatment of GAE, EDA, BAE, and PAM. At the end, this review aims to provide a useful tool for physicians in their choice to optimize the treatment of FLA infections.

Drug discovery for primary amebic meningoencephalitis: from screen to identification of leads.

Introduction: Naegleria fowleri is responsible for primary amebic meningoencephalitis (PAM) which has a fatality rate of >97%. Because of the rarity of the disease, pharmaceutical companies do not pursue new drug discovery for PAM. Yet, it is possible that the infection is underreported and finding a better drug would have an impact on people suffering from this deadly infection.Areas covered: This paper reports the efforts undertaken by different academic groups over the last 20 years to test different compounds against N. fowleri. The drug discovery research encompassed synthesis of new compounds, development and use of high-throughput screening methods and attempts to repurpose clinically developed or FDA-approved compounds for the treatment of PAM.Expert opinion: In absence of economic investment to develop new drugs for PAM, repurposing the FDA-approved drugs has been the best strategy so far to identify new leads against N. fowleri. Increasing use of high-throughput phenotypic screening has the potential to accelerate the identification of new leads, either in monotherapy or in combination treatment. Since phase II clinical trial is not possible for PAM, it is critical to demonstrate in vivo efficacy of a clinically safe compound to translate the discovery from lab to the clinic.

Synthetic nanoparticle-conjugated bisindoles and hydrazinyl arylthiazole as novel antiamoebic agents against brain-eating amoebae.

Balamuthia mandrillaris and Naegleria fowleri are free-living amoebae that can cause life-threatening infections involving the central nervous system. The high mortality rates of these infections demonstrate an urgent need for novel treatment options against the amoebae. Considering that indole and thiazole compounds possess wide range of antiparasitic properties, novel bisindole and thiazole derivatives were synthesized and evaluated against the amoebae. The antiamoebic properties of four synthetic compounds i.e., two new bisindoles (2-Bromo-4-(di (1H-indol-3-yl)methyl)phenol (denoted as A1) and 2-Bromo-4-(di (1H-indol-3-yl)methyl)-6-methoxyphenol (A2)) and two known thiazole (4-(3-Nitrophenyl)-2-(2-(pyridin-3-ylmethylene)hydrazinyl)thiazole (A3) and 4-(Biphenyl-4-yl)-2-(2-(1-(pyridin-4-yl)ethylidene)hydrazinyl)thiazole (A4)) were evaluated against B. mandrillaris and N. fowleri. The ability of silver nanoparticle (AgNPs) conjugation to enrich antiamoebic activities of the compounds was also investigated. The synthetic heterocyclic compounds demonstrated up to 53% and 69% antiamoebic activities against B. mandrillaris and N. fowleri respectively, while resulting in up to 57% and 68% amoebistatic activities, respectively. Antiamoebic activities of the compounds were enhanced by up to 71% and 51% against B. mandrillaris and N. fowleri respectively, after conjugation with AgNPs. These compounds exhibited potential antiamoebic effects against B. mandrillaris and N. fowleri and conjugation of synthetic heterocyclic compounds with AgNPs enhanced their activity against the amoebae.