Despite the amount of awareness created, waterborne disease still poses threat, especially in developing countries. Due to the scarcity of reported data on waterborne parasites, the consumption of unsafe water prolongs. Thus, the occurrences of waterborne parasites from various samples were investigated from one of the Southeast Asian country, the Philippines.

Background and Aim: Acanthamoeba, an opportunistic protozoan, exists widely in natural sources and can cause infections in humans and animals. The absence of effective monotherapy after the initial infection leads to chronic disease and recurrence. Tubulin protein is a vital target for design-targeted drug discovery. Anti-tubulin drugs are also used to treat Acanthamoeba infection, although resistance to these drugs has been observed. Therefore, it is necessary to identify a new targeted drug for Acanthamoeba infection. Therefore, this study aimed to assess the in vitro activity of ethanol extracts of Rhizophora apiculata extracts (RAE) against Acanthamoeba spp. and to predict its chemical compound on β-tubulin interaction. Materials and Methods: Acanthamoeba, an opportunistic protozoan, exists widely in natural sources and can cause infections in humans and animals. The absence of effective monotherapy after the initial infection leads to chronic disease and recurrence. Tubulin protein is a vital target for design-targeted drug discovery. Anti-tubulin drugs are also used to treat Acanthamoeba infection, although resistance to these drugs has been observed. Therefore, it is necessary to identify a new targeted drug for Acanthamoeba infection. Therefore, this study aimed to assess the in vitro activity of ethanol extracts of Rhizophora apiculata extracts (RAE) against Acanthamoeba spp. and to predict its chemical compound on β-tubulin interaction. Results: The results revealed that A. triangularis and A. polyphaga trophozoites had the highest inhibition at 90% at a MIC of 8 mg/mL after treatment with RALE and RABE, respectively, at 24 h. Those MPC values were exhibited at 16 mg/mL against A. triangularis trophozoites. In addition, both extracts inhibited the adhesive properties of all Acanthamoeba approximately 80%–90% at 4 mg/ mL, as well as adherent structural acanthopodia loss. MCC was 0.25 mg/mL, provided to be harmless to mammalian cells. GC-MS analysis supported that 8 and 11 major phytochemicals were from RABE and RALE, respectively. Molecular docking and molecular dynamics demonstrated that Acanthamoeba-β-tubulin exhibited potent root-mean-square deviation, root mean square fluctuation, and binding free energy values with clionasterol (from RABE and RALE) and stigmasterol (from RALE). Based on our results, ethanolic RABE and RALE exhibited anti-Acanthamoeba activity in reducing adhesion. In silico showed that promising clionasterol and stigmasterol interacted with a targeting β-tubulin. Conclusion: The RABE and RALE exhibited a potential adherent effect on A. triangularis, low toxicity, and the clionasterol and stigmasterol in RABE and RALE predicted to interact the targeted β-tubulin. These agents may be used as alternative therapeutic agents in the management of disease using a sustainable one-heath approach. Keywords: Acanthamoeba triangularis, anti-adherent activity, molecular docking, molecular dynamic simulation, Rhizophora, tubulin.

Background and Aim: Keratitis is a serious ocular infection often caused by pathogenic micro-organisms such as Acanthamoeba spp. Among other harmful microbes, Acanthamoeba keratitis presents a particular challenge due to its resistance to conventional antimicrobial agents. Piper betle Linn., commonly known as betel leaf, has been traditionally used for its medicinal properties. This study aimed to assess the potential of the leaf ethanol extract of P. betle Linn. in the treatment of Acanthamoeba triangularis in monoculture and co-culture with two prevalent pathogenic bacteria, Staphylococcus aureus and Pseudomonas aeruginosa, associated with keratitis. Materials and Methods: Minimum inhibitory concentrations (MICs) of A. triangularis, S. aureus, and P. aeruginosa extracts in monoculture and coinfected conditions were examined. In addition, this study explored the potential of the extract in preventing Acanthamoeba adherence in both monoculture and co-culture environments. Scanning electron microscopy (SEM) analysis confirmed the impact of the extract on Acanthamoeba cell membranes, including acanthopodia. Furthermore, a time-kill kinetic assay was used to validate the amoebicidal activity of the extract against A. triangularis and the tested bacteria. Results: MICs for trophozoites, cysts, P. aeruginosa, and S. aureus in the monoculture were 0.25, 0.25, 0.51, and 0.128 mg/mL, respectively, whereas the MICs for Acanthamoeba coinfected with bacteria were higher than those in the monoculture. This extract inhibited the growth of A. triangularis trophozoites and cysts for up to 72 h. Moreover, P. betle extract effectively prevented the adherence of Acanthamoeba to contact lenses under monoculture conditions. SEM analysis confirmed that P. betle extract affects the cell membrane of Acanthamoeba, including Acanthopodia. In addition, the time-kill kinetic assay confirmed that the extract contained amoebicidal activity against A. triangularis, including the tested bacteria. Notably, S. aureus was more susceptible than A. triangularis and P. aeruginosa to P. betle extract treatment. Unexpectedly, our study revealed that S. aureus negatively affected A. triangularis in the co-culture after 3 days of incubation, whereas P. aeruginosa facilitated the growth of A. triangularis in the presence of the extract. Conclusion: This study provides compelling evidence of the anti-adhesive and anti-Acanthamoeba properties of P. betle leaf extract against A. triangularis under monoculture and co-culture conditions. The observed impact on Acanthamoeba cell membranes, coupled with the time-kill kinetic assay results, underscores the potential of P. betle leaf extract as a promising agent for combating Acanthamoeba-related infections in humans and animals. Keywords: Piper betle extract, Acanthamoeba triangularis, co-infection, keratitis, Pseudomonas aeruginosa, Staphylococcus aureus.

Plasmodium knowlesi infections can progress rapidly to life-threatening complications, such as acute respiratory distress syndrome (ARDS) and acute kidney injury (AKI), which are driven by the ability of the zoonotic parasite to rapidly replicate towards high parasitemia and the cytoadherence properties of the infected erythrocytes (IRBCs). This study evaluated the anti-parasitic and vasoprotective potential of Annona muricata (soursop) leaf ethanol extract against P. knowlesi. In vitro assays using the P. knowlesi A1-H.1 reference strain revealed significant blood stage schizogony inhibition (IC₅₀: 9.65 µg/mL), specifically targeting the trophozoites. The anti-parasitic activity was concentrated in the <30 kDa subfraction of the extract. Washout assays confirmed that the effect was parasitostatic rather than parasiticidal, where the malaria parasites underwent developmental arrest but remained morphologically normal and resumed growth post-removal. Furthermore, priming human endothelial cell lines with the extract significantly reduced IRBC–endothelial binding. These results demonstrate that A. muricata extract exerts a dual-action effect by arresting P. knowlesi asexual replication and inhibiting IRBC–endothelial cytoadherence. While clinical translation would require exhaustive standardization and chemotypic profiling due to the natural variability of plant compositions, these findings provide a foundational academic framework for the potential of A. muricata leaf extract in mitigating severe knowlesi malaria complications.

Acanthamoeba spp., free‐living amoebae (FLA), are the causative agents of Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE). These diseases have limited treatment options; therefore, drug discovery remains a critical priority. This study evaluated the leaf extract of Annona muricata L., which contains quercetin, formulated in silica nanoparticles against Acanthamoeba species. The anti‐ Acanthamoeba effect of the nanoformulation was determined by its MIC and percentage viability. Scanning electron microscopy confirmed the nanocompound‐directed effect on trophozoites and cysts. The nanoquercetin exhibited anti‐ Acanthamoeba activity, showing greater effectiveness against the trophozoites of Acanthamoeba polyphaga ATCC 30461 (AP) (MIC > 2 mg/mL) compared to cysts (MIC > 8 mg/mL) and against A. triangularis WU19001 (AT) trophozoites (MIC > 4 mg/mL) compared to cysts (MIC > 8 mg/mL). It also showed reduced activity against Acanthamoeba castellanii ATCC 30010 (AC3) trophozoites (MIC > 4 mg/mL) compared to cysts (MIC > 8 mg/mL) and A. castellanii ATCC50739 (AC5) trophozoites (MIC > 4 mg/mL) compared to cysts (MIC > 8 mg/mL). The anti‐ Acanthamoeba activity of A. muricata on all four species was compared to chlorhexidine, which had MIC values of 0.008 mg/mL for AC3 and AT trophozoites and 0.016 mg/mL for AC5 and AP trophozoites. For cysts, chlorhexidine exhibited MIC values of > 0.0128 mg/mL for AC3 and AP, 0.032 mg/mL for AC5, and 0.064 mg/mL for AT. All MIC values demonstrated inhibition of Acanthamoeba species with reduced viability exceeding 90%. SEM findings revealed morphological changes in Acanthamoeba , indicating parasitic inhibition. Quercetin‐loaded silica nanoparticles demonstrated anti‐ Acanthamoeba activity with low cytotoxicity. This indicates the potential of quercetin as a nano‐enhanced natural therapeutic or contact lens‐care agent. This study is limited by its in vitro design, small parasite strain panel, and potential nanoparticle variability. This highlights the need for in vivo validation, ocular/contact lens model testing, and formulation optimization for improved cysticidal efficacy.

Background and Aim: Acanthamoeba spp. is free-living protozoa capable of causing severe infections, notably Acanthamoeba keratitis, which is difficult to manage due to cyst resistance and the cytotoxicity of current treatments. Plant-derived com­pounds represent a promising alternative strategy. This study investigated the amoebicidal, anti-adhesive, and cytotoxic properties of Mangifera indica L. (mango) leaf extract against ocularly relevant Acanthamoeba spp. Materials and Methods: Crude ethanolic leaf extract of M. indica was prepared and evaluated against Acanthamoeba polyphaga American Type Culture Collection (ATCC) 30461 and Acanthamoeba castellanii ATCC 50739. Minimum inhibitory concentration (MIC) and minimum parasiticidal concentration were determined for trophozoites and cysts. Morphological changes were analyzed by scanning electron microscopy (SEM). Anti-adhesion assays were conducted using polystyrene surfaces, with a commercial multipurpose contact lens (CL) solution as a control. Cytotoxicity was tested in Vero cells using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide assay to establish the minimum cytotoxic concentration. Results: The extract inhibited trophozoite growth at 2 mg/mL and demonstrated cysticidal activity at 4 mg/mL for A. polyph­aga and 32 mg/mL for A. castellanii. SEM revealed disruption of trophozoite morphology, loss of acanthopodia, and sur­face perforations in cysts. At MIC levels, adhesion was reduced by >70%, and even at 1/8 MIC, inhibition remained above 50%, comparable to a commercial multipurpose solution. Cytotoxicity assessment showed >80% Vero cell viability at 0.125 mg/mL, indicating a favorable therapeutic window. Conclusion: This is the first report demonstrating amoebicidal and anti-adhesive effects of M. indica L. leaf extract against ocular Acanthamoeba species. The dual trophozoiticidal and anti-adhesive actions, combined with low cytotoxicity, high­light its potential for development as a plant-based therapeutic agent, particularly in ocular formulations or CL disinfectants. Future work should focus on phytochemical isolation, mechanistic studies, and novel delivery systems to enhance efficacy and safety. Keywords: Acanthamoeba, amoebicidal activity, anti-adhesion, cytotoxicity, Mangifera indica L., ocular infection.