Displaying publications 1 - 20 of 39 in total

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  1. Self-assembled micelles loaded with itraconazole as anti-Acanthamoeba nano-formulation
    Rao K, Abdullah M, Ahmed U, Wehelie HI, Shah MR, Siddiqui R, et al.
    Arch Microbiol, 2024 Mar 04;206(4):134.
    PMID: 38433145 DOI: 10.1007/s00203-024-03854-3
    Acanthamoeba castellanii are opportunistic pathogens known to cause infection of the central nervous system termed: granulomatous amoebic encephalitis, that mostly effects immunocompromised individuals, and a sight threatening keratitis, known as Acanthamoeba keratitis, which mostly affects contact lens wearers. The current treatment available is problematic, and is toxic. Herein, an amphiphilic star polymer with AB2 miktoarms [A = hydrophobic poly(ℇ-Caprolacton) and B = hydrophilic poly (ethylene glycol)] was synthesized by ring opening polymerization and CuI catalyzed azide-alkyne cycloaddition. Characterization by 1H and 13C NMR spectroscopy, size-exclusion chromatography and fluorescence spectroscopy was accomplished. The hydrophobic drug itraconazole (ITZ) was incorporated in self-assembled micellar structure of AB2 miktoarms through co-solvent evaporation. The properties of ITZ loaded (ITZ-PCL-PEG2) and blank micelles (PCL-PEG2) were investigated through zeta sizer, scanning electron microscopy and Fourier-transform infrared spectroscopy. Itraconazole alone (ITZ), polymer (DPB-PCL), empty polymeric micelles (PCL-PEG2) alone, and itraconazole loaded in polymeric micelles (ITZ-PCL-PEG2) were tested for anti-amoebic potential against Acanthamoeba, and the cytotoxicity on human cells were determined. The polymer was able to self-assemble in aqueous conditions and exhibited low value for critical micelle concentration (CMC) 0.05-0.06 µg/mL. The maximum entrapment efficiency of ITZ was 68%. Of note, ITZ, DPB, PCL-PEG2 and ITZ-PCL-PEG2 inhibited amoebae trophozoites by 37.34%, 36.30%, 35.77%, and 68.24%, respectively, as compared to controls. Moreover, ITZ-PCL-PEG2 revealed limited cytotoxicity against human keratinocyte cells. These results are indicative that ITZ-PCL-PEG2 micelle show significantly better anti-amoebic effects as compared to ITZ alone and thus should be investigated further in vivo to determine its clinical potential.
    Matched MeSH terms: Acanthamoeba castellanii*
  2. Apoptosis in Acanthamoeba castellanii belonging to the T4 genotype
    Baig AM, Lalani S, Khan NA
    J Basic Microbiol, 2017 Jul;57(7):574-579.
    PMID: 28466971 DOI: 10.1002/jobm.201700025
    Here we describe features of apoptosis in unicellular Acanthamoeba castellanii belonging to the T4 genotype. When exposed to apoptosis-inducing compounds such as doxorubicin, A. castellanii trophozoites exhibited cell shrinkage and membrane blebbing as observed microscopically, DNA fragmentation using agarose gel electrophoresis, and phosphatidylserine (PS) externalization using annexin V immunostaining. Overall, these findings suggest the existence of apoptosis in A. castellanii possibly mediated by intrinsic apoptotic cascade. Further research in this field could provide avenues to selectively induce apoptosis in A. castellanii by triggering intrinsic apoptotic cascade.
    Matched MeSH terms: Acanthamoeba castellanii/cytology*; Acanthamoeba castellanii/drug effects; Acanthamoeba castellanii/genetics; Acanthamoeba castellanii/physiology*
  3. Fulltext Camellia sinensis solvent extract, epigallocatechin gallate and caffeine confer trophocidal and cysticidal effects against Acanthamoeba castellanii
    Fakae LB, Harun MSR, Ting DSJ, Dua HS, Cave GWV, Zhu XQ, et al.
    Acta Trop, 2023 Jan;237:106729.
    PMID: 36280206 DOI: 10.1016/j.actatropica.2022.106729
    We examined the anti-acanthamoebic efficacy of green tea Camellia sinensis solvent extract (SE) or its chemical constituents against Acanthamoeba castellanii by using anti-trophozoite, anti-encystation, and anti-excystation assays. C. sinensis SE (625-5000 µg/mL) inhibited trophozoite replication within 24-72 h. C. sinensis SE exhibited a dose-dependent inhibition of encystation, with a marked cysticidal activity at 2500-5000 µg/mL. Two constituents of C. sinensis, namely epigallocatechin-3-gallate and caffeine, at 100 μM and 200 μM respectively, significantly inhibited both trophozoite replication and encystation. Cytotoxicity analysis showed that 156.25-2500 µg/mL of SE was not toxic to human corneal epithelial cells, while up to 625 µg/mL was not toxic to Madin-Darby canine kidney cells. This study shows the anti-acanthamoebic potential of C. sinensis SE against A. castellanii trophozoites and cysts. Pre-clinical studies are required to elucidate the in vivo efficacy and safety of C. sinensis SE.
    Matched MeSH terms: Acanthamoeba castellanii*
  4. Targeting Acanthamoeba proteins interaction with flavonoids of Propolis extract by in vitro and in silico studies for promising therapeutic effects
    Sama-Ae I, Sangkanu S, Siyadatpanah A, Norouzi R, Chuprom J, Mitsuwan W, et al.
    F1000Res, 2022;11:1274.
    PMID: 36936052 DOI: 10.12688/f1000research.126227.1
    Background : Propolis is a natural resinous mixture produced by bees. It provides beneficial effects on human health in the treatment/management of many diseases. The present study was performed to demonstrate the anti- Acanthamoeba activity of ethanolic extracts of Propolis samples from Iran. The interactions of the compounds and essential proteins of Acanthamoeba were also visualized through docking simulation. Methods: The minimal inhibitory concentrations (MICs) of Propolis extract against Acanthamoeba trophozoites and cysts was determined in vitro. In addition, two-fold dilutions of each of the agents were tested for encystment, excystment and adhesion inhibitions. Three major compounds of Propolis extract such as chrysin, tectochrysin and pinocembrin have been selected in molecular docking approach to predict the compounds that might be responsible for encystment, excystment and adhesion inhibitions of A. castellanii. Furthermore, to confirm the docking results, molecular dynamics (MD) simulations were also carried out for the most promising two ligand-pocket complexes from docking studies. Results : The minimal inhibitory concentrations (MICs) 62.5 and 125 µg/mL of the most active Propolis extract were assessed in trophozoites stage of Acanthamoeba castellanii ATCC30010 and ATCC50739, respectively. At concentrations lower than their MICs values (1/16 MIC), Propolis extract revealed inhibition of encystation. However, at 1/2 MIC, it showed a potential inhibition of excystation and anti-adhesion. The molecular docking and dynamic simulation revealed the potential capability of Pinocembrin to form hydrogen bonds with A. castellanii Sir2 family protein (AcSir2), an encystation protein of high relevance for this process in Acanthamoeba. Conclusions : The results obtained provided a candidate for the development of therapeutic drugs against Acanthamoeba infection. In vivo experiments and clinical trials are necessary to support this claim.
    Matched MeSH terms: Acanthamoeba castellanii*
  5. Malabaricones from the fruit of Myristica cinnamomea King as potential agents against Acanthamoeba castellanii
    Ahmed U, Sivasothy Y, Khan KM, Khan NA, Wahab SMA, Awang K, et al.
    Acta Trop, 2023 Dec;248:107033.
    PMID: 37783284 DOI: 10.1016/j.actatropica.2023.107033
    Acanthamoeba castellanii is an opportunistic free-living amoeba (FLA) pathogen which can cause fatal central nervous system (CNS) infection, granulomatous amoebic encephalitis (GAE) and potentially blinding ocular infection, Acanthamoeba keratitis (AK). Acanthamoeba species remain a challenging protist to treat due to the unavailability of safe and effective therapeutic drugs and their ability to protect themselves in the cyst stage. Natural products and their secondary metabolites play a pivotal role in drug discovery against various pathogenic microorganisms. In the present study, the ethyl acetate extract of Myristica cinnamomea King fruit was evaluated against A. castellanii (ATCC 50492), showing an IC50 of 45.102 ± 4.62 µg/mL. Previously, the bio-guided fractionation of the extract resulted in the identification of three active compounds, namely Malabaricones (A-C). The isolated and thoroughly characterized acylphenols were evaluated for their anti-amoebic activity against A. castellanii for the first time. Among tested compounds, Malabaricone B (IC50 of 101.31 ± 17.41 µM) and Malabaricone C (IC50 of 49.95 ± 6.33 µM) showed potent anti-amoebic activity against A. castellanii trophozoites and reduced their viability up-to 75 and 80 %, respectively. Moreover, both extract and Malabaricones also significantly (p < 0.05) inhibit the encystation and excystation of A. castellanii, while showed minimal toxicity against human keratinocyte cells (HaCaT cells) at lower tested concentrations. Following that, the explanation of the possible mechanism of action of purified compounds were assessed by detection of the state of chromatin. Hoechst/PI 33342 double staining showed that necrotic cell death occurred in A. castellanii trophozoites after 8 h treatment of Malabaricones (A-C). These findings demonstrate that Malabaricones B and C could serve as promising therapeutic options against A. castellanii infections.
    Matched MeSH terms: Acanthamoeba castellanii*
  6. Acanthamoeba castellanii interactions with Streptococcus pneumoniae and Streptococcus pyogenes
    Siddiqui R, Yee Ong TY, Jung SY, Khan NA
    Exp Parasitol, 2017 Dec;183:128-132.
    PMID: 28823705 DOI: 10.1016/j.exppara.2017.08.005
    Among the genus Streptococcus, S. pyogenes and S. pneumoniae are the major causes of pharyngitis, impetigo, pneumonia and meningitis in humans. Streptococcus spp. are facultative anaerobes that are nutritionally fastidious, yet survive in the environment and target the predisposed population. Antibacterial disinfectants have been partially effective only, indicating the need for novel preventative measures and to understand mechanisms of bacterial resistance. Acanthamoeba is a free-living protist that is known to harbour microbial pathogens, provide shelter, and assist in their transmission to susceptible population. The overall aim of this study was to determine whether S. pyogenes and S. pneumoniae can interact with A. castellanii by associating, invading, and surviving inside trophozoites and cysts. It was observed that both S. pyogenes and S. pneumoniae were able to associate as well as invade and/or taken up by the phagocytic A. castellanii trophozoite. Notably, S. pyogenes and S. pneumoniae survived the encystation process, avoided phagocytosis, multiplied, and exhibited higher recovery from the mature cysts, compared with the trophozoite stage (approximately 2 bacteria per amoebae ratio for cyst stage versus 0.02 bacteria per amoeba ration for trophozoite stage). As Acanthamoeba cysts are resilient and can disperse through the air, A. castellanii can act as a vector in providing shelter, facilitating growth and possibly genetic exchanges. In addition, these interactions may contribute to S. pyogenes and S. pneumoniae survival in harsh environments, and transmission to susceptible population and possibly affecting their virulence. Future studies will determine the molecular mechanisms associated with Acanthamoeba interactions with Streptococcus and the evolution of pathogenic bacteria and in turn expedite the discovery of novel therapeutic and/or preventative measures.
    Matched MeSH terms: Acanthamoeba castellanii/growth & development; Acanthamoeba castellanii/microbiology*; Acanthamoeba castellanii/physiology*
  7. Fulltext A transmission electron microscopy study on effects of a modified Glutaraldehyde fixation on Acanthamoeba castellanii
    Santhana Raj L, Teh Hamidah Z, Nor Asiha CP, Paramasvaran S
    Trop Biomed, 2006 Jun;23(1):69-74.
    PMID: 17041554 MyJurnal
    Transmission electron microscopy (TEM) can provide high resolution imaging of biological specimens. The study is to establish the effects of a modified glutaraldehyde (GA) compare to the standard GA fixation on Acanthamoeba castellanii from TEM perspectives and thus provide precise and accurate information on the ultrastructure studies of the parasite. By increasing the contrast, the ultrastructures of the parasite were more evident. The TEM images were obtained from parasites fixed with the modified GA and the standard GA and then the area of the nucleus and the gray values of the image of the nucleus of the parasites were measured. The mean areas of the nucleus were found to be significantly reduced in the standard GA fixed parasites (12210.4 nm2) compared to the modified GA fixed parasites (8676.3 nm2) (p < 0.05). The mean gray values of the image were significantly reduced from 2024 in the standard GA fixed parasites (2024) to the modified GA fixed parasites (1636) (p < 0.05). The study shows that the modified GA produced significantly better contrast on TEM images of the A. castellanii compared to the standard GA. This was because the modified GA generated more free water molecules during fixation and the uptake of modified GA by the nucleus of the parasite organizing all protein constituents in the cell into a more closely packed configuration than that of the standard GA. With such properties, the modified GA is a better fixative providing better images for ultrastructures of the parasite.
    Matched MeSH terms: Acanthamoeba castellanii/ultrastructure*
  8. Lactase can target cellular differentiation of Acanthamoeba castellanii belonging to the T4 genotype
    Simau FA, Ahmed U, Khan KM, Khan NA, Siddiqui R, Alharbi AM, et al.
    Parasitol Res, 2024 Jan 31;123(2):117.
    PMID: 38294565 DOI: 10.1007/s00436-024-08131-2
    The free living Acanthamoeba spp. are ubiquitous amoebae associated with potentially blinding disease known as Acanthamoeba keratitis (AK) and a fatal central nervous system infection granulomatous amoebic encephalitis (GAE). With the inherent ability of cellular differentiation, it can phenotypically transform to a dormant cyst form from an active trophozoite form. Acanthamoeba cysts are highly resistant to therapeutic agents as well as contact lens cleaning solutions. One way to tackle drug resistance against Acanthamoeba is by inhibiting the formation of cysts from trophozoites. The biochemical analysis showed that the major component of Acanthamoeba cyst wall is composed of carbohydrate moieties such as galactose and glucose. The disaccharide of galactose and glucose is lactose. In this study, we analyzed the potential of lactase enzyme to target carbohydrate moieties of cyst walls. Amoebicidal assessment showed that lactase was ineffective against trophozoite of A. castellanii but enhanced amoebicidal effects of chlorhexidine. The lactase enzyme did not show any toxicity against normal human keratinocyte cells (HaCaT) at the tested range. Hence, lactase can be used for further assessment for development of potential therapeutic agents in the management of Acanthamoeba infection as well as formulation of effective contact lens disinfectants.
    Matched MeSH terms: Acanthamoeba castellanii*
  9. Anti-amoebic properties of a Malaysian marine sponge Aaptos sp. on Acanthamoeba castellanii
    Nakisah MA, Ida Muryany MY, Fatimah H, Nor Fadilah R, Zalilawati MR, Khamsah S, et al.
    World J Microbiol Biotechnol, 2012 Mar;28(3):1237-44.
    PMID: 22805843 DOI: 10.1007/s11274-011-0927-8
    Crude methanol extracts of a marine sponge, Aaptos aaptos, collected from three different localities namely Kapas, Perhentian and Redang Islands, Terengganu, Malaysia, were tested in vitro on a pathogenic Acanthamoeba castellanii (IMR isolate) to examine their anti-amoebic potential. The examination of anti-Acanthamoebic activity of the extracts was conducted in 24 well plates for 72 h at 30 °C. All extracts possessed anti-amoebic activity with their IC(50) values ranging from 0.615 to 0.876 mg/mL. The effect of the methanol extracts on the surface morphology of A. castellanii was analysed under scanning electron microscopy. The ability of the extracts to disrupt the amoeba cell membrane was indicated by extensive cell's blebbing, changes in the surface morphology, reduced in cell size and with cystic appearance of extract-treated Acanthamoeba. Number of acanthapodia and food cup was also reduced in this Acanthamoeba. Morphological criteria of apoptosis in Acanthamoeba following treatment with the sponge's extracts was determined by acridine orange-propidium iodide staining and observed by fluorescence microscopy. By this technique, apoptotic and necrotic cells can be visualized and quantified. The genotoxic potential of the methanol extracts was performed by the alkaline comet assay. All methanol extracts used were significantly induced DNA damage compared to untreated Acanthamoeba by having high percentage of scores 1, 2, and 3 of the DNA damage. Results from cytotoxicity and genotoxicity studies carried out in the present study suggest that all methanol extracts of A. aaptos have anti-amoebic properties against A. castellanii.
    Matched MeSH terms: Acanthamoeba castellanii/drug effects*; Acanthamoeba castellanii/growth & development*; Acanthamoeba castellanii/ultrastructure
  10. Fulltext Acanthamoeba genotype T4 detected in naturally-infected feline corneas found to be in homology with those causing human keratitis
    Ithoi I, Mahmud R, Abdul Basher MH, Jali A, Abdulsalam AM, Ibrahim J, et al.
    Trop Biomed, 2013 Mar;30(1):131-40.
    PMID: 23665719 MyJurnal
    A total of 10 out of 65 cornea swab samples from cats with eye symptoms showed Acanthamoeba-like morphology after cultivation. By PCR and DNA sequencing of Acanthamoeba diagnostic fragment 3 (DF3), all 10 isolates from the positive samples were categorized into two homologous groups of AfC1 (PM1, PM2, PM3, PF6, KM7, KF8, KMK9) and AfC2 (PM4, PM5, KFK10) due to the presence of bases A(354) and G(354), respectively. Furthermore, DF3 of AfC1 and AfC2 showed 100% similarity with Genbank reference isolates with the accession numbers DQ087314, EU146073 and U07401, GU808323, which were Acanthamoeba castellanii strains genotype T4 originating from human keratitis. This finding suggests that A. castellani strains have the capability to infect cats and human under favorable conditions.
    Matched MeSH terms: Acanthamoeba castellanii/classification*; Acanthamoeba castellanii/genetics*; Acanthamoeba castellanii/isolation & purification
  11. Antiamoebic activity of synthetic tetrazoles against Acanthamoeba castellanii belonging to T4 genotype and effects of conjugation with silver nanoparticles
    Anwar A, Yi YP, Fatima I, Khan KM, Siddiqui R, Khan NA, et al.
    Parasitol Res, 2020 Jun;119(6):1943-1954.
    PMID: 32385711 DOI: 10.1007/s00436-020-06694-4
    Acanthamoeba causes diseases such as Acanthamoeba keratitis (AK) which leads to permanent blindness and granulomatous Acanthamoeba encephalitis (GAE) where there is formation of granulomas in the brain. Current treatments such as chlorhexidine, diamidines, and azoles either exhibit undesirable side effects or require immediate and prolonged treatment for the drug to be effective or prevent relapse. Previously, antifungal drugs amphotericin B, nystatin, and fluconazole-conjugated silver with nanoparticles have shown significantly increased activity against Acanthamoeba castellanii. In this study, two functionally diverse tetrazoles were synthesized, namely 5-(3-4-dimethoxyphenyl)-1H-tetrazole and 1-(3-methoxyphenyl)-5-phenoxy-1H-tetrazole, denoted by T1 and T2 respectively. These compounds were evaluated for anti-Acanthamoeba effects at different concentrations ranging from 5 to 50 μM. Furthermore, these compounds were conjugated with silver nanoparticles (AgNPs) to enhance their efficacy. Particle size analysis showed that T1-AgNPs and T2-AgNPs had an average size of 52 and 70 nm respectively. After the successful synthesis and characterization of tetrazoles and tetrazole-conjugated AgNPs, they were subjected to anti-Acanthamoeba studies. Amoebicidal assay showed that at concentration 10 μM and above, T2 showed promising antiamoebic activities between the two compounds while encystation and excystation assays reveal that both T1 and T2 have inhibited differentiation activity against Acanthamoeba castellanii. Conjugation of T1 and T2 to AgNP also increased efficacy of tetrazoles as anti-Acanthamoeba agents. This may be due to the increased bioavailability as AgNP allows better delivery of treatment compounds to A. castellanii. Human cell cytotoxicity assay revealed that tetrazoles and AgNPs are significantly less toxic towards human cells compared with chlorhexidine which is known to cause undesirable side effects. Cytopathogenicity assay also revealed that T2 conjugated with AgNPs significantly reduced cytopathogenicity of A. castellanii compared with T2 alone, suggesting that T2-conjugated AgNP is an effective and safe anti-Acanthamoeba agent. The use of a synthetic azole compound conjugated with AgNPs can be an alternative strategy for drug development against A. castellanii. However, mechanistic and in vivo studies are needed to explore further translational values.
    Matched MeSH terms: Acanthamoeba castellanii/drug effects*; Acanthamoeba castellanii/genetics; Acanthamoeba castellanii/isolation & purification
  12. Strategies to counter transmission of "superbugs" by targeting free-living amoebae
    Siddiqui R, Khan NA
    Exp Parasitol, 2017 Dec;183:133-136.
    PMID: 28807757 DOI: 10.1016/j.exppara.2017.08.006
    Bacterial infections have remained significant despite our advances in the development of a plethora of disinfectants as well as antimicrobial chemotherapy. This is in part due to our incomplete understanding of the prevalence of bacterial pathogens in the environmental and clinical settings. Several lines of evidence suggest that Acanthamoeba is one of the most ubiquitous/resilient protists that also acts as a host/reservoir for pathogenic microbes. Thus targeting the hardy host, which harbour microbial pathogens, offer a potential avenue to counter infection transmission, particularly hospital/community-acquired infections. This will complement existing approach of applying disinfectants that are targeted against bacterial pathogens directly.
    Matched MeSH terms: Acanthamoeba castellanii/microbiology*; Acanthamoeba castellanii/ultrastructure
  13. Amoebicidal activity of Cassia angustifolia extract and its effect on Acanthamoeba triangularis autophagy-related gene expression at the transcriptional level
    Boonhok R, Sangkanu S, Norouzi R, Siyadatpanah A, Mirzaei F, Mitsuwan W, et al.
    Parasitology, 2021 Aug;148(9):1074-1082.
    PMID: 33966667 DOI: 10.1017/S0031182021000718
    Cassia angustifolia Vahl. plant is used for many therapeutic purposes, for example, in people with constipation, skin diseases, including helminthic and parasitic infections. In our study, we demonstrated an amoebicidal activity of C. angustifolia extract against Acanthamoeba triangularis trophozoite at a micromolar level. Scanning electron microscopy (SEM) images displayed morphological changes in the Acanthamoeba trophozoite, which included the formation of pores in cell membrane and the membrane rupture. In addition to the amoebicidal activity, effects of the extract on surviving trophozoites were observed, which included cyst formation and vacuolization by a microscope and transcriptional expression of Acanthamoeba autophagy in response to the stress by quantitative polymerase chain reaction. Our data showed that the surviving trophozoites were not transformed into cysts and the trophozoite number with enlarged vacuole was not significantly different from that of untreated control. Molecular analysis data demonstrated that the mRNA expression of AcATG genes was slightly changed. Interestingly, AcATG16 decreased significantly at 12 h post treatment, which may indicate a transcriptional regulation by the extract or a balance of intracellular signalling pathways in response to the stress, whereas AcATG3 and AcATG8b remained unchanged. Altogether, these data reveal the anti-Acanthamoeba activity of C. angustifolia extract and the autophagic response in the surviving trophozoites under the plant extract pressure, along with data on the formation of cysts. These represent a promising plant for future drug development. However, further isolation and purification of an active compound and cytotoxicity against human cells are needed, including a study on the autophagic response at the protein level.
    Matched MeSH terms: Acanthamoeba castellanii/drug effects*; Acanthamoeba castellanii/genetics
  14. Anaerobic respiration: In vitro efficacy of Nitazoxanide against mitochondriate Acanthamoeba castellanii of the T4 genotype
    Aqeel Y, Siddiqui R, Farooq M, Khan NA
    Exp Parasitol, 2015 Oct;157:170-6.
    PMID: 26297676 DOI: 10.1016/j.exppara.2015.08.007
    Acanthamoeba is an opportunistic protist pathogen that is responsible for serious human and animal infection. Being one of the most frequently isolated protists from the environment, it is likely that it readily encounters microaerophilic environments. For respiration under anaerobic or low oxygen conditions in several amitochondriate protists, decarboxylation of pyruvate is catalyzed by pyruvate ferredoxin oxidoreductase instead of pyruvate dehydrogenase. In support, Nitazoxanide, an inhibitor of pyruvate ferredoxin oxidoreductase, is effective and non-mutagenic clinically against a range of amitochondriate protists, Giardia intestinalis, Entamoeba histolytica and Trichomonas vaginalis. The overall aim of the present study was to determine in vitro efficacy of Nitazoxanide against Acanthamoeba castellanii. At micromolar concentrations, the findings revealed that Nitazoxanide neither affected A. castellanii growth or viability nor amoeba-mediated host cell monolayer damage in vitro or extracellular proteolytic activities. Similarly, microaerophilic conditions alone had no significant effects. In contrast, microaerophilic conditions together with Nitazoxanide showed amoebicidal effects and inhibited A. castellanii-mediated host cell monolayer damage as well as extracellular proteases. Using encystation assays, it was observed that Nitazoxanide inhibited trophozoite transformation into cysts both under aerophilic and microaerophilic conditions. Furthermore, pre-treatment of cysts with Nitazoxanide inhibited A. castellanii excystation. These findings are important in the identification of potential targets that could be useful against parasite-specific respiration as well as to understand the basic biology of the life cycle of Acanthamoeba.
    Matched MeSH terms: Acanthamoeba castellanii/classification; Acanthamoeba castellanii/drug effects*; Acanthamoeba castellanii/genetics; Acanthamoeba castellanii/physiology
  15. Clinically Approved Drugs against CNS Diseases as Potential Therapeutic Agents To Target Brain-Eating Amoebae
    Anwar A, Rajendran K, Siddiqui R, Raza Shah M, Khan NA
    ACS Chem Neurosci, 2019 01 16;10(1):658-666.
    PMID: 30346711 DOI: 10.1021/acschemneuro.8b00484
    Central nervous system (CNS) infections caused by free-living amoebae such as Acanthamoeba species and Naegleria fowleri are rare but fatal. A major challenge in the treatment against the infections caused by these amoebae is the discovery of novel compounds that can effectively cross the blood-brain barrier to penetrate the CNS. It is logical to test clinically approved drugs against CNS diseases for their potential antiamoebic effects since they are known for effective blood-brain barrier penetration and affect eukaryotic cell targets. The antiamoebic effects of clinically available drugs for seizures targeting gamma-amino butyric acid (GABA) receptor and ion channels were tested against Acanthamoeba castellanii belonging to the T4 genotype and N. fowleri. Three such drugs, namely, diazepam (Valium), phenobarbitone (Luminal), phenytoin (Dilantin), and their silver nanoparticles (AgNPs) were evaluated against both trophozoites and cysts stage. Drugs alone and drug conjugated silver nanoparticles were tested for amoebicidal, cysticidal, and host-cell cytotoxicity assays. Nanoparticles were synthesized by sodium borohydride reduction of silver nitrate with drugs as capping agents. Drug conjugated nanoconjugates were characterized by ultraviolet-visible (UV-vis) and Fourier transform infrared (FT-IR) spectroscopies and atomic force microscopy (AFM). In vitro moebicidal assay showed potent amoebicidal effects for diazepam, phenobarbitone, and phenytoin-conjugated AgNPs as compared to drugs alone against A. castellanii and N. fowleri. Furthermore, both drugs and drug conjugated AgNPs showed compelling cysticidal effects. Drugs conjugations with silver nanoparticles enhanced their antiacanthamoebic activity. Interestingly, amoeba-mediated host-cell cytotoxicity was also significantly reduced by drugs alone as well as their nanoconjugates. Since, these drugs are being used to target CNS diseases, their evaluation against brain-eating amoebae seems feasible due to advantages such as permeability of the blood-brain barrier, established pharmacokinetics and dynamics, and United States Food and Drug Administration (FDA) approval. Given the limited availability of effective drugs against brain-eating amoebae, the clinically available drugs tested here present potential for further in vivo studies.
    Matched MeSH terms: Acanthamoeba castellanii/drug effects; Acanthamoeba castellanii/parasitology*
  16. Antiamoebic activity of 3-aryl-6,7-dimethoxyquinazolin-4(3H)-one library against Acanthamoeba castellanii
    Shahbaz MS, Anwar A, Saad SM, Kanwal, Anwar A, Khan KM, et al.
    Parasitol Res, 2020 Jul;119(7):2327-2335.
    PMID: 32476058 DOI: 10.1007/s00436-020-06710-7
    Acanthamoeba castellanii is a free-living amoeba which can cause a blinding keratitis and fatal granulomatous amoebic encephalitis. The treatment of Acanthamoeba infections is challenging due to formation of cyst. Quinazolinones are medicinally important scaffold against parasitic diseases. A library of nineteen new 3-aryl-6,7-dimethoxyquinazolin-4(3H)-one derivatives was synthesized to evaluate their antiamoebic activity against Acanthamoeba castellanii. One-pot synthesis of 3-aryl-6,7-dimethoxyquinazolin-4(3H)-ones (1-19) was achieved by reaction of 2-amino-4,5-dimethoxybenzoic acid, trimethoxymethane, and different substituted anilines. These compounds were purified and characterized by standard chromatographic and spectroscopic techniques. Antiacanthamoebic activity of these compounds was determined by amoebicidal, encystation, excystation and host cell cytopathogenicity in vitro assays at concentrations of 50 and 100 μg/mL. The IC50 was found to be between 100 and 50 μg/mL for all the compounds except compound 5 which did not exhibit amoebicidal effects at these concentrations. Furthermore, lactate dehydrogenase assay was also performed to evaluate the in vitro cytotoxicity of these compounds against human keratinocyte (HaCaT) cells. The results revealed that eighteen out of nineteen derivatives of quinazolinones significantly decreased the viability of A. castellanii. Furthermore, eighteen out of nineteen tested compounds inhibited the encystation and excystation, as well as significantly reduced the A. castellanii-mediated cytopathogenicity against human cells. Interestingly, while tested against human normal cell line HaCaT keratinocytes, all compounds did not exhibit any overt cytotoxicity. Furthermore, a detailed structure-activity relationship is also studied to optimize the most potent hit from these synthetic compounds. This report presents several potential lead compounds belonging to 3-aryl-6,7-dimethoxyquinazolin-4(3H)-one derivatives for drug discovery against infections caused by Acanthamoeba castellanii.
    Matched MeSH terms: Acanthamoeba castellanii/drug effects*; Acanthamoeba castellanii/growth & development
  17. Silver nanoparticle conjugation affects antiacanthamoebic activities of amphotericin B, nystatin, and fluconazole
    Anwar A, Siddiqui R, Hussain MA, Ahmed D, Shah MR, Khan NA
    Parasitol Res, 2018 Jan;117(1):265-271.
    PMID: 29218442 DOI: 10.1007/s00436-017-5701-x
    Infectious diseases are the leading cause of morbidity and mortality, killing more than 15 million people worldwide. This is despite our advances in antimicrobial chemotherapy and supportive care. Nanoparticles offer a promising technology to enhance drug efficacy and formation of effective vehicles for drug delivery. Here, we conjugated amphotericin B, nystatin (macrocyclic polyenes), and fluconazole (azole) with silver nanoparticles. Silver-conjugated drugs were synthesized successfully and characterized by ultraviolet-visible spectrophotometry, Fourier transform infrared spectroscopy, and atomic force microscopy. Conjugated and unconjugated drugs were tested against Acanthamoeba castellanii belonging to the T4 genotype using amoebicidal assay and host cell cytotoxicity assay. Viability assays revealed that silver nanoparticles conjugated with amphotericin B (Amp-AgNPs) and nystatin (Nys-AgNPs) exhibited significant antiamoebic properties compared with drugs alone or AgNPs alone (P 
    Matched MeSH terms: Acanthamoeba castellanii/drug effects; Acanthamoeba castellanii/genetics
  18. 'Targeting the feast of a sleeping beast': Nutrient and mineral dependencies of encysted Acanthamoeba castellanii
    Baig AM, Khan NA, Katyara P, Lalani S, Baig R, Nadeem M, et al.
    Chem Biol Drug Des, 2021 01;97(1):18-27.
    PMID: 32602961 DOI: 10.1111/cbdd.13755
    Acanthamoeba spp. cause a corneal infection, Acanthamoeba keratitis (AK), and a cerebral infection, granulomatous amoebic encephalitis (GAE). Though aggressive chemotherapy has been able to kill the active trophozoite form of Acanthamoeba, the encysted form of this parasite has remained problematic to resist physiological concentrations of drugs. The emergence of encysted amoeba into active trophozoite form poses a challenge to eradicate this parasite. Acanthamoeba trophozoites have active metabolic machinery that furnishes energy in the form of ATPs by subjecting carbohydrates and lipids to undergo pathways including glycolysis and beta-oxidation of free fatty acids, respectively. However, very little is known about the metabolic preferences and dependencies of an encysted trophozoite on minerals or potential nutrients that it consumes to live in an encysted state. Here, we investigate the metabolic and nutrient preferences of the encysted trophozoite of Acanthamoeba castellanii and the possibility to target them by drugs that act on calcium ion dependencies of the encysted amoeba. The experimental assays, immunostaining coupled with bioinformatics tools show that the encysted Acanthamoeba uses diverse nutrient pathways to obtain energy in the quiescent encysted state. These findings highlight potential pathways that can be targeted in eradicating amoebae cysts successfully.
    Matched MeSH terms: Acanthamoeba castellanii/drug effects; Acanthamoeba castellanii/growth & development; Acanthamoeba castellanii/metabolism*
  19. Fulltext Cobalt nanoparticles as novel nanotherapeutics against Acanthamoeba castellanii
    Anwar A, Numan A, Siddiqui R, Khalid M, Khan NA
    Parasit Vectors, 2019 Jun 03;12(1):280.
    PMID: 31159839 DOI: 10.1186/s13071-019-3528-2
    BACKGROUND: Species of Acanthamoeba are facultative pathogens which can cause sight threatening Acanthamoeba keratitis and a rare but deadly brain infection, granulomatous amoebic encephalitis. Due to conversion of Acanthamoeba trophozoites to resistant cyst stage, most drugs are found to be ineffective at preventing recurrence of infection. This study was designed to test the antiacanthamoebic effects of different cobalt nanoparticles (CoNPs) against trophozoites and cysts, as well as parasite-mediated host cell cytotoxicity.

    METHODS: Three different varieties of CoNPs were synthesized by utilizing hydrothermal and ultrasonication methods and were thoroughly characterized by X-ray diffraction and field emission scanning electron microscopy. Amoebicidal, encystation, excystation, and host cell cytopathogenicity assays were conducted to study the antiacanthamoebic effects of CoNPs.

    RESULTS: The results of the antimicrobial evaluation revealed that cobalt phosphate Co3(PO4)2 hexagonal microflakes, and 100 nm large cobalt hydroxide (Co(OH)2) nanoflakes showed potent amoebicidal activity at 100 and 10 µg/ml against Acanthamoeba castellanii as compared to granular cobalt oxide (Co3O4) of size 35-40 nm. Furthermore, encystation and excystation assays also showed consistent inhibition at 100 µg/ml. CoNPs also inhibited amoebae-mediated host cell cytotoxicity as determined by lactate dehydrogenase release without causing significant damage to human cells when treated alone.

    CONCLUSIONS: To our knowledge, these findings determined, for the first time, the effects of composition, size and morphology of CoNPs against A. castellanii. Co3(PO4)2 hexagonal microflakes showed the most promising antiamoebic effects as compared to Co(OH)2 nanoflakes and granular Co3O4. The results reported in the present study hold potential for the development of antiamoebic nanomedicine.

    Matched MeSH terms: Acanthamoeba castellanii/drug effects*
  20. Novel antiacanthamoebic compounds belonging to quinazolinones
    Anwar A, Shahbaz MS, Saad SM, Kanwal, Khan KM, Siddiqui R, et al.
    Eur J Med Chem, 2019 Nov 15;182:111575.
    PMID: 31415900 DOI: 10.1016/j.ejmech.2019.111575
    We report one-pot synthesis of a series of new 3-aryl-8-methylquinazolin-4(3H)-ones (QNZ) and their antimicrobial activity against Acanthamoeba castellanii belonging to T4 genotype. A library of fifteen synthetic derivatives of QNZs was synthesized, and their structural elucidation was performed by using nuclear magnetic resonance (NMR) spectroscopy and electron impact mass spectrometry (EI-MS). Elemental analyses and high-resolution mass spectrometry data of all derivatives were found to be in agreeable range. Amoebicidal assays performed at concentrations ranging from 50 to 100 μg/mL revealed that all derivatives of QNZ significantly decreased the viability of A. castellanii and QNZ 2, 5, 8, and 13 were found to have efficient antiamoebic effects. Field emission scanning electron microscopy (FESEM) imaging of amoeba treated with compounds 5 and 15 showed that these compounds cause structural alterations on the walls of A. castellanii. Furthermore, several QNZs inhibited the encystation and excystationas as well as abolished A. castellanii-mediated host cells cytopathogenicity in human cells. Whereas, these QNZs showed negligible cytotoxicity when tested against human cells in vitro. Hence, this study identified potential lead molecules having promising properties for drug development against A. castellanii. A brief structure-activity relationship is also developed to optimize the hit of most potent compounds from the library. To the best of our knowledge, it is first of its kind medicinal chemistry approach on a single class of compounds i.e., quinazolinone against keratitis and brain infection causing free-living amoeba, A. castellanii.
    Matched MeSH terms: Acanthamoeba castellanii/drug effects*
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