Brain-eating Amoeba Research Articles

The potential of nanocomposites (patuletin-conjugated with gallic acid-coated zinc oxide) against free-living amoebae pathogens.

Free-living amoebae infections are on the rise while the prognosis remains poor. Current therapies are ineffective, and there is a need for novel effective drugs which can target Naegleria, Balamuthia, and Acanthamoeba species. In this study, we determined the effects of a nano-formulation based on flavonoid patuletin-loaded gallic acid functionalized zinc oxide nanoparticles (PA-GA-ZnO) against Acanthamoeba, Balamuthia, and Naegleria trophozoites. Characterization of the nano-formulation was accomplished utilizing analytical tools, namely Fourier-transform infrared spectroscopy, drug entrapment efficiency, polydispersity index, dimensions, and surface morphologies. Anti-amoebic effects were investigated using amoebicidal assay, cytopathogenicity assay, and cytotoxicity of the nano-formulation on human cells. The findings revealed that nano-formulation (PA-GA-ZnO) displayed significant anti-amoebic properties and augmented effects of patuletin alone against all three brain-eating amoebae. When tested alone, patuletin nano-formulations showed minimal toxicity effects against human cells. In summary, the nano-formulations evaluated herein depicts efficacyversusAcanthamoeba, Balamuthia, and Naegleria. Nonetheless, future studies are needed to comprehend the molecular mechanisms of patuletin nano-formulations versus free-living amoebae pathogens, in addition to animal studies to determine their potential value for clinical applications.

Recurring seasonal cases of Naegleria fowleri (Brain-Eating Amoeba) in Pakistan: A rapidly growing threat.

Recurring seasonal cases of Naegleria fowleri (Brain-Eating Amoeba) in Pakistan: A rapidly growing threat.

Brain-eating amoeba: Recognition and treatment of primary amebic meningoencephalitis.

Brain-eating amoeba: Recognition and treatment of primary amebic meningoencephalitis.

Next generation imidazothiazole and imidazooxazole derivatives as potential drugs against brain-eating amoebae.

Managing primary amoebic meningoencephalitis, induced by Naegleria fowleri poses a complex medical challenge. There is currently no specific anti-amoebic drug that has proven effectiveness against N. fowleri infection. Ongoing research endeavours are dedicated to uncovering innovative treatment strategies, including the utilization of drugs and immune modulators targeting Naegleria infection. In this study, we explored the potential of imidazo[2,1-b]thiazole and imidazooxazole derivatives that incorporate sulfonate and sulfamate groups as agents with anti-amoebic properties against N. fowleri. We assessed several synthesized compounds (1f, 1m, 1q, 1s,and 1t) for their efficacy in eliminating amoebae, their impact on cytotoxicity, and their influence on the damage caused to human cerebral microvascular endothelial (HBEC-5i) cells when exposed to the N. fowleri (ATCC 30174) strain. The outcomes revealed that, among the five compounds under examination, 1m, 1q, and 1t demonstrated notable anti-parasitic effects against N. fowleri (P ≤ 0.05). Compound 1t exhibited the highest anti-parasitic activity, reducing N. fowleri population by 80%. Additionally, three compounds, 1m, 1q, and 1t, significantly mitigated the damage inflicted on host cells by N. fowleri. However, the results of cytotoxicity analysis indicated that while 1m and 1q had minimal cytotoxic effects on endothelial cells, compound 1t caused moderate cytotoxicity (34%). Consequently, we conclude that imidazo[2,1-b]thiazole and imidazooxazole derivatives containing sulfonate and sulfamate groups exhibit a marked capacity to eliminate amoebae viability while causing limited toxicity to human cells. In aggregate, these findings hold promise that could potentially evolve into novel therapeutic options for treating N. fowleri infection.

Naegleria fowleri 98 Percent Fatal a Comprehensive Survey

Naegleria fowleri, colloquially termed the "brain-eating amoeba," presents a formidable public health concern due to its astonishingly high fatality rate of 98%. This abstract provides a thorough examination of the various aspects surrounding Naegleria fowleri, encompassing its morphology, life cycle, transmission routes, clinical manifestations, diagnostic methods, treatment strategies, and preventive measures. Naegleria fowleri, an amoeboid protist, thrives in warm freshwater environments, predominantly in regions with elevated temperatures. Infections occur when contaminated water enters the nasal passages, leading to primary amebic meningoencephalitis (PAM), an acute and often fatal condition affecting the central nervous system. The clinical course of PAM is rapid, with symptoms escalating from initial flu-like manifestations to severe neurological impairment within days. Diagnosis of PAM remains challenging, relying on specialized laboratory techniques such as polymerase chain reaction (PCR) and immunofluorescence assays. Despite efforts to develop effective treatments, mortality rates remain alarmingly high, emphasizing the critical importance of preventive measures. Avoiding freshwater activities in warm, stagnant water bodies, utilizing nose clips or plugs during water-related activities, and ensuring proper disinfection of recreational water sources are key strategies in mitigating the risk of the risk of Naegleria fowleri infections and reducing its devastating impact on public health.

Molecular mechanisms and therapeutic strategies of Naegleria fowleri Carter (1970): a review of the fatal brain-eating amoeba

Molecular mechanisms and therapeutic strategies of Naegleria fowleri Carter (1970): a review of the fatal brain-eating amoeba

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.

Exploring therapeutic approaches against Naegleria fowleri infections through the COVID box

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.

Exploring the extrachromosomal plasmid rDNA of Naegleria fowleri AY27 genotype II: A human brain-eating amoeba via high-throughput sequencing

Naegleria fowleri, also known as brain-earing amoeba, causes severe and rapidly fatal CNS infection in humans called primary amebic meningoencephalitis (PAM). The DNA from the N. fowleri clinical isolate was sequenced for circular extrachromosomal ribosomal DNA (CERE - rDNA). The CERE contains 18 S, 5.8 S, and 28 S ribosomal subunits separated by internal transcribed spacers, 5 open reading frames (ORFs), and mostly repeat elements comprising 7268 bp out of 15,786 bp (46%). A wide variety of variations and recombination events were observed. Finally, the ORFs that comprised only 4 hypothetical proteins were modeled and screened against Zinc drug-like compounds. Two compounds [ZINC77564275 (ethyl 2-(((4-isopropyl-4 H-1,2,4-triazol-3-yl) methyl) (methyl)amino) oxazole-4-carboxylate) and ZINC15022129 (5-(2-methoxyphenoxy)-[2,2’-bipyrimidine]-4,6(1 H,5 H)-dione)] were finalized as potential druggable compounds based on ADME toxicity analysis. We propose that the compounds showing the least toxicity would be potential drug candidates after laboratory experimental validation is performed.

Naegleria fowleri in 2023: A rising threat to Pakistan.

Madam, Naegleria fowleri, i.e., brain-eating amoeba, is a single-cell, free-living parasite that resides in soil and warm freshwater bodies such as lakes, rivers, and hot springs. It enters the nasal cavity from contaminated water, uses the olfactory nerve to reach the brain, and causes a deadly infection called primary amoebic meningoencephalitis (PAM), with a mortality rate higher than 97% even with treatment. (1,2) It causes symptoms similar to those of other bacterial and viral meningitis, such as fever, headache, vomiting, altered level of consciousness and coma, followed by death in the span of a week to 10 days. (1) There are 20 strains of Naegleria world worldwide, but only N. fowleri is dangerous to humans. (1) In 2008, the first case was reported from Karachi, Pakistan. (2) By 2019, the number had reached 146, (2) and the cases have been increasing until now. In 2023, eight cases have been reported from May to August, among which seven are from the province of Sindh and one from Punjab. (3, 4, 6) The available data shows that two deaths were due to recreational water activities and others because of contaminated tap water.(3,4) This pathogenic amoeba commonly infects men, and the majority of cases were in people aged 26 to 45 years.(1) Of all the reported cases this year, one was a 32-year-old female, and seven were males in the aged 19-45 years.(3,4, 6) The increasing global warming and higher authorities’ negligence towards WHO recommended chlorination (0.1-10 mg/L) of domestic water supply are contributing factors to the higher burden of Naegleria fowleri cases in Karachi, the largest metropolitan city of Pakistan .(5) The new lethal strain of N. fowleri, named as Karachi-NF001, has been found in one patient who traveled from Saudi Arabia to Karachi in 2019. (2) These emerging cases concern strict actions against this problem and the need to alert the Karachi Water and Sewerage Board (KWSB) regarding consistent chlorination from April to October. (5) The monsoon season (May to September) is the best time to spread awareness regarding its prevention and update the current status of N. fowleri in our country. The general public should be encouraged to use boiled or sterilised water for ablution and avoid aquatic activities in non-chlorinated water during the summer season. (3) Besides that, all underground and overhead water tanks in homes, hospitals, shopping malls, schools, offices, etc. should be cleaned once a year. (3)

In silico discovery of diagnostic/vaccine candidate antigenic epitopes and a multi-epitope peptide vaccine (NaeVac) design for the brain-eating amoeba Naegleria fowleri causing human meningitis

Naegleria fowleri, the brain-eating amoeba, is a free-living amoeboflagellate with three different life cycles (trophozoite, flagellated, and cyst) that lives in a variety of habitats around the world including warm freshwater and soil. It causes a disease called naegleriasis leading meningitis and primary amoebic meningoencephalitis (PAM) in humans. N. fowleri is transmitted through contaminated water sources such as insufficiently chlorinated swimming pool water or contaminated tap water, and swimmers are at risk. N. fowleri is found all over the world, and most infections were reported in both developed and developing countries with high mortality rates and serious clinical findings. Until now, there is no FDA approved vaccine and early diagnosis is urgent against this pathogen. In this study, by analyzing the N. fowleri vaccine candidate proteins (Mp2CL5, Nfa1, Nf314, proNP-A and proNP-B), it was aimed to discover diagnostic/vaccine candidate epitopes and to design a multi-epitope peptide vaccine against this pathogen. After the in silico evaluation, three prominent diagnostic/vaccine candidate epitopes (EAKDSK, LLPHIRILVY, and FYAKLLPHIRILVYS) with the highest antigenicities were discovered and a potentially highly immunogenic/antigenic multi-epitope peptide vaccine (NaeVac) was designed against the brain-eating amoeba N. fowleri causing human meningitis.

Addressing The Alarming Surge Of Naegleria And Other Water-Borne Diseases In Karachi: A Call For Urgent Action.

Naegleria fowleri, a thermophilic, free-living organism causes a life-threatening condition called primary amoebic meningoencephalitis (PAM). Naegleria thrives in warm freshwater but the rising number of cases in Karachi has raised concerns about the quality of the city’s domestic water supply. Pakistan has dealt with numerous healthcare burdens in the past, including outbreaks of diseases such as diphtheria, and cholera, and is persistently dealing with the challenge of polio. Moreover, bacterial contamination and improper water infrastructure pose a significant public health risk to several other water-borne diseases, prevalent in the city. Furthermore, the surge in cases of Naegleria adds to these ongoing health concerns. This brain-eating amoeba has the potential to spread beyond its current location, posing a significant threat to the nation's healthcare system. While the world is struggling to find an effective treatment for PAM, implementing preventive measures to improve water management, and surveillance can save lives and lessen the burden. The addition of chlorine to the water supply of the city by KWSB is a crucial step toward keeping this infection under control.

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.

Characterization of the extracellular vesicles, ultrastructural morphology, and intercellular interactions of multiple clinical isolates of the brain-eating amoeba, Naegleria fowleri.

As global temperatures rise to unprecedented historic levels, so too do the latitudes of habitable niches for the pathogenic free-living amoeba, Naegleria fowleri. This opportunistic parasite causes a rare, but >97% fatal, neurological infection called primary amoebic meningoencephalitis. Despite its lethality, this parasite remains one of the most neglected and understudied parasitic protozoans. To better understand amoeboid intercellular communication, we elucidate the structure, proteome, and potential secretion mechanisms of amoeba-derived extracellular vesicles (EVs), which are membrane-bound communication apparatuses that relay messages and can be used as biomarkers for diagnostics in various diseases. Herein we propose that N. fowleri secretes EVs in clusters from the plasma membrane, from multivesicular bodies, and via beading of thin filaments extruding from the membrane. Uptake assays demonstrate that EVs are taken up by other amoebae and mammalian cells, and we observed a real-time increase in metabolic activity for mammalian cells exposed to EVs from amoebae. Proteomic analysis revealed >2,000 proteins within the N. fowleri-secreted EVs, providing targets for the development of diagnostics or therapeutics. Our work expands the knowledge of intercellular interactions among these amoebae and subsequently deepens the understanding of the mechanistic basis of PAM.

Naegleria Fowleri - Prevention is the best cure

Respected Editor, With the temperatures rising every summer and the quality of water deteriorating in Pakistan and specially Karachi the ‘brain-eating amoeba’ Naegleria fowleri was on rise during this summer too. 6 cases of primary amebic meningoencephalitis (PAM) caused by N. fowleri were reported which took at least 5 lives in 2022.1 Naegleria fowleri is a non-parasitic, thermophilic, disease-causing flagellate amoeba. N. fowleri can tolerate high temperatures (up to 45%) and mainly consumes bacteria due to which it is usually found in warm freshwater bodies.2 The stage in which it is considered most infective is the trophozoite stage which is also its reproductive stage and it requires favourable conditions for this stage to be exhibited.2 The main route of entry for N. Fowleri is the olfactory route when water contaminated with this pathogen is forcefully inhaled during water-related activities like swimming and ablution etc.2 Signs and symptoms of PAM are similar to those of viral or bacterial meningitis including fever, headache, stiff neck, vomiting, anorexia, and seizures. This is the reason Naegleria fowleri is also commonly known as “brain-eating amoeba”. This similarity makes it difficult for physicians to diagnose it early. PAM can cause death within 3 to 7 days after the symptoms appear.3 After the first case of PAM which was reported in 2008, approximately 150 cases have been reported from Karachi alone. In only a decade, the number of PAM cases in Pakistan exceeded those reported in the USA in half a century (i.e. 142 cases between 1968 and 2019)4. Aga Khan University Hospital, Karachi, Pakistan has observed a death rate of approximately 20 deaths per year due to PAM in Pakistan.5 Naegleria fowleri can be killed by chlorinating water and the ensuing disease and deaths can be eliminated but due to the failure of the Karachi Water and Sewerage Board before supplying it to houses and then the use of that water for bathing and ablution, this pathogen continues to cause deaths every year.6 According to the WHO, free chlorine having residual concentration equal to or more than 0.5 mg/L, at pH less than 8.0 and 20°C, after a contact time of at least 30 minutes is effective for chlorine disinfection. The government should make sure that this level of chlorine is maintained throughout the distribution system.2 ---Continue

Kaempferol induces programmed cell death in Naegleria fowleri

BackgroundNaegleria fowleri is a brain-eating amoeba causing a fatal brain infection called primary amoebic meningoencephalitis (PAM). Despite its high mortality over 95%, effective therapeutic drug for PAM has not been developed yet. Therefore, development of an effective and safe therapeutic drug for PAM is urgently needed. In this study, we investigated anti-amoebic effect of kaempferol (KPF) against N. fowleri and its underlying anti-amoebic molecular mechanisms. MethodsAnti-amoebic activity of KPF against N. fowleri trophozoites, as well as cytotoxicity of KPF in C6 glial cells and CHO-K1 cells were investigated. The programmed cell death mechanisms in KPF-treated N. fowleri were also analyzed by apoptosis-necrosis assay, mitochondrial dysfunction assay, TUNEL assay, RT-qPCR, and CYTO-ID assay. ResultsKPF showed anti-amoebic activity against N. fowleri trophozoites with an IC50 of 29.28 ± 0.63 μM. However, it showed no significant cytotoxicity to mammalian cells. KPF induced significant morphological alterations of the amoebae, resulting in death. Signals associated with apoptosis were detected in the amoebae upon treatment with KPF. KPF induced an increase of intracellular reactive oxygen species level, loss of mitochondrial membrane potential, increases of expression levels of genes associated with mitochondria dysfunction, and reduction of ATP levels in the amoebae. Autophagic vacuole accumulations with increased expression levels of autophagy-related genes were also detected in KPF-treated amoebae. ConclusionKPF induces programmed cell death in N. fowleri trophozoites via apoptosis-like pathway and autophagy pathway. KPF could be used as a candidate of anti-amoebic drug or supplement compound in the process of developing or optimizing therapeutic drug for PAM.

Naegleria fowleri: An emerging issue of significant concern in Pakistan

There is thermophilic flagellate amoeba that is known as Naegleria fowleri and it is labelled as brain-eating amoeba. This is a causative agent for primary amoebic meningoencephalitis (PAM), a dangerous waterborne disease affecting humans and animals. Pakistan is ranked the 2nd most affected country, where most of the cases are reported from the biggest city of Pakistan i.e. Karachi. An immediate epidemiologic examination of the relevant environmental issues is required in order to determine the causes of this abrupt increase in PAM cases. Furthermore, many people in Pakistan do not have access to medical services, and those that do exist are inaccessible. Lack of potable water, sanitation, and knowledge exacerbates water-borne illness outbreaks. Authorities must detect widespread health issues and take action to alleviate suffering. Furthermore, there is an immediate need to educate individuals on the significance of using boiling water for nasal cleaning, particularly after swimming.

Azole and 5-nitroimidazole based nanoformulations are potential antiamoebic drug candidates against brain eating amoebae.

Herein, the anti-parasitic activity of azoles (fluconazole and itraconazole) and 5-nitroimdazole (metronidazole) against brain eating amoebae: Naegleria fowleri and Balamuthia mandrillaris was elucidated. Azoles and 5-nitroimdazole based nanoformulations were synthesized and characterized using UV-visible spectrophotometer, atomic force microscopy and fourier transform infrared spectroscopy. H1-NMR, EI-MS and ESI-MS were performed to determine their molecular mass and elucidate their structures. Their size, zeta potential, size distribution and polydispersity index were assessed. Amoebicidal assays revealed that all the drugs and their nanoformulations, (except itraconazole) presented significant anti-amoebic effects against B. mandrillaris, while all the treatments indicated notable amoebicidal properties against N. fowleri. Amoebicidal effects were radically enhanced upon conjugating the drugs with nanoparticles. The IC50 values for KM-38-AgNPs-F, KM-20-AgNPs-M and KM-IF were 65.09µg.mL-1, 91.27µg.mL-1 and 72.19µg.mL-1 respectively against B. mandrillaris. Whereas against N. fowleri, IC50 values were: 71.85µg.mL-1, 73.95µg.mL-1, and 63.01µg.mL-1 respectively. Additionally, nanoformulations significantly reduced N. fowleri mediated host cell death, while nanoformulations along with fluconazole and metronidazole considerably reduced Balamuthia mediated human cell damage. Finally, all the tested drugs and their nanoformulations revealed limited cytotoxic activity against human cerebral microvascular endothelial cell (HBEC-5i) cells. These compounds should be developed into novel chemotherapeutic options for use against these distressing infections due to free-living amoebae, as currently there are no effective treatments.

Intranasal Route for the Delivery of Antiamebic Drugs Against brain-eating Amoeba

Tweetable abstract Nebulized emanator for intranasal delivery of antiamebic drugs to the brain.