A novel montmorillonite clay-cetylpyridinium chloride complex as a potential antiamoebic composite material in contact lenses disinfection.

BACKGROUND: Acanthamoeba keratitis is a painful, sight-threatening infection. It is commonly associated with the use of contact lens. Several lines of evidence suggest inadequate contact lens solutions especially against the cyst forms of pathogenic Acanthamoeba, indicating the need to develop effective disinfectants. OBJECTIVE: In this work, the application and assessment of montmorillonite clay (Mt-clay), cetylpyridinium chloride (CPC) and cetylpyridinium chloride-montmorillonite clay complex (CPC-Mt) against keratitis-causing A. castellanii belonging to the T4 genotype was studied. METHODS: Adhesion to human cells and amoeba-mediated cytopathogenicity assays were conducted to determine the impact of Mt-clay, CPC and CPC-Mt complex on amoeba-mediated binding and host cell death. Furthermore, assays were also performed to determine inhibitory effects of Mt-clay, CPC and CPC-Mt complex on encystment and excystment. In addition, the cytotoxicity of Mt-clay, CPC and CPC-Mt complex against human cells was examined. RESULTS: The results revealed that CPC and CPC-Mt complex presented significant antiamoebic effects against A. castellanii at microgram dose. Also, the CPC and CPC-Mt complex inhibited amoebae binding to host cells. Furthermore, CPC and CPC-Mt complex, were found to inhibit the encystment and excystment processes. Finally, CPC and CPC-Mt complex showed minimal host cell cytotoxicity. These results show that CPC and CPC-Mt complex exhibit potent anti-acanthamoebic properties. CONCLUSION: Given the ease of usage, safety, cost-effectiveness and long-term stability, CPC and CPC-Mt complex can prove to be an excellent choice in the rational development of contact-lens disinfectants to eradicate pathogenic Acanthamoeba effectively.

Antiamoebic properties of salicylic acid-based deep eutectic solvents for the development of contact lens disinfecting solutions against Acanthamoeba.

Acanthamoeba castellanii is a protist pathogen that can cause sight-threatening keratitis and a fatal infection of the central nervous system, known as granulomatous amoebic encephalitis. In this study, effects of five malonic acid and salicylic acid-based deep eutectic solvents (DES) on A. castellanii were investigated. These are salicylic acid-trioctylphosphine (DES 1), salicylic acid- trihexylamine (DES 2), salicylic acid-trioctylamine (DES 3), malonic acid-trioctylphosphine (DES 4) and malonic acid-trihexylamine (DES 5). The experiments were done by performing amoebicidal, encystment, excystment, cytopathogenicity, and cytotoxicity assays. At micromolar dosage, the solvents DES 2 and DES 3 displayed significant amoebicidal effects (P < 0.05), inhibited encystment and excystment, undermined the cell-mediated cytopathogenicity of A. castellanii, and also displayed minimal cytotoxicity to human cells. Conversely, the chemical components of these solvents: salicylic acid, trihexylamine, and trioctylamine showed minimal effects when tested individually. These results are very promising and to the best of our knowledge, are reported for the first time on the effects of deep eutectic solvents on amoebae. These results can be applied in the development of new formulations of novel contact lens disinfectants against Acanthamoeba castellanii.

Cationic Surfactant-Natural Clay Complex as a Novel Agent Against Acanthamoeba castellanii Belonging to the T4 Genotype.

BACKGROUND: Acanthamoeba is a protozoan pathogen that is widely distributed in the environment. Given the opportunity, it can cause a serious eye infection known as Acanthamoeba keratitis as well as a fatal brain infection known as granulomatous amoebic encephalitis. Inappropriate use of contact lenses can contribute to contracting Acanthamoeba keratitis, and contact lens disinfectants are not always effective in eradicating Acanthamoeba. Therefore, there is a need to develop novel antimicrobial agents with efficient antiamoebic properties. OBJECTIVE: In this study, we tested octadecyltrimethylammonium (ODTMA)-clay (montmorillonite) complex as a novel antiamoebic agent. METHODS: Using A. castellanii belonging to the T4 genotype of keratitis origin, amobicidal assays were performed to determine the effects of ODTMA-cay complex on the viability of parasites at various concentrations ranging from 10 to 100 µg. Adhesion and cytopathogenicity assays were performed to investigate ODTMA effects on A. castellanii-mediated binding and damage to human cells. Encystation and excystation assays were conducted to establish ODTMA-mediated inhibitory effects against the cyst stage of A. castellanii. RESULTS: Using cell survival assays, the results revealed that ODTMA-clay complex exhibited amobicidal activity against keratitis-causing A. castellanii in a dose-dependent manner. Pretreatment of A. castellanii with ODTMA-clay complex inhibited parasite adhesion to as well as parasite-mediated human cell damage. Using encystation and excystation assays, it was revealed that ODTMA-clay complex inhibited A. castellanii cysts at 100 µg (P<0.05). CONCLUSION: To the best of our knowledge, for the first time, it was shown that ODTMA-clay complex exhibited anti-Acanthamoebic activities. The possibility of adding ODTMA-clay in a contact lens cleaning solution to formulate effective disinfectants is discussed further.

Tannic acid-modified silver nanoparticles enhance the anti-Acanthamoeba activity of three multipurpose contact lens solutions without increasing their cytotoxicity.

BACKGROUND: Free-living amoebae of the genus Acanthamoeba are cosmopolitan, widely distributed protozoans that cause a severe, vision-threatening corneal infection known as Acanthamoeba keratitis (AK). The majority of the increasing number of AK cases are associated with contact lens use. Appropriate eye hygiene and effective contact lens disinfection are crucial in the prevention of AK because of the lack of effective therapies against it. Currently available multipurpose contact lens disinfection systems are not fully effective against Acanthamoeba trophozoites and cysts. There is an urgent need to increase the disinfecting activity of these systems to prevent AK infections. Synthesized nanoparticles (NPs) have been recently studied and proposed as a new generation of anti-microbial agents. It is also known that some plant metabolites, including tannins, have anti-parasitic activity. The aim of this study was to evaluate the anti-amoebic activity and cytotoxicity of tannic acid-modified silver NPs (AgTANPs) conjugated with selected multipurpose contact lens solutions. METHODS: The anti-amoebic activities of pure contact lens care solutions, and NPs conjugated with contact lens care solutions, were examined in vitro by a colorimetric assay based on the oxido-reduction of alamarBlue. The cytotoxicity assays were performed using a fibroblast HS-5 (ATCC CRL-11882) cell line. The results were statistically analysed by ANOVA and Student-Newman-Keuls test using P < 0.05 as the level of statistical significance. RESULTS: We show that the NPs enhance the anti-Acanthamoeba activities of the tested contact lens solutions without increasing their cytotoxicity profiles. The activities are enhanced within the minimal disinfection time recommended by the manufacturers. CONCLUSIONS: The conjugation of the selected contact lens solutions with AgTANPs might be a novel and promising approach for the prevention of AK infections among contact lens users.

Anti-Acanthamoeba disinfection: hands, surfaces and wounds.

Acanthamoebae are facultative parasites causing rare but serious infections such as keratitis and encephalitis and are also known as vectors for several bacterial pathogens, including legionellae and pseudomonads. Acanthamoeba cysts are particularly resilient and enable the amoebae to withstand desiccation and to resist disinfection and therapy. While the search for new therapeutic options has been intensified in the past years, hand and surface disinfectants as well as topical antiseptics for preventing infections have not been studied in detail to date. The aim of this study was to screen well-known and commonly used antimicrobial products in various formulations and different concentrations for their efficacy against Acanthamoeba trophozoites and cysts, including aliphatic alcohols, quaternary ammonium compounds (QACs), peracetic acid (PAA), potassium peroxymonosulfate sulfate (PPMS) and octenidine dihydrochloride (OCT). Of all products tested, OCT and QACs showed the highest efficacy, totally eradicating both trophozoites and cysts within 1 min. The determined 50% effective concentration (EC50) for cysts was 0.196 mg/mL for OCT and 0.119 mg/mL for QACs after 1 min of exposure. PAA and PPMS showed reliable cysticidal efficacies only with prolonged incubation times of 30 min and 60 min, respectively. Aliphatic alcohols generally had limited efficacy, and only against trophozoites. In conclusion, OCT and QACs are potent actives against Acanthamoeba trophozoites and cysts at concentrations used in commercially available products, within contact times suitable for surface and hand disinfection as well as topical antisepsis.

Evaluation of in vitro effect of selected contact lens solutions conjugated with nanoparticles in terms of preventive approach to public health risk generated by Acanthamoeba strains.

INTRODUCTION: Various Acanthamoeba species are free-living organisms widely distributed in the human environment. Amphizoic amoebae as facultative parasites may cause vision-threatening eye disease - Acanthamoeba keratitis, mostly among contact lens wearers. As the number of cases is increasing, and applied therapy often unsuccessful, proper hygienic measures and effective contact lenses disinfection are crucial for the prevention of this disease. Available contact lens solutions are not fully effective against amphizoic amoebae; there is a need to enhance their disinfecting activity to prevent amoebic infections. The use of developing nanotechnology methods already applied with success in the prevention, diagnostic and therapy of other infectious diseases might be helpful regarding amoebic keratitis. This study assesses the in vitro effect of selected contact lens solutions conjugated with nanoparticles against Acanthamoeba trophozoites. MATERIAL AND METHODS: Three selected contact lens solutions conjugated with silver and gold nanoparticles in concentration of 0.25-2.5 ppm were used in vitro against the axenically cultured ATCC 30010 type Acanthamoeba castellanii strain. The anti-amoebic efficacy was examined based on the oxido-reduction of AlamarBlue. The cytotoxicity tests based on the measurement of lactate dehydrogenase (LDH) activity were performed using a fibroblast HS-5 cell line. RESULTS: Enhancement of the anti-amoebic activity of contact lens solutions conjugated with selected nanoparticles expressed in the dose dependent amoebic growth inhibition with a low cytotoxicity profile was observed. CONCLUSIONS: Results of the study showed that conjugation of selected contact lens solutions with silver nanoparticles might be a promising approach to prevent Acanthamoeba keratitis among contact lens users.

Chloroquine as a possible disinfection adjunct of disinfection solutions against Acanthamoeba.

Acanthamoeba keratitis is commonly encountered by contact lens wearers. Contact lens solution plays an important role in the safe use of contact lenses. The most popular products for disinfecting lenses are multipurpose disinfecting solutions (MPDS). However, almost all MPDS retailed in Korea are ineffective in killing Acanthamoeba. The objective of this study was to determine the possibility of using autophagy inhibitor chloroquine as a disinfecting agent to improve the amoebicidal activity of MPDS against Acanthamoeba, especially the cyst. Amoebicidal effects of eight different MPDSs combined with chloroquine (CQ), an autophagy inhibitor, and their cytotoxicities to human corneal epithelium cells were determined. Almost all MPDS showed strong amoebicidal effect on trophozoites after 8 h of exposure. However, they showed inadequate amoebicidal effect on cysts even after 24 h of exposure. MPDSs combined with 100 µM CQ increased their amoebicidal effects on immature cyst by inhibiting formation of mature cysts. Incubation with 100 µM CQ for 30 min did not have cytotoxicity to human corneal epithelial cells.

Optimized Protocol for Testing Multipurpose Contact Lens Solution Efficacy Against Acanthamoeba.

OBJECTIVES: To evaluate the interlaboratory and intralaboratory reproducibility of a proposed protocol for multipurpose contact lens solution (MPS) disinfection efficacy against Acanthamoeba. METHODS: Acanthamoeba castellanii and Acanthamoeba polyphaga and four MPS with different biocidal agents were used to evaluate the protocol in two different laboratories. In addition to the negative control, a positive control and neutralization control were used. One experiment was performed in triplicate, and all other experiments were performed in duplicate in each laboratory. Acanthamoeba trophozoites were grown axenically, and cysts were generated using the starvation method. Trophozoites and cysts at a concentration of 2.0 × 10 to 2.0 × 10 organisms per milliliter were exposed to the test MPS for 0, 4 or 6 (manufacturer's recommended soak time [MRST]), 8, and 24 hr. Survivors were determined by a limiting dilution method that used a most probable number evaluation. RESULTS: The positive and negative controls displayed consistent results and trends both within each laboratory and between each laboratory for trophozoites and cysts of both A. castellanii and A. polyphaga. The neutralization control consistently demonstrated the ability of the neutralizing agents to neutralize the MPS and the positive control and demonstrated no inhibition of Acanthamoeba by the negative control. Testing in triplicate and duplicate demonstrated the reproducibility of the protocol both within each laboratory and between the laboratories. Our results demonstrated that the MPS at the MRST and at 8 hr (likely overnight soak time) are generally more effective against trophozoites than they are against cysts. Only the MPS with hydrogen peroxide as the biocidal agent was able to provide a greater than three-log kill of cysts at the MRST and longer. Among the MPS we tested, trophozoites of A. castellanii and A. polyphaga showed similar responses. Some variability was observed when testing cysts of both species. In both laboratories, one nonhydrogen peroxide containing MPS had some effect (>1 log kill) on A. polyphaga cysts. This solution had no effect (<1 log kill) on A. castellanii cysts, A. castellanii trophozoites, and A. polyphaga trophozoites. CONCLUSIONS: The protocol that we have revised and evaluated is a well-controlled and reproducible procedure that can effectively evaluate the efficacy of MPS against Acanthamoeba trophozoites. Some variability was observed when testing the cyst stage.

Status of the effectiveness of contact lens disinfectants in Malaysia against keratitis-causing pathogens.

The aim of this study was (i) to assess the antimicrobial effects of contact lens disinfecting solutions marketed in Malaysia against common bacterial eye pathogens and as well as eye parasite, Acanthamoeba castellanii, and (ii) to determine whether targeting cyst wall would improve the efficacy of contact lens disinfectants. Using ISO 14729 Stand-Alone Test for disinfecting solutions, bactericidal and amoebicidal assays of six different contact lens solutions including Oxysept®, AO SEPT PLUS, OPTI-FREE® pure moist®, Renu® fresh™, FreshKon® CLEAR and COMPLETE RevitaLens™ were performed using Manufacturers Minimum recommended disinfection time (MRDT). The efficacy of contact lens solutions was determined against keratitis-causing microbes, namely: Pseudomonas aeruginosa, Methicillin-resistant Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, and Acanthamoeba castellanii. In addition, using chlorhexidine as an antiamoebic compound and cellulase enzyme to disrupt cyst wall structure, we determined whether combination of both agents can enhance efficacy of marketed contact lens disinfectants against A. castellanii trophozoites and cysts, in vitro. The results revealed that all contact lens disinfectants tested showed potent bactericidal effects exhibiting 100% kill against all bacterial species tested. In contrast, none of the contact lens disinfectants had potent effects against Acanthamoeba cysts viability. When tested against trophozoites, two disinfectants, Oxysept Multipurpose and AO-sept Multipurpose showed partial amoebicidal effects. Using chlorhexidine as an antiamoebic compound and cellulase enzyme to disrupt cyst wall structure, the findings revealed that combination of both agents in contact lens disinfectants abolished viability of A. castellanii cysts and trophozoites. Given the inefficacy of contact lens disinfectants tested in this study, these findings present a significant concern to public health. These findings revealed that targeting cyst wall by using cyst wall degrading molecules in contact lens disinfecting solutions will enhance their efficacy against this devastating eye infection.

Fibrous Catalyst-Enhanced Acanthamoeba Disinfection by Hydrogen Peroxide.

SIGNIFICANCE: Hydrogen peroxide (H2O2) disinfection systems are contact-lens-patient problem solvers. The current one-step, criterion-standard version has been widely used since the mid-1980s, without any significant improvement. This work identifies a potential next-generation, one-step H2O2, not based on the solution formulation but rather on a case-based peroxide catalyst. PURPOSE: One-step H2O2 systems are widely used for contact lens disinfection. However, antimicrobial efficacy can be limited because of the rapid neutralization of the peroxide from the catalytic component of the systems. We studied whether the addition of an iron-containing catalyst bound to a nonfunctional propylene:polyacryonitrile fabric matrix could enhance the antimicrobial efficacy of these one-step H2O2 systems. METHODS: Bausch + Lomb PeroxiClear and AOSept Plus (both based on 3% H2O2 with a platinum-neutralizing disc) were the test systems. These were tested with and without the presence of the catalyst fabric using Acanthamoeba cysts as the challenge organism. After 6 hours' disinfection, the number of viable cysts was determined. In other studies, the experiments were also conducted with biofilm formed by Stenotrophomonas maltophilia and Elizabethkingia meningoseptica bacteria. RESULTS: Both control systems gave approximately 1-log10 kill of Acanthamoeba cysts compared with 3.0-log10 kill in the presence of the catalyst (P < .001). In the biofilm studies, no viable bacteria were recovered following disinfection in the presence of the catalyst compared with ≥3.0-log10 kill when it was omitted. In 30 rounds' recurrent usage, the experiments, in which the AOSept Plus system was subjected to 30 rounds of H2O2 neutralization with or without the presence of catalytic fabric, showed no loss in enhanced biocidal efficacy of the material. The catalytic fabric was also shown to not retard or increase the rate of H2O2 neutralization. CONCLUSIONS: We have demonstrated the catalyst significantly increases the efficacy of one-step H2O2 disinfection systems using highly resistant Acanthamoeba cysts and bacterial biofilm. Incorporating the catalyst into the design of these one-step H2O2 disinfection systems could improve the antimicrobial efficacy and provide a greater margin of safety for contact lens users.

Evaluation of the anti-Acanthamoeba activity of two commercial eye drops commonly used to lower eye pressure.

Efficient treatments against Acanthamoeba Keratitis (AK), remains until the moment, as an issue to be solved due to the existence of a cyst stage which is highly resistant to most chemical and physical agents. In this study, two antiglaucoma eye drops were tested for their activity against Acanthamoeba. Moreover, this study was based on previous data which gave us evidence of a possible link between the absences of Acanthamoeba at the ocular surface in patients treated with beta blockers for high eye pressure both containing timolol as active principle. The amoebicidal activity of the tested eye drops was evaluated against four strains of Acanthamoeba using Alamar blue method. For the most active drug the cysticidal activity against A. castellanii Neff cysts and further experiments studying changes in chromatin condensation levels, in the permeability of the plasmatic membrane, the mitochondrial membrane potential and the ATP levels in the treated amoebic strains were done. Even though both eye drops were active against the different tested strains of Acanthamoeba, statistical analysis revealed that one of them (Timolol Sandoz) was the most effective one against all the tested strains presenting IC50s ranging from 0.529% ± 0.206 for the CLC 16 strain to 3.962% ± 0.150 for the type strain Acanthamoeba castellanii Neff. Timolol Sandoz 0.50% seems to induce amoebic cell death by damaging the amoebae at the mitochondrial level. Considering its effect, Timolol Sandoz 0.50% could be used in the case of contact lens wearers and patients with glaucoma.

Effects of Lead Phytochemicals of Radix Scutellariae on Acanthamoeba.

Purpose: Acanthamoeba keratitis (AK), which is associated with noncompliant use of contact lenses, remains difficult to treat due to delayed diagnosis and paucity of therapeutic agents. Although improvements in activity against Acanthamoeba infection have been achieved in disinfecting solutions for soft contact lenses, such modifications have not been extended to those for special rigid gas permeable (RGP) contact lenses, which are increasingly used for myopia control in children. Phytochemicals present in herbs used for traditional Chinese medicine may be effective as therapeutic or preventive agents. The purpose of this study was to investigate amoebicidal properties of lead phytochemicals of Radix scutellariae alone and in combination with multipurpose (disinfecting) solutions (MPS) for RGP lenses. Methods: Viability of Acanthamoeba castellani and A. polyphaga trophozoites was determined following exposure to four phytochemicals: baicalin, baicalein, wogonoside, and oroxylin A and both alone and in combination with four RGP MPS, using a modified stand-alone technique. Results: As individual agents, wogonoside and oroxylin A showed highest activity against A. castellani and A. polyphaga trophozoites, respectively. For both organisms, the combination of baicalein and oroxylin A was superior. Effectiveness of MPS alone did not exceed 0.27 log reduction, but addition of combined baicalein and oroxylin A resulted in 0.92 and 0.64 log reductions of A. castellani and A. polyphaga, respectively. Conclusions: The combination of baicalein and oroxylin A enhanced the activity of MPS for RGP contact lenses against trophozoites of two pathogens, A. castellani, and A. polyphaga, and offers a potential therapeutic and/or preventative agent for AK.

Microwave Irradiation as a Promising Method of Sterilization for Acanthamoeba polyphaga in Cultures.

PURPOSE: To determine the killing effect of microwave irradiation on Acanthamoeba polyphaga. METHODS: The trophozoites and cysts of A. polyphaga both in water and on agar were exposed to microwave irradiation with a capacity of 750 W for 0, 1, 3, 5, and 10 minutes, respectively. Furthermore, the trophozoites and cysts of A. polyphaga in water were exposed to microwave irradiation with a capacity of 100, 300, and 500 W for 1 minute, respectively. RESULTS: The trophozoites and cysts of A. polyphaga on agar were completely killed by 3 minutes of microwave irradiation with a capacity of 750 W. The trophozoites and cysts of A. polyphaga in water were completely killed by microwave irradiation with a capacity of 300 W for 1 minute. CONCLUSIONS: We demonstrate that microwave treatment is effective in killing A. polyphaga both in water and on agar and may be a helpful modality to prevent Acanthamoeba keratitis.

The use of dimethyl sulfoxide in contact lens disinfectants is a potential preventative strategy against contracting Acanthamoeba keratitis.

Acanthamoeba castellanii is the causative agent of blinding keratitis. Though reported in non-contact lens wearers, it is most frequently associated with improper use of contact lens. For contact lens wearers, amoebae attachment to the lens is a critical first step, followed by amoebae binding to the corneal epithelial cells during extended lens wear. Acanthamoeba attachment to surfaces (biological or inert) and migration is an active process and occurs during the trophozoite stage. Thus retaining amoebae in the cyst stage (dormant form) offers an added preventative measure in impeding parasite traversal from the contact lens onto the cornea. Here, we showed that as low as 3% DMSO, abolished A. castellanii excystation. Based on the findings, it is proposed that DMSO should be included in the contact lens disinfectants as an added preventative strategy against contracting Acanthamoeba keratitis.

Targeting cyst wall is an effective strategy in improving the efficacy of marketed contact lens disinfecting solutions against Acanthamoeba castellanii cysts.

Acanthamoeba cysts are highly resistant to contact lens disinfecting solutions. Acanthamoeba cyst wall is partially made of 1,4 ß-glucan (i.e., cellulose) and other complex polysaccharides making it a hardy shell that protects the resident amoeba. Here, we hypothesize that targeting the cyst wall structure in addition to antiamoebic compound would improve the efficacy of marketed contact lens disinfecting solutions. Using chlorhexidine as an antiamoebic compound and cellulase enzyme to disrupt cyst wall structure, the findings revealed that combination of both agents abolished viability of Acanthamoeba castellanii cysts and trophozoites. When tested alone, none of the agents nor contact lens disinfecting solutions completely destroyed A. castellanii cysts and trophozoites. The absence of cyst wall-degrading enzymes in marketed contact lens disinfecting solutions render them ineffective against Acanthamoeba cysts. It is concluded that the addition of cyst wall degrading molecules in contact lens disinfecting solutions will enhance their efficacy in decreasing the incidence of Acanthamoeba effectively.

Strategies for the prevention of contact lens-related Acanthamoeba keratitis: a review.

PURPOSE: Acanthamoeba keratitis is a severe, often sight threatening, corneal infection which in Western countries is predominantly seen in daily wear of contact lenses. This review aims to summarise the pathobiology and epidemiology of contact lens-related Acanthamoeba keratitis, and to present strategies for prevention, particularly with respect to modifiable risk factors in contact lens wear. RECENT FINDINGS: The virulence of Acanthamoeba and resistance to treatment in keratitis appears to be linked with the production of a low molecular weight protease MIP133 by the organism, in response to binding to corneal epithelial cells through a mannose binding protein, and to the ability of the organism to convert from the trophozoite to the resistant cyst form. Recent epidemiological studies in contact lens relate disease have confirmed the link between solution topping up and Acanthamoeba keratitis and have reinforced the importance of avoidance of tap water, either as part of the care for the contact lens or storage case, handling lenses with wet hands or showering while wearing lenses. In the most recent analysis from the USA, there were no strong effects for solution type, water source or water disinfection process. Wearer age, lens wear time and history to appear to be linked with Acanthamoeba keratitis. Daily disposable contact lens use would be expected to reduce the prevalence of Acanthamoeba disease although this is unproven. SUMMARY: While Acanthamoeba keratitis remains challenging to diagnose and manage, strategies to limit the disease severity in contact lens wearers should include attention to recently identified risk factors, particularly those related to water contact. Public health awareness measures, the use of daily disposable contact lenses, a better understanding of the contribution of the host immunity and the development of standardised methods for culture of amoeba and testing of contact lens care systems against Acanthamoeba in the licensing process may be of value. Alternative treatments for the future may include those which target the mannose binding protein or the genes which control conversion to the cyst form.

Contact lens care solution killing efficacy against Acanthamoeba castellanii by in vitro testing and live-imaging.

In the past decade there has been an increased incidence of Acanthamoeba keratitis, particularly in contact lens wearers. The aim of this study was to utilize in vitro killing assays and to establish a novel, time-lapse, live-cell imaging methodology to demonstrate the efficacy of contact lens care solutions in eradicating Acanthamoeba castellanii (A. castellanii) trophozoites and cysts. Standard qualitative and quantitative in vitro assays were performed along with novel time-lapse imaging coupled with fluorescent dye staining that signals cell death. Quantitative data obtained demonstrated that 3% non-ophthalmic hydrogen peroxide demonstrated the highest percent killing at 87.4% corresponding to a 4.4 log kill. The other contact lens care solutions which showed a 72.9 to 29.2% killing which was consistent with 4.3-2.8 log reduction in trophozoite viability. Both analytical approaches revealed that polyquaternium/PHMB-based was the least efficacious in terms of trophicidal activity. The cysticidal activity of the solutions was much less than activity against trophozoites and frequently was not detected. Live-imaging provided a novel visual endpoint for characterizing the trophocidal activity of the care solutions. All solutions caused rapid rounding or pseudocyst formation of the trophozoites, reduced motility and the appearance of different morphotypes. Polyquaternium/alexidine-based and peroxide-based lens care system induced the most visible damage indicated by significant accumulation of debris from ruptured cells. Polyquaternium/PHMB-based was the least effective showing rounding of the cells but minimal death. These observations are in keeping with care solution biocides having prominent activity at the plasma membrane of Acanthamoeba.

Miraflow, Soft Contact Lens Cleaner: Activity Against Acanthamoeba Spp.

OBJECTIVE: Miraflow is a cleaner for soft contact lens which contains 20% isopropyl alcohol. The purpose of this study was to determine the activity against Acanthamoeba trophozoites and cysts for Miraflow. In addition, to determine the activity of combined Miraflow and multipurpose solutions (MPSs) against Acanthamoeba cysts. METHODS: Two simulated-use studies were conducted. The significance in the log reduction in the number of trophozoites and cysts of A. castellanii strains ATCC 50514 and ATCC 50370 or A. polyphaga ATCC 30461 after exposure to Miraflow alone was determined by the Spearman-Karber method. To examine the activity against Acanthamoeba of combined Miraflow and an MPS, the log reduction in the number of cysts after a 1-min exposure to Miraflow followed by a 4-hr exposure to MPS (ReNu fresh) was also determined. RESULTS: Short-time exposure of 30 sec to Miraflow demonstrated activity against the Acanthamoeba trophozoites. However, a 1-min treatment was only relatively effective (1.1 log reduction) against the cysts of A. castellanii ATCC 50514, but no statistically significant reduction was observed for the cysts of the other 2 strains. The combined use with Miraflow and MPS demonstrated activity against the cysts, and a 3.0, 1.0, or 1.5 log reduction in the numbers was obtained for A. castellanii ATCC 50514, A. castellanii ATCC 50370, and A. polyphaga ATCC 30461, respectively. CONCLUSIONS: Exposure to combined Miraflow and MPS resulted in reductions in the number of Acanthamoeba cysts.

Proteomic profiling of the infective trophozoite stage of Acanthamoeba polyphaga.

Acanthamoeba polyphaga is a free-living protozoan pathogen, whose infective trophozoite form is capable of causing a blinding keratitis and fatal granulomatous encephalitis in humans. The damage caused by A. polyphaga trophozoites in human corneal or brain infections is the result of several different pathogenic mechanisms that have not yet been elucidated at the molecular level. We performed a comprehensive analysis of the proteins expressed by A. polyphaga trophozoites, based on complementary 2-DE MS/MS and gel-free LC-MS/MS approaches. Overall, 202 non-redundant proteins were identified. An A. polyphaga proteomic map in the pH range 3-10 was produced, with protein identification for 184 of 370 resolved spots, corresponding to 142 proteins. Additionally, 94 proteins were identified by gel-free LC-MS/MS. Functional classification revealed several proteins with potential importance for pathogen survival and infection of mammalian hosts, including surface proteins and proteins related to defense mechanisms. Our study provided the first comprehensive proteomic survey of the trophozoite infective stage of an Acanthamoeba species, and established foundations for prospective, comparative and functional studies of proteins involved in mechanisms of survival, development, and pathogenicity in A. polyphaga and other pathogenic amoebae.