Acanthamoeba spp. merupakan ameba hidup bebas yang tersebar luas di persekitaran. Ameba ini menyebabkan penyakit
keratitis terutamanya kepada individu yang memakai kanta sentuh terkontaminasi atau mempunyai sejarah trauma
pada mata. Tujuh puluh empat sampel kikisan kornea pesakit keratitis daripada 4 buah hospital di sekitar Lembah
Kelang telah dikultur untuk menemukan Acanthamoeba spp. Keputusan kajian mendapati Acanthamoeba spp. berjaya
dipencilkan daripada 14.87% sampel kikisan kornea pesakit keratitis. Berdasarkan kajian ini, kesemua pesakit keratitis
Acanthamoeba adalah merupakan pemakai kanta sentuh yang majoritinya (90.9%) terdiri daripada kaum wanita. Kanta
sentuh lembut terlibat dalam 90.9% kes manakala jenis kanta separa keras cuma satu kes 9.1%. Kesemua 11 pencilan
Acanthamoeba yang dipencilkan adalah daripada kumpulan polyphagids yang biasanya patogenik kepada manusia dan
menyebabkan jangkitan ini. Kegagalan pengguna kanta sentuh mengamalkan tahap kebersihan kanta sentuh yang baik
merupakan faktor risiko yang penting dalam kejadian keratitis Acanthamoeba di Malaysia.
Acanthamoeba is a free living protozoa that can cause keratitis and granulomatous amoebic encephalitis. Physiological characteristics of this amoeba are found to have a medical importance and related to the pathogenic potential of the organism. This study was carried out to investigate the physiological characteristic from the aspect of temperature tolerance. Six Acanthamoeba strains from three clinical isolates (HSB 1, HKL 48 and HKL 95) and three environmental isolates (PHS 2, PHS 11 and PHS 15) were used in this study. Test was done by culturing cysts at 30°C, 37°C and 42°C for two weeks and the ability of cysts to change to trophozoites were observed. The result showed all strain was able to change to trophozoites at 30°C and 37°C. However, no trophozoites were observed at 42°
C. This indicate that there is a similarity in the physiological trait of strains from both isolates are the same and strains from the environment are able to show the pathogenic potential thus capable of causing infection to human.
Keywords: Acanthamoeba; temperature tolerance; clinical; environmental isolates.
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.
Effective disinfection of contact lenses is of paramount importance in the prevention of Acanthamoeba (AK). Therefore, this study was undertaken to evaluate the effectiveness of the multi-purpose contact lens disinfecting solutions against Acanthamoeba cysts of four isolates comprising two clinical isolates; HS 5, HTH 73, and two environmental isolates; TTA 1 and TKA 14. Five multi-purpose contact lens disinfecting solutions; Complete®, Renu® fresh™, RevitaLens OcuTec®, Opti-Free® Express® and Solo Care Aqua® were tested on cyst of the four Acanthamoeba isolates. The soaking times were based on manufactures recommendations (4 and 6 h), 8 h and 24 h. Each mixture of the cysts and disinfecting solution was transferred onto non-nutrient agar seeded with Escherichia coli. The agar plates were examined under inverted microscope daily until day 14 to detect the presence of Acanthamoeba trophozoites. All 5 multi-purpose contact lens disinfecting solutions tested were ineffective at killing all Acanthamoeba cyst isolates tested. This indicates that most multi-purpose contact lens disinfecting solutions do not have anti-Acanthamoeba activity necessary to prevent keratitis amongst the contact lens wearers.
Key words: Acanthamoeba; effectiveness; Malaysia; multi-purpose contact lens disinfecting solution
The present study aims to identify the Acanthamoeba genotypes and their pathogenic potential in three recreational lakes in Malaysia. Thirty water samples were collected by purposive sampling between June and July 2022. Physical parameters of water quality were measured in situ while chemical and microbiological analyses were performed in the laboratory. The samples were vacuum filtered through nitrate filter, cultured onto non-nutrient agar and observed microscopically for amoebic growth. DNAs from positive samples were extracted and made to react with polymerase chain reaction using specific primers. Physiological tolerance tests were performed for all Acanthamoeba-positive samples. The presence of Acanthamoeba was found in 26 of 30 water samples by PCR. The highest rate in lake waters contaminated with amoeba was in Biru Lake (100%), followed by Titiwangsa Lake (80%) and Shah Alam Lake (80%). ORP, water temperature, pH and DO were found to be significantly correlated with the presence of Acanthamoeba. The most common genotype was T4. Temperature- and osmo-tolerance tests showed that 8 (30.8%) of the genotypes T4, T9 and T11 were highly pathogenic. The presence of genotype T4 in habitats related to human activities supports the relevance of this amoeba as a potential public health concern.
Acanthamoeba spp. can cause amoebic keratitis (AK). Chlorhexidine is effective for AK treatment as monotherapy, but with a relative failure on drug bioavailability in the deep corneal stroma. The combination of chlorhexidine and propamidine isethionate is recommended in the current AK treatment. However, the effectiveness of treatment depends on the parasite and virulence strains. This study aims to determine the potential of Garcinia mangostana pericarp extract and α-mangostin against Acanthamoeba triangularis, as well as the combination with chlorhexidine in the treatment of Acanthamoeba infection. The minimal inhibitory concentrations (MICs) of the extract and α-mangostin were assessed in trophozoites with 0.25 and 0.5 mg/mL, for cysts with 4 and 1 mg/mL, respectively. The MIC of the extract and α-mangostin inhibited the growth of A. triangularis trophozoites and cysts for up to 72 h. The extract and α-mangostin combined with chlorhexidine demonstrated good synergism, resulting in a reduction of 1/4-1/16 of the MIC. The SEM results showed that Acanthamoeba cells treated with a single drug and its combination caused damage to the cell membrane and irregular cell shapes. A good combination displayed by the extract or α-mangostin and chlorhexidine, described for the first time. Therefore, this approach is promising as an alternative method for the management of Acanthamoeba infection in the future.
Acanthamoeba is a free living protozoa that can cause keratitis and granulomatous amoebic encephalitis. Physiological characteristics of this amoeba are found to have a medical importance in which it can be related to the pathogenicity potential of the organism. This study was carried out to investigate the physiological characteristics of survivability during axenization. Six Acanthamoeba strains from three clinical isolates (HSB 1, HKL 48 and HKL 95) and three environmental isolates (PHS 2, PHS 11 and PHS 15) were used in this study. Axenization test was done by treating cysts with hydrochloric acid (3%) and Page saline containing Gentamicin (100 μg/ml). Cysts were then cultured into PYG enrich media, incubated at 30oC and the presence and proliferation of trophozoites of Acanthamoeba were observed. This study showed that PHS 15, HSB 1, HKL 48 and HKL 95 could be axenized but they have poor proliferation rate in PYG enrich media. The result showed that the difference between both clinical and environmental isolates was observed in two strains; PHS 2 and PHS 11. This indicates that there is a possibility that the physiological traits of strains from both isolates are the same and strains from the environment are able to show the pathogenic potential and capable of causing infection to human.
Keywords: Axenization, Survivability, Acanthamoeba, Clinical and environmental strains
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.
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.
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.
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.
This study aimed to identify the Acanthamoeba genotypes and their pathogenic potential in five recreational hot springs in Peninsular Malaysia. Fifty water samples were collected between April and September 2018. Physical parameters of water quality were measured in situ while chemical and microbiological analyses were performed in the laboratory. All samples were filtered through the nitrocellulose membrane and tested for Acanthamoeba using both cultivation and polymerase chain reaction (PCR) by targeting the 18S ribosomal RNA gene. The pathogenic potential of all positive isolates was identified using thermo- and osmotolerance tests. Thirty-eight (76.0%) samples were positive for Acanthamoeba. Water temperature (P = 0.035), chemical oxygen demand (P = 0.026), sulphate (P = 0.002) and Escherichia coli (P < 0.001) were found to be significantly correlated with the presence of Acanthamoeba. Phylogenetic analysis revealed that 24 samples belonged to genotype T4, nine (T15), two (T3) and one from each genotype T5, T11 and T17. Thermo- and osmotolerance tests showed that 6 (15.79%) of the Acanthamoeba strains were highly pathogenic. The existence of Acanthamoeba in recreational hot springs should be considered as a health threat among the public especially for high-risk people. Periodic surveillance of hot spring waters and posting warning signs by health authorities is recommended to prevent disease related to pathogenic Acanthamoeba.
Curcuma longa and Curcumin have been documented to have a wide spectrum of pharmacological effects, including anti-Acanthamoeba activity. Hence, this study sought to explore the anti-adhesion activity of C. longa extract and Curcumin against Acanthamoeba triangularis trophozoites and cysts in plastic and contact lenses. Our results showed that C. longa extract and Curcumin significantly inhibited the adhesion of A. triangularis trophozoites and cysts to the plastic surface, as investigated by the crystal violet assay (P
Acanthamoeba keratitis is a serious ocular problem and can cause blindness if not treated. This study was therefore performed to evaluate the effectiveness of eyedrop antibiotics on eight Acanthamoeba spp. isolates, of which four
were clinical isolates and the remaining four from the environment. Three different eyedrop antibiotics (neomycin, ciprofloxacin and gentamicin) currently available in the market and ready for use were tested. Cyst suspension from all strains were tested against eyedrop antibiotics, respectively. After 48 hours of incubation period, the solutions were filtered and the filtered membranes were put onto non-nutrient agar lawn with E. coli. The plates were examined daily for Acanthamoeba trophozoites under inverted microscope until day 14. Neomycin, ciprofloxacin and gentamicin were found
to be effective against Acanthamoeba spp. cysts for all test strains.
Key words: Acanthamoeba Keratitis, Eyedrop Antibiotics, Effectiveness
Keratitis Acanthamoeba merupakan sejenis inflamasi kornea yang dikaitkan dengan penggunaan kanta sentuh. la disebabkan oleh Acanthamoeba spp., ameba hidup bebas yang tersebar luas di pelbagai persekitaran manusia. Kontaminasi Acanthamoeba spp. pada bekas penyimpanan kanta sentuh merupakan faktor kehadiran ameba pada kanta seterusnya menjangkiti mata. Kajian ini bertujuan untuk melihat kehadiran Acanthamoeba spp. pada bekas penyimpanan kanta sentuh pengguna asimptomatik. Seramai 90 orang pengguna kanta sentuh asimptomatik terlibat dalam kajian ini. Sampel diambil secara swab pada bekas kanta sentuh dan dikulturkan ke atas agar bukan nutrien yang dilapisi Escherichia coli. Plat agar diperiksa setiap hart bagi mengesan kehadiran ameba. Kultur positif seterusnya disahkan di bawah 'Image Analysis with Video TesT 4.0'. Acanthamoeba spp. didapati positif pada lapan daripada 90 sampel (8. 7%) dan kesemua strain adalah kumpulan II (polyphagids). Penemuan ini membuktikan Acanthamoeba spp. boleh Nadir pada bekas penyimpanan kanta sentuh pengguna asimptomatik dan boleh menjadi risiko jangkitan keratitis Acanthamoeba.
Acanthamoeba spp. merupakan ameba hidup bebas yang biasa ditemui
di persekitaran. Ia merupakan agen penyebab keratitis Acanthamoeba (AK)
dan ensefalitis ameba bergranuloma (GAE). Ameba ini juga mampu menjadi
perumah kepada pelbagai bakteria termasuklah yang bersifat patogenik seperti
Mycobacterium, Legionella dan Staphylococcus aureus rintang metisilin (MRSA).
Berdasarkan maklumat ini, satu kajian dijalankan untuk mengesan kehadiran tiga
bakteria endosimbion berkepentingan perubatan di dalam Acanthamoeba spp. yang
telah dipencilkan dari bolong penghawa dingin yang terdapat di wad and dewan
bedah di Pusat Perubatan Universiti Kebangsaan Malaysia. Kehadiran bakteria
endosimbion ini disaring menggunakan pasangan primer khusus bagi setiap genus
menggunakan reaksi rantai polimerase (PCR) konvensional dan disahkan dengan
analisis penjujukan. Dua puluh sembilan (80.56%) pencilan Acanthamoeba spp.
didapati mengandungi bakteria endosimbion patogenik yang disasarkan dengan
sekurang-kurangnya satu genus bakteria bagi setiap pencilan. Mycobacterium
(82.76 %) adalah bakteria yang paling banyak dikesan, diikuti dengan Legionella sp.
(65.52 %) dan Pseudomonas spp. (62.07 %). Tiada bakteria MRSA dikesan daripada
mana-mana pencilan dalam kajian ini. Dua endosimbion Mycobacterium yang
dikenalpasti telah dikelompokkan ke dalam strain Mycobacterium tuberculosis.
Kami membuat kesimpulan bahawa, kebanyakan Acanthamoeba berpotensi untuk
menjadi perumah bagi pelbagai bakteria patogenik, namun implikasi interaksi ini
terhadap patogenisiti kedua-dua organisma masih kurang jelas dan memerlukan
penyelidikan yang lebih lanjut.
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.
Garcinia mangostana L., also known as the mangosteen tree, is a native medicinal plant in Southeast Asia having a wide variety of pharmacologically active compounds, including xanthonoid mangostin. In this study, we examined the pharmacological activities of the selected semi-synthetic mangostin derivative, namely, amoebicidal activity, encystation inhibition, excystation activity, and removal capacity of adhesive Acanthamoeba from the surface of contact lens (CL). Among the three derivatives, C1 exhibited promising anti-Acanthamoeba activity against Acanthamoeba triangularis WU19001 trophozoites and cysts. SEM images displayed morphological changes in Acanthamoeba trophozoites, including the loss of acanthopodia, pore formation in the cell membrane, and membrane damage. In addition, the treated cyst was shrunken and adopted an irregular flat cyst shape. Under a fluorescence microscope, acridine orange and propidium iodide (AO/PI) staining revealed C1 induced condensation of cytoplasm and chromatin with the loss of cell volume in the treated trophozoites, while calcofluor white staining demonstrated the leakage of cell wall in treated cysts, leading to cell death. Interestingly, at the concentration ranges in which C1 showed the anti-Acanthamoeba effects (IC50 values ranging from 0.035-0.056 mg/mL), they were not toxic to Vero cells. C1 displayed the highest inhibitory effect on A. triangularis encystation at 1/16×MIC value (0.004 mg/mL). While C1 demonstrated the excystation activity at 1/128×MIC value with a high rate of 89.47%. Furthermore, C1 exhibited the removal capacity of adhesive Acanthamoeba from the surface of CL comparable with commercial multipurpose solutions (MPSs). Based on the results obtained, C1 may be a promising lead agent to develop a therapeutic for the treatment of Acanthamoeba infections and disinfectant solutions for CL.
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.