Naegleria fowleri and Balamuthia mandrillaris are free-living, opportunistic protists, distributed widely in the environment. They are responsible for primary amoebic meningoencephalitis (PAM) and granulomatous amoebic encephalitis (GAE), the fatal central nervous infections with mortality rates exceeding 90%. With the rise of global warming and water shortages resulting in water storage in tanks (where these amoebae may reside), the risk of infection is increasing. Currently, as a result of a lack of awareness, many cases may be misdiagnosed. Furthermore, the high mortality rate indicates the lack of effective drugs available. In this study, secondary metabolites from the plants Rinorea vaundensis and Salvia triloba were tested for their anti-amoebic properties against N. fowleri and B. mandrillaris. Three of the nine compounds showed potent and significant anti-amoebic activities against both N. fowleri and B. mandrillaris: ursolic acid, betulinic acid, and betulin. Additionally, all compounds depicted limited or minimal toxicity to human cells and were capable of reducing amoeba-mediated host cell death. Moreover, the minimum inhibitory concentration required to inhibit 50% of amoebae growth, the half-maximal effective concentration, and the maximum non-toxic dose against human cells of the compounds were determined. These effective plant-derived compounds should be utilized as potential therapies against infections due to free-living amoebae, but future research is needed to realize these expectations.

The free-living amoeba Acanthamoeba castellanii is responsible for the central nervous infection granulomatous amoebic encephalitis and sight-threatening infection Acanthamoeba keratitis. Moreover, no effective treatment is currently present, and a combination drug therapy is used. In this study, twelve DNA minor groove binders (MGBs) were synthesized and tested for their antiamoebic activity via amoebicidal, encystation, excystation, and cytopathogenicity assays. It was found that the compounds MGB3, MGB6, MGB22, MGB24, and MGB16 significantly reduce amoeba viability to 76.20%, 59.45%, 66.5%, 39.32%, and 43.21%, respectively, in amoebicidal assays. Moreover, the compounds MGB6, MGB20, MGB22, MGB28, MGB30, MGB32, and MGB16 significantly inhibit Acanthamoeba cysts, leading to the development of only 46.3%, 39%, 30.3%, 29.6%, 27.8%, 41.5%, and 45.6% cysts. Additionally, the compounds MGB3, MGB4, MGB6, MGB22, MGB24, MGB28, MGB32, and MGB16 significantly reduce the re-emergence of cysts to trophozoites, with viable trophozoites being only 64.3%, 47.3%, 41.4%, 52.9%, 55.4%, 40.6%, 62.1%, and 51.7%. Moreover, the compounds MGB3, MGB4, and MGB6 exhibited the greatest reduction in amoeba-mediated host-cell death, with cell death reduced to 41.5%, 49.4%, and 49.5%. With the following determined, future in vivo studies can be carried out to understand the effect of the compounds on animal models such as mice.

We examined the antiamoebic effect of several imidazothiazole derivatives on Acanthamoeba castellanii of the T4 genotype. Trypan blue exclusion assays and haemocytometer counting were used to determine the reduction in A. castellanii trophozoite proliferation, in response to treatment with these compounds. To determine the effects of these compounds on host cells, lactate dehydrogenase assay was performed using HeLa cell lines. Amoebicidal assays revealed that the tested compounds at concentrations of 50 µM significantly inhibited amoebae trophozoites compared to controls. Compounds 1m and 1zb showed the highest amoebicidal effects eradicating 70% and 67% of A. castellanii, respectively. The compounds blocked both the encystation and excystation process in A. castellanii. Compounds 1m and 1zb blocked 61% and 55%, respectively, of amoeba binding to human cells. Moreover, the compounds showed minimal cytotoxic effects against host cells and considerably reduced amoeba-mediated host cell death. Overall, our study revealed that compounds 1m and 1zb have excellent antiamoebic potential, and should be considered in the development of curative antiamoebic medications in future studies. Further work is critical to determine the translational value of these findings.

Naegleria fowleri (N. fowleri) is a free-living, unicellular, opportunistic protist responsible for the fatal central nervous system infection, primary amoebic meningoencephalitis (PAM). Given the increase in temperatures due to global warming and climate change, it is estimated that the cases of PAM are on the rise. However, there is a current lack of awareness and effective drugs, meaning there is an urgent need to develop new therapeutic drugs. In this study, the target compounds were synthesized and tested for their anti-amoebic properties against N. fowleri. Most compounds exhibited significant amoebicidal effects against N. fowleri; for example, 1h, 1j, and 1q reduced N. fowleri's viability to 15.14%, 17.45% and 28.78%, respectively. Furthermore, the majority of the compounds showed reductions in amoeba-mediated host death. Of interest are the compounds 1f, 1k, and 1v, as they were capable of reducing the amoeba-mediated host cell death to 52.3%, 51%, and 56.9% from 100%, respectively. Additionally, these compounds exhibit amoebicidal properties as well; they were found to decrease N. fowleri's viability to 26.41%, 27.39%, and 24.13% from 100%, respectively. Moreover, the MIC50 values for 1e, 1f, and 1h were determined to be 48.45 µM, 60.87 µM, and 50.96 µM, respectively. Additionally, the majority of compounds were found to exhibit limited cytotoxicity, except for 1l, 1o, 1p, 1m, 1c, 1b, 1zb, 1z, 1y, and 1x, which exhibited negligible toxicity. It is anticipated that these compounds may be developed further as effective treatments against these devastating infections due to brain-eating amoebae.

The pathogenic free-living amoeba, Acanthamoeba castellanii, is responsible for a rare but deadly central nervous system infection, granulomatous amoebic encephalitis and a blinding eye disease called Acanthamoeba keratitis. Currently, a combination of biguanides, amidine, azoles and antibiotics are used to manage these infections; however, the host cell cytotoxicity of these drugs remains a challenge. Furthermore, Acanthamoeba species are capable of transforming to the cyst form to resist chemotherapy. Herein, we have developed a nano drug delivery system based on iron oxide nanoparticles conjugated with isoniazid, which were further loaded with amphotericin B (ISO-NPs-AMP) to cause potent antiamoebic effects against Acanthamoeba castellanii. The IC50 of isoniazid conjugated with magnetic nanoparticles and loaded with amphotericin B was found to be 45 μg/mL against Acanthamoeba castellanii trophozoites and 50 μg/mL against cysts. The results obtained in this study have promising implications in drug discovery as these nanomaterials exhibited high trophicidal and cysticidal effects, as well as limited cytotoxicity against rat and human cells.

Acanthamoeba castellanii is a ubiquitous free-living amoeba capable of instigating keratitis and granulomatous amoebic encephalitis in humans. Treatment remains limited and inconsistent. Accordingly, there is a pressing need for novel compounds. Nanotechnology has been gaining attention for enhancing drug delivery and reducing toxicity. Previous work has shown that various antibiotic classes displayed antiamoebic activity. Herein, we employed two antibiotics: ampicillin and ceftriaxone, conjugated with the nanocarrier zinc oxide and β-cyclodextrin, and tested them against A. castellanii via amoebicidal, amoebistatic, encystment, excystment, cytopathogenicity, and cytotoxicity assays at a concentration of 100 μg/mL. Notably, zinc oxide β-cyclodextrin ceftriaxone significantly inhibited A. castellanii growth and cytopathogenicity. Additionally, both zinc oxide β-cyclodextrin ceftriaxone and ceftriaxone markedly inhibited A. castellanii encystment. Furthermore, all the tested compounds displayed negligible cytotoxicity. However, minimal anti-excystment or amoebicidal effects were observed for the compounds. Accordingly, this novel nanoconjugation should be employed in further studies in hope of discovering novel anti-Acanthamoeba compounds.

Due to global warming, coupled with global water shortages and the reliance of the public on household water tanks, especially in developing countries, it is anticipated that infections caused by free-living amoebae such as Acanthamoeba will rise. Thus, the development of novel disinfectant(s) which can target pathogenic free-living amoebae effectively is warranted. Herein, we extracted and isolated several plant-based secondary metabolites as novel disinfectants for use against pathogenic Acanthamoeba. The identity of the compounds was confirmed by nuclear magnetic resonance and tested for antiamoebic activities against clinical isolate of A. castellanii, belonging to the T4 genotype. Amoebicidal assays revealed that the compounds tested showed antiamoebic properties. Betulinic acid and betulin exhibited parasite killing of more than 65%. When tested against the cyst stage, betulinic acid, betulin, and vanillic acid inhibited both encystation and excystation processes. Furthermore, the plant-based metabolites significantly inhibited the binding capability of A. castellanii to host cells. Finally, most of the tested compounds displayed minimal cytotoxic activities against human cells and noticeably perturbed amoeba-mediated host cell cytotoxicity. Notably, both alkaloid and betulinic acid showed 20% cytotoxic effects, whereas betulin and lupeol had cytotoxic effects of 24% and 30%, respectively. Overall, our findings indicate that plant-based natural compounds demonstrate anti-Acanthamoebic properties, and they have potential candidates for water disinfectants or contact lens disinfecting solutions, as well as possible therapeutic drugs against Acanthamoeba infections.

Naegleria fowleri and Balamuthia mandrillaris are opportunistic protists, responsible for fatal central nervous system infections such as primary amoebic meningoencephalitis (PAM) and granulomatous amoebic encephalitis (GAE) with mortality rates higher than 90%. Threatening a rise in cases is the increase in temperature due to global warming. No effective treatment is currently available. Herein, nanotechnology was used to conjugate Zinc oxide with Ampicillin, Ceftrixon, Naringin, Amphotericin B, and Quericitin, and the amoebicidal activity and host cell cytotoxicity of these resulting compounds were investigated. The compounds ZnO-CD-AMPi, ZnO-CD-CFT, ZnO-CD-Nar, ZnO-CD-AMB, and ZnO-CD-QT were found to reduce N. fowleri viability to 35.5%, 39.6%, 52.0%, 50.8%, 35.9%, and 69.9%, respectively, and B. mandrillaris viability to 40.9%, 48.2%, 51.6%, 43.8%, and 62.4%, respectively, when compared with their corresponding controls. Furthermore, the compounds reduced N. fowleri-mediated and B. mandrillaris-mediated host cell death significantly. Additionally, the compounds showed limited cytotoxicity against human cells; cell toxicity was 35.5%, 36.4%, 30.9%, 36.6%, and 35.6%, respectively, for the compounds ZnO-CD-AMPi, ZnO-CD-CFT, ZnO-CD-Nar, ZnO-CD-AMB, and ZnO-CD-QT. Furthermore, the minimum inhibitory concentrations to inhibit amoeba growth by 50% were determined for N. fowleri and B. mandrillaris. The MIC50 for N. fowleri were determined to be 69.52 µg/mL, 82.05 µg/mL, 88.16 µg/mL, 95.61 µg/mL, and 85.69 µg/mL, respectively; the MIC50 of the compounds for B. mandrillaris were determined to be 113.9 µg/mL, 102.3 µg/mL, 106.9 µg/mL, 146.4 µg/mL, and 129.6 µg/mL, respectively. Translational research to further develop therapies based on these compounds is urgently warranted, given the lack of effective therapies currently available against these devastating infections.